Poster Abstracts

P01. Alzheimer and Related

P01‐1

A role for microglia in ageing and dementia

Boche D

University of Southampton, Southampton, UK

Genetic risk factors for AD have been recently identified in genome wide association studies which involve the innate inflammatory response implying that inflammation plays a causal role in AD. Using the brain donor resource of the MRC Cognitive Function and Ageing Study (CFAS), we aim to clarify the role of microglia in human brain ageing and dementia.

Frontal cortex from 298 CFAS cases (age at death: 77–93; 267 with known APOE genotype), Mini‐Mental State Examination (MMSE) and Alzheimer pathology (scores) were analyzed for microglial phagocytic activity (CD68); a cell surface lipoprotein receptor involved in direct ligand recognition specific for amyloid (macrophage scavenger receptor (MSR)‐A) and the Fcg receptor I (CD64) a cell surface receptor with high affinity for monomeric IgG‐type antibody. All analyses were adjusted for age of death and sex.

Overall, MMSE was associated negatively with CD68 (β = –8.55; 95% CI (β) = –11.09; –6.01) and positively with CD64 (β = –1.14; 95% CI (β) = –2.45; 0.17). No significant association was observed with MSR‐A. Among the cases without dementia, positive associations were observed for CD64 and CD68 with diffuse plaques, negative associations with neuritic plaques and none with tangles. In the cases with dementia and AD pathology, diffuse plaques were associated with all microglial markers, the strongest association being with CD64 (OR 21.59, 95% CI (β) = 14.22; 32.78); neuritic plaques were also associated with all microglial markers with the strongest association with CD68 (OR 49.28, 95% CI (β) = 11.54; 86.81). Tangles were significantly associated with CD68 (OR 49.28, 95% CI (β) = 19.43; 124.99) and MSR‐A (OR 3.24, 95% CI (β) = 1.74; 6.04). With regard to the APOE polymorphism, ε2 seems associated with the expression of CD64 and MSR‐A and ε4 with CD68 and CD64.

These data suggest that microglia may respond differently to Aβ and tau in subjects with and without dementia so that the microglial response may influence the likelihood of developing dementia. Interestingly the findings also suggest that APOE polymorphisms may influence the microglial profile. Additional data are being generated for Iba1 (resting and activated microglia), and also for TREM2 (gateway influencing phagocytic vs proinflammatory microglial activity) and HLA‐DR (surface antigen presenting function) both recently identified as genetic risk factors for AD.

P01‐2

Aβ immunotherapy for Alzheimer's disease: effects on apoE and cerebral vasculopathy

Nicoll J

University of Southampton, Southampton, UK

Aβ immunotherapy for Alzheimer's disease (AD) results in the removal of Ab plaques and increased cerebral amyloid angiopathy (CAA). In current clinical trials amyloid‐related imaging abnormalities (ARIA), putatively due to exacerbation of CAA, are concerning side effects. We aimed to assess the role of the Ab transporter apolipoprotein E (apoE) in the exacerbation of CAA and development of CAA‐associated vasculopathy after Aβ immunotherapy. 12 Aβ42‐immunized AD (iAD; AN1792, Elan Pharmaceuticals) cases were compared with 28 unimmunized AD (cAD) cases. Immunohistochemistry was quantified for Aβ42, apoE, apoE E4 and smooth muscle actin, and CAA‐associated vasculopathy was analyzed. Aβ immunotherapy was associated with re‐distribution of apoE from cortical plaques to cerebral vessel walls, mirroring the altered distribution of Aβ42. Concentric vessel wall splitting was increased threefold in leptomeningeal vessels after immunotherapy (cAD 6.3% vs iAD 20.6%, P < 0.001), but smooth muscle cell abnormalities did not differ. The findings suggest that apoE is involved in the removal of plaques and transport of Aβ to the cerebral vasculature induced by Aβ immunotherapy. Immunotherapy was not associated with CAA‐related vascular smooth muscle damage, but was accompanied by increased splitting of the vessel wall, perhaps reflecting enhanced deposition and subsequent removal of Aβ. ARIA occurring in some current trials of Ab immunotherapy may reflect an extreme form of these vascular changes.

P01‐3

AB42 senile plaques and blood brain barrier associated vasoactive and amyloid transport proteins in Alzheimer's disease – a correlation analysis

Provias J¹ and Jeynes B²

¹ McMaster University HHS; ² Brock University, Canada

The accumulation of Aβ₄₂ within senile plaques [SP] is characteristic of these lesions in Alzheimer's disease. The superior temporal [ST] cortex is particularly susceptible. The pathogenesis of B amyloid accumulation and SP development is still a matter to be resolved. The accumulation of Aβ₄₂ in the ST cortical interstitium may result from overproduction from amyloid precursor protein (APP) or an inability of the blood brain barrier (BBB) to homeostatically regulate the trans‐endothelial transport and clearance of the amyloid. Lipoprotein receptor related protein (LRP) and P‐glycoprotein (P‐gp) facilitate the efflux of β‐amyloid out of the brain as specific B amyloid transport proteins, whereas receptor for advanced glycation end products (RAGE) facilitates the influx. In addition, both vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) influence endothelial cell function and possibly the trans‐BBB transport of B amyloid. In this study, in ST samples, we examined and compared SP burden of all types with the capillary immunohistochemical expression of LRP, p‐gp, RAGE, VEGF and e‐NOS in samples from 15 normative and 15 Alzheimer brains. LRP, P‐gp RAGE, VEGF and eNOS positive capillaries and Aβ₄₂ plaques [SP] were quantified and statistical analysis of the non‐parametric data was performed using the Mann–Whitney and Kruskal–Wallis tests. In the Alzheimer condition P‐gp, eNOS and VEGF positive capillaries were negatively correlated with SP burden; but LRP and RAGE were positively correlated with SP burden. These results support the role of an altered BBB clearance system in the pathogenesis of SPs and Aβ₄₂ senile plaque burden in Alzheimer brains.

P01‐4

Alzheimer's disease: the beginning

Graeber MB

Brain and Mind Research Institute, The University of Sydney, Camperdown, NSW, Australia

Alzheimer's disease (AD) is set to become the most important human disease due to its increasing frequency and economic impact. Alois Alzheimer discovered neurofibrillary tangles in the brain of Auguste D., his original patient (1). It is likely that observing Alzheimer's second case, Johann F., convinced Kraepelin to name the disease after Alzheimer. The rediscovered histological slides of Auguste D. and Johann F. (2) are of great practical value. They allow the rejection of ill‐founded etiological hypotheses published before the original slides were rediscovered, e.g. Auguste D. representing a case of metachromatic leukodystrophy or vascular dementia. It is of great interest today that there is no evidence of inflammation in Alzheimer's original cases. This 20 years‐long research project would not have been possible without the timely contributions of K. Fujisawa (Japan) who stimulated the original search (2), the late H. De F. Webster (USA) who mediated his contact to Munich, the late P. Mehraein (Germany) who rescued the “anonymous” material, G. W. Kreutzberg (Germany) whose department assisted with reagents from Spielmeyer's time, R. B. Banati (now Australia) who provided a crucial piece of information, A. Hirano (USA) whose enthusiasm for Johann F.'s slides in Perth (at the ISN's 1997 meeting) made me start a new and eventually successful search for the sections of Auguste D.'s brain, and last but not least U. Muller (Germany) whose laboratory identified the presenilin 1 mutation in her brain (3). A promised online atlas has been delayed due to events in London but the project is still alive.

References:  1.  Alzheimer A. Übereineeigenartige Erkrankung der Hirnrinde. Allg Zeitschr Psychiatr 1907; 64:146–148.

2.  Graeber MB. No man alone: the rediscovery of Alois Alzheimer's original cases. Brain Pathol 1999; 9:237–240.

3.  Müller U, Winter P, Graeber MB. A presenilin 1 mutation in the first case of Alzheimer's disease. Lancet Neurol 2013; 12:129–130.

P01‐5

Density of metabotropic glutamate receptor type 5 (mGluR5) in Alzheimer's disease

Guiot M‐C¹, Minuzzy L² and Rosa‐Neto P²

¹ Montreal Neurological Hospital and Institute, McGill University, Montreal, Canada; ² Douglas Research Institute, McGill University, Montreal, Canada

Introduction: Quantification of molecular processes directly affected by Alzheimer's disease (AD) has potential application for monitoring disease progression. Preclinical work has demonstrated that oligomeric forms of beta amyloid peptide impose deleterious effects on glutamatergic neurotransmission, particularly on metabotropic glutamate receptor type 5 (mGluR5). We aim to demonstrate abnormal mGLUR5 binding in AD and hypothesize decline in mGLUR5 concentration in AD and mild cognitive impairment patients.

Methods: We conducted a cross sectional study with banked tissue, comparing mGLUR5 availability in two populations: (1) postmortem AD patients and controls and (2) patients with no cognitive impairment and mild cognitive impairment. A saturation binding study was carried out on frozen sections using [3H]ABP688. Non‐specific binding was determined with addition of the mGLUR5 antagonist MPEP in adjacent sections. The binding of [3H]ABP688 was calculated by saturation binding analysis and compared using F‐test (p < 0.05).

Results: Single concentration binding using [3H]ABP688 revealed higher binding in the control group (Ctrl 1.31 ± 0.06; AD 0.711 ± 0.03 pmol/mg of tissue). Binding of [3H]ABP688 in hippocampus was blocked after addition of 10 μM MPEP, around 50% reduction in total concentration of receptors in AD hippocampi was statistically significant (p < 0.001).

Conclusion: Decline of mGluR5 found in AD patients are part of neuroreceptor changes in early AD, they result in downregulation of signaling pathways involved in neuronal excitability and cell survival.

P01‐6

Distinct patterns of sirtuin expression during progression of Alzheimer's disease

Kovacs GG, Lutz MI, Milenkovic I and Regelsberger G

Institute of Neurology, Medical University of Vienna, Vienna, Austria

Aging is one of the major risk factors for Alzheimer's disease (AD). Sirtuins are associated with prolonged lifespan. We were interested whether the expression levels of sirtuins associate with the progression of AD. We performed a comparative immunoblotting and immunohistochemical study of SIRT1, 3, and 5 in the entorhinal cortex and hippocampal subregions and white matter in 45 cases grouped according to Braak and Braak stages of neurofibrillary degeneration. We evaluated the load of tau and amyloid‐beta deposits with morphometry and compared with the expression levels of sirtuins. Our study revealed that the neuronal subcellular redistribution of SIRT1 parallels the decrease of its expression suggesting step‐wise loss of neuroprotection dependent on the neuronal population. In contrast to SIRT1 and 3, expression of SIRT5 increases during the progression of AD. This associates with the appearance of SIRT5 expression in microglia. The complex patterns of the expression of sirtuins in relation to tissue damage should be taken into account when searching for therapies interacting with sirtuins.

Disclosure: Supported by European Commission's 7th Framework Programme under GA No 278486, “DEVELAGE”.

P01‐7

Failure of elimination of Aβ from the brain in Alzheimer's disease and cerebral amyloid angiopathy

Weller RO, Hawkes CA, Jayakody N and Carare RO

Clinical Neurosciences, Faculty of Medicine, University of Southampton, UK

Introduction: Failure of elimination of Aβ from the brain is reflected in the accumulation of Aβ in brain parenchyma and in artery walls as cerebral amyloid angiopathy (CAA). Degradation of Aβ by neprilysin, absorption of Aβ into the blood, and drainage of Aβ along perivascular lymphatic drainage pathways all appear to fail with age. Age and ApoEε4 (APOE4) are major risk factors for sporadic Alzheimer's disease. Here we review the reasons why perivascular drainage of Aβ fails with age and APOE4 and examine the effects of CAA on the anatomy of cerebral arteries.

Methods: (a) Perivascular drainage of soluble Aβ was assessed in aged mice and in APOE4 mice (b) human CAA was examined by confocal microscopy using immunocytochemistry for Aβ and basement membrane proteins.

Results: Significant impairment of perivascular drainage was observed in the hippocampus, but not the thalamus of aged mice, and in the presence of APOE4 and CAA. In human arteries, deposits of fibrillar Aβ separate the layers of smooth muscle basement membrane.

Conclusions: Reduction in the motive force for perivascular drainage of Aβ with age‐related stiffening of artery walls and changes in the composition of basement membranes is associated with impaired elimination of Aβ. Similar impairment is seen in the presence of APOE4. Aβ progressively accumulates in basement membranes of artery walls leading to disruption of perivascular drainage pathways and of artery walls themselves. Facilitating the drainage of Aβ along ageing arteries may prevent the accumulation of Aβ in the brain in Alzheimer's disease.

Disclosure: None.

P01‐08

Familial dementia associated with the novel PSEN1 F176V mutation

Ghetti B, Oblak A, Richardson R, Epperson F and Murrell J

Division of Neuropathology, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

Auguste Deter, Alzheimer's first patient had the PSEN1 F176L mutation. We now report a novel mutation at the same codon. The subject, a female, developed memory impairment at 59 years and, on neurological examination, it was concluded that she was in the early stages of Alzheimer disease. Short‐term memory progressively deteriorated in the ensuing years. At age 69, her mini mental status exam score was 20. At age 70, a computed tomography scan revealed mild diffuse cerebral atrophy and at age 71, PET scan with fluorodeoxyglucose revealed hypometabolism in the posterior parietal region of the left cerebral hemisphere. Her cognitive status continued to deteriorate and she died at age 77. Presenile dementia was traced back three generations preceding that of the proband. At autopsy, the brain weighed 838 grams. Sequencing of PSEN1 gene revealed a TTT to GTT point mutation predicting a substitution of phenylalanine to valine at residue 176. There was moderate‐to‐severe atrophy of the head of the caudate nucleus and severe atrophy of the hippocampus and parahippocampal cortex. For neuropathology, Luxol fast blue/H&E, Bielschowsky and thioflavin S stains were obtained. For immunohistochemistry, antibodies against tau, beta‐amyloid and alpha‐synuclein were used. Numerous beta‐amyloid‐immunopositive plaques, tau‐immunopositive neurons and neuropil threads were seen throughout the cerebral cortex. The hemispheric white matter showed marked loss of myelinated fibers. Amyloid angiopathy and parenchymal beta‐amyloid deposition were severe in the cerebellum. Additional studies on these kindred are needed to clarify the clinical and pathologic phenotypes associated with the PSEN1 F176V mutation.

P01‐09

In vitro properties of [¹⁸F]NAV4694: dynamic range, displacement and white‐matter binding

Guiot M‐C¹, Zimmer ER², Leuzy A², Cheewakriengkrai L², Mohades S², Schilling L², Benedet AL², Schirrmacher ES², Soucy J‐P², Kostikov A², Reininger C³, Gauthier S² and Rosa‐Neto P²

¹ Montreal Neurological Hospital and Institute, McGill University, Montreal, Canada; ² Douglas Research Institute, McGill University, Montreal, Canada; ³ Navidea Biopharmaceuticals, Dublin, OH, USA

Introduction: [¹¹C]PIB is the benchmark PET agent for quantifying amyloid in Alzheimer's disease; however, carbon‐eleven short half‐life limits its use. We analyzed in vitro properties of a new agent [¹⁸F]NAV4694 (longer half‐life) using autoradiography. We compared [¹⁸F]NAV4694 and [¹¹C]PIB dynamic range and non‐specific binding, investigated binding site similarities through competitive binding assay between [¹⁸F]NAV4694 and PiB. We predict wider dynamic range and lower non‐specific binding.

Methods: Autoradiography was performed using [¹⁸F]NAV4694 (60 Ci/mmol) and [¹¹C]PIB (302 Ci/mmol), along with a competition study using [¹⁸F]NAV4694 and cold‐PiB on controls and AD samples (prefrontal, hippocampus, parietal, cingulate and cerebellum). Specific binding was calculated using cerebellum as reference region.

Results: [¹⁸F]NAV4694 showed a wider dynamic range than [¹¹C]PiB (prefrontal: 5.3 vs. 3.2; hippocampus: 1.35 vs. 0.59; parietal 6.13 vs. 1.24; cingulate: 4.66 vs. 2.5 z‐scores). No differences were found in cerebellum. The ratio between cerebellum and white matter was 0.61 ± 0.06 for [¹⁸F]NAV4694 and 0.46 ± 0.07 for [¹¹C]PIB. Competition with cold‐PiB (range: –9.30 to –7.49 log M) almost fully displaced specific binding in the prefrontal (∼96%; Ki ∼ 3.692e^–10M), parietal (∼97%; Ki ∼2.923e^–10M) and cingulate (∼95%; Ki ∼3.587e^–10M), but not in hippocampus (∼76%; Ki ∼3.263e^–10M).

Conclusion: Despite molecular similarities, [18F]NAV4694 presents wider dynamic range than [11C]PIB possibly due to higher affinity (K_D = 2.4 nM). The competition study shows that [¹⁸F]NAV4694 cannot be fully displaced by [¹¹C]PIB in hippocampus, which suggests binding to early forms of amyloid. These robust in vitro properties support the unique binding properties of [¹⁸F]NAV4694.

P01‐10

Presenile dementia associated with PSEN1 N135S mutation: coexistence of Alzheimer disease and diffuse Lewy body disease

Ghetti B¹, Murrell J¹, Farlow M², Unverzagt F³, Oblak A¹, Epperson F¹, Richardson R¹, Risacher S⁴, Saykin A⁴, Morris J⁵, Cairns N⁵

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine; ² Department of Neurology; ³ Department of Psychiatry and ⁴ Department of Radiology, Indiana University School of Medicine, Indianapolis, IN, USA; ⁵ Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA

The phenotype of Alzheimer disease (AD) associated with PSEN1 mutations is not uniform. A male presented with gait difficulties at age 39. He became repetitive and had word finding difficulties at age 41. Subsequently, he developed generalized seizures and had a rapid decline. At age 42, he became apraxic; tremor was absent. Spasticity predominantly involved the lower extremities; he had bilateral Babinski signs. His speech was strained and spastic. His Clinical Dementia Rating was 3.0, consistent with severe dementia. At baseline and follow‐up, [18F]FDG and [11C]PiB PET showed generalized hypometabolism and a positive amyloid signal, most evident in the striatum. He died at age 43. At autopsy, the brain weighed 1530 grams. For histology, Luxol fast blue/H&E and thioflavin S were used. For immunohistochemistry, antibodies against tau, beta‐amyloid, glial fibrillary acidic protein, and alpha‐synuclein were used. Beta‐amyloid cored plaques and diffuse deposits were seen in the neocortex and hippocampus, while diffuse beta‐amyloid deposits were predominant in the striatum and cerebellum. Amyloid angiopathy was severe. Tau‐immunopositive neurons and neuropil threads were numerous in the cerebral cortex and the pons. Alpha‐synuclein‐immunopositive Lewy bodies and neurites were abundant in frontal and cingulate cortices, and substantia nigra. Sequencing of PSEN1 gene revealed an A to G point mutation predicting an amino acid substitution of asparagine to serine at residue 135. The neuropathologic phenotype associated with the PSEN1 N135S mutation was reported previously; however, the present case is noteworthy for the coexistence of diffuse Lewy body disease with severe AD. (Funding sources: P30AG010133, U19AG032438).

P01‐11

Protein expression of Alzheimer's disease – and reduced hippocampal volume – risk genetic loci in human hippocampus

Delalle I¹, Norman T¹, Tilton K¹ and Seshadri S²

Departments of ¹ Pathology and ² Neurology, Boston University School of Medicine, Boston, MA, USA

Aging is associated with the occurrence of Alzheimer's disease (AD) and reduction in hippocampal volume. We examined the protein expression of novel genes implicated in both events using immunohistochemistry on paraffin‐embedded postmortem hippocampal CA1 sections with no or mild‐to‐moderate AD‐associated pathological changes, accompanied with the CDR (Clinical Dementia Rating) scale scores. Genome‐wide association studies (GWAS) have established BIN1 as the most important AD susceptibility locus after APOE. We found BIN1 protein expressed strongly in CA1 glial somata of all the examined cases while the punctate signal in neuropil varied substantially. EPHA1, another confirmed AD risk locus, is an ephrin receptor implicated in brain‐region specific apoptosis. We found EphA1 in the cytoplasm of neurons and glia; however, the quality of signal (homogeneous vs. granular) appeared to vary with AD changes. Methionine sulfoxide reductase B3 (MSRB3) locus, critical for oxidative stress, was identified GWAS studies on hippocampal volume. We found MSRB3 signal as discrete cytoplasmic puncta in CA1 pyramidal neurons from all of the examined cases, albeit in various amounts. We also examined the expression of LEMD3, a possible causal gene at the MSRB3 locus, and observed it in either some or virtually all CA1 neurons and glia, depending on the degree of AD changes. Our data provide first insight into the expression of AD – and hippocampal volume reduction – risk associated genes in human hippocampi in health or early degeneration. Further quantitative analysis of these distinctly expressed genes is underway to examine the correlation with cognitive decline.

P01‐12

Pyroglutamylated amyloid‐β correlates with hyperphosphorylated tau and severity of Alzheimer's disease

Walker L¹, Mandler M², Santic R², Hanson P³, Upadhaya AR⁴, Colloby SJ², Morris CM³, Thal DR⁴, Thomas AJ², Schneeberger A¹ and Attems J²

¹ Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK; ² AFFiRiS AG, Vienna Biocenter, Vienna, Austria; ³ Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; ⁴ Laboratory of Neuropathology, Institute of Pathology, Center for Clinical Research, University of Ulm, Ulm, Germany

Objectives: Pyroglutamylated amyloid‐β (pE‐Aβ) has been suggested to play a major role in Alzheimer's disease (AD) pathogenesis as it might initiate tau‐dependent cytotoxicity. We aimed to further elucidate the associations between pE‐Aβ, Aβ and hyperphosphorylated tau (HPT) in human brain tissue.

Methods: We examined 41 postmortem brains (mean age 79.6 years, SE: ±1.5) of both AD (n = 18) and controls. Adjacent sections from each frontal and entorhinal cortex, were stained with pE‐Aβ, HPT and Aβ specific antibodies. We used image analysis to quantitatively assess loads for pE‐Aβ, HPT and non‐pE‐Aβ.

Results: All loads were significantly higher in AD as compared to controls (p ≤ 0.01). However, regression analysis revealed that only frontal pE‐Aβ load independently predicted the presence of AD (p = 0.01). In frontal and entorhinal cortices pE‐Aβ load independently predicted HPT load (p < 0.001). All loads correlated with neurofibrillary tangle Braak stages and Thal Aβ phases (p < 0.01). Mini Mental State Examination scores were predicted by entorhinal HPT load independent of other entorhinal loads (p < 0.001) and by frontal pE‐Aβ load independent of frontal non‐pE‐Aβ (p = 0.01).

Conclusion: Here, we report an association between pE‐Aβ and HPT in human brain tissue and an influence of frontal pE‐Aβ on both the severity of AD neuropathological change and clinical dementia. Our findings further support the notion that pE‐Aβ may represent an important link between Aβ and HPT and investigations into its role as diagnostic and therapeutic target in AD are warranted.

P01‐13

Quantification of pathological lesions detects clinico‐pathological phenotypes of mixed AD/LBD

Walker L, Thomas AJ and Attems J

Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK

Objectives: Cases that neuropathologically fulfill criteria for both Alzheimer's disease (AD) and Lewy body disease (LBD – dementia with Lewy bodies (DLB) or Parkinson's disease dementia (PDD)) are classified as mixed AD/LBD. Using semi‐quantitative scoring, no differences were detected in mixed AD/LBD between cases that presented clinically with AD or LBD. We investigated whether a quantitative approach could reveal differences in the amounts of AD and LBD pathology in mixed AD/LBD cases.

Methods: 19 mixed AD/LBD cases, which presented clinically as AD, DLB or PDD, 5 pure AD and 5 pure DLB cases were stained for antibodies against tau, β amyloid and α‐synuclein and percentage burden of each pathology was quantified in neocortical, limbic and brainstem regions.

Results: A significant increase in the amount of tau pathology was observed in the hippocampus (p < 0.05), striatum (p < 0.05), neocortex (p < 0.05) and locus coeruleus (p < 0.01) in clinically AD compared to LBD patients, respectively. When comparing neuropathologically mixed AD/LBD with pure AD and DLB cases we observed an increase in all 3 protein aggregates in the substantia nigra in the mixed AD/LBD group. Moreover, we found a significant correlation between hyperphosphorylated tau and αsynuclein (p < 0.01 and R ² = 0.616) in mixed AD/LBD group.

Conclusion: Our findings suggest that cases neuropathologically diagnosed as mixed AD/LBD can be separated into different clinico‐pathological phenotypes when quantitative neuropathological methods are applied. However, the question whether the clinical diagnosis reflects the primary underlying pathology, which may be confounded by concomitant pathologies, warrants further investigation.

P01‐14

Raft derived tau associated vesicles are component of neurofibrillary tangle

Nishikawa T, Takahashi T, Nakamori M, Nagano Y, Maruyama H and Matsumoto M

Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences, Japan

Introduction: Alzheimer's disease (AD) pathologically manifests amyloid plaques and neurofibrillary tangles (NFTs). Granulovacuolar degeneration (GVD) bodies are also pathological hallmarks of AD. We reported that GVD bodies are immunopositive for charged multivesicular body protein 2B (CHMP2B), suggesting that GVD body is related to multivesicular body. We also reported that both cyclin‐dependent kinase 5, one of tau kinases, and phosphatidylinositol 4,5‐bisphosphate[PtdIns(4,5)P2], one of lipid raft associated materials, exist in NFTs as well as GVD bodies. These results suggest that the generation of GVD bodies and NFTs could have the common mechanisms.

Materials and Methods: Brain specimens from five AD cases, 17 other neurodegenerative disease cases and four control cases were subjected to immunohistochemical staining, using anti‐PtdIns(4,5)P2, anti‐CHMP2B, anti‐RD3 and anti‐RD4 antibodies, and Gallyas–Braak method. In addition, we investigated the relationship between the number of GVD bodies and the area of PtdIns(4,5)P2‐positive structures within individual neuron in CA1 at GVD stage 1 and 5. Moreover, we observed the PtdIns(4,5)P2‐positive structures in detail using electron microscopy.

Results: PtdIns(4,5)P2‐positive small structures were close to phosphorylated tau and immunonegative for CHMP2B. Gallyas–Braak method revealed that these vesicles existed in pretangle and early stage of NFTs. We also observed that paired helical filaments were intermingled with these vesicles using electron microscopy. Statistical analysis showed the number of GVD bodies and the area of PtdIns(4,5)P2‐positive structures were inverse correlation.

Conclusion: We identified the raft derived tau associated vesicles within pretangle and early stage of NFTs. These vesicles could be precursor of GVD bodies.

P01‐15

Sphingolipids deregulation in Alzheimer's disease

Delisle MB¹, Ceccom J¹, Loukh N¹, Lauwers‐Cances V¹, Touriol C¹, Nicaise Y¹, Gentil C¹, Uro‐Coste E¹, Pitson S², Maurage CA³, Duyckaerts C⁴ and Cuvillier O⁵

¹ Neuropathology Department, CHU Toulouse, University of Toulouse III, Toulouse, France; ² Center for Cancer Biology, Australia; CHU Lille, University of Lille, France; ⁴ Salpetrière Hospital, Paris, France; ⁵ University of Toulouse, France

The anti‐apoptotic sphingosine 1‐phosphate (S1P) and the pro‐apoptotic ceramide are involved in cellular fate set by sphingolipid balance. Exposition of cells to amyloid peptide (Aβ) induces a downregulation of sphingosine kinase 1 (SphK1) activity (Gomez‐Brouchet et al., 2007) which is involved in S1P synthesis. Downregulation of SphK1 led to S1P level reduction, increase of ceramide level and cellular death. Our main objective was to determine in vivo, the relation between amyloid deposits and the expression of SphK1 within neurons. Subsequent objectives focused on targets involved in S1P metabolism: S1P receptor (S1P1), an activator of SphK1 (IGF‐1R), sphingosine kinase 2 (SphK2) and sphingosine 1‐phosphate lyase (SPL) which eliminate S1P. Immunohistochemical study on 56 late stage Alzheimer's disease (AD) patients showed a negative correlation between the expression of SphK1 and the density of amyloid deposits while SPL showed the inverse relation. Immunoblot assays showed decreased expression of SphK1 and increased expression of SPL in AD compared to controls. SphK2 expression did not vary but this need to be refined as this kinase relies on 3 isoforms, not yet investigated. Decreased IGF‐1R expression in AD cases corroborated our results from in vitro study. Finally, S1P1 expression was dramatically decreased in AD cases. These original data reveal an imbalance in S1P synthesis and elimination systems, linked to Aβ deposits, which might play a crucial role in AD (Ceccom et al., 2014) and identified promising targets which could serve as therapeutic levers to reverse the imbalance of sphingolipid rheostat and in‐fine redirect cells to survival behavior.

P01‐16

The interactive role of capillary VEGF and eNOS in the pathogenesis of AB42 senile plaques in Alzheimer's disease

John Provias¹ and Brian Jeynes²

¹ McMaster University HHS; ² Brock University, Canada

Fifteen Alzheimer [AD] and fifteen normative [NM] age‐matched autopsy brains were examined within hippocampal [HC], brainstem [BS] and superior temporal cortex [ST] samples. VEGF‐ and eNOS‐positive capillaries and β‐amyloid₄₂ senile plaques were quantified. The data were statistically analyzed using Mann–Whitney, Kruskal–Wallis and non‐parametric Spearman's test. There was a significantly different expression of capillary VEGF [p < 0.05] between the NM and AD conditions when comparing comparable inter‐condition site pairs; the VEGF expression being consistently greater in the normative condition. There were no significant differences between any comparably paired sites in either condition for VEGF capillary expression. There was no significant difference with respect to eNOS expression when comparing the NM and AD conditions in any condition. In both conditions there were significant differences between BS samples and both ST and HC sample pairs [p < 0.018–0.001]. Both VEGF and eNOS expression were negatively correlated with the presence of SP₄₂ senile plaques in the ST site [p < 0.01] in the AD condition. There was a strong positive correlation for eNOS and VEGF capillary expression only in the AD condition and only in the ST and HC sites (p < 0.01 and p < 0.01, respectively). These two cerebral capillary regulators are likely contributory, and likely interactive, in AD pathogenesis by altering beta amyloid burden.

P01‐17

Transition from 4R to 3R tau underlies dendrosomatic and regional progression of neurofibrillary pathology

Uchihara T¹, Hara M¹, Kamei S² and Hirokawa K³

¹ Structural Neuropathology, Tokyo Metropolitan Inst Medical Sci; ² Neurology, Nihon Univ, School of Medicine; ³ Pathology, Nakano General Hospital, Japan

Regional progression of neurofibrillary tangles (NFTs) around the hippocampus was traced on sections double immunofluorolabeled with RD3 and RD4 antibodies (Brain Pathol 2011; 21:180–188; Histochem Cell Biol 2012; 137:261–267), specific for 3R and 4R tau, respectively. Density of tau‐positive neurons was predominant in the entorhinal cortex and cornu ammonis (CA)1, and decreased progressively to the CA2–4 subregions. This regional gradient was replicated with RD3+/4– and RD3+/4+ neurons, while RD3–/4+ neurons exhibited the reverse gradient. Comparison of the subregion pairs confirmed a consistent profile shift along this gradient. To clarify the underlying mechanism of this profile shift, intraneuronal intensity of RD3 and RD4 immunoreactivity (IR) was quantified. Although their intensities were both lower in dendrites than in the soma, this gradient was steeper with RD4, leaving RD3 IR in dendrites. Dendritic arborization was abundant in RD3–/4+ pretangles, attenuated in RD3+/4+ neurons, and further attenuated in RD3+/4– ghost tangles, suggesting that dendritic RD4 IR retracts first, leaving RD3 IR in the dendrites. Taken together, this dendrite‐oriented retraction initiates the gradual shift from RD3–/4+ pretangle neurons to RD3+/4– ghost tangles by way of RD3+/4+ NFTs. This intraneuronal profile shift may be a basis for the regional gradation featured by the similar profile shift during progression of NFT pathology (Acta Neuropathologica 2013; 125:565–579).

P02. Ataxia and Related

P02‐1

The pathology of CANVAS – a new sensory neuronopathy

McLean CA¹, Szmulewicz DJ², Rodriguez M³, Pollard J⁴, Roberts L⁵, Halmagyi M⁶ and Storey E¹

¹ Alfred Health, Melbourne; ² The Eye and Ear Hospital, Melbourne; ³ Forensic Institute, Sydney; ⁴ University of Sydney; ⁵ St. Vincents Hospital, Melbourne; ⁶ Royal Prince Alfred Hospital, Sydney; ⁷ Alfred Health, Melbourne, Australia

Cerebellar ataxia with neuronopathy and bilateral vestibular areflexia syndrome (CANVAS) is a recently described slowly progressive ataxic disorder presumed to be autosomal recessive.

From a cohort of 80 patients including 13 kindred, 8 sural nerves were examined during life and four autopsies performed on a 71, 81, 85, and 90 year old with a 10–40 year history. Patients developed; imbalance, cerebellar dysarthria, cerebellar oculomotor abnormalities and appendicular ataxia with decreased vestibular reflexes, patchy to global decreased somatic sensory perception and decreased to absent deep tendon reflexes. Gene testing excluded Friedreich ataxia, SCA 1, 2, 3, 6 and 7.

Sural nerve examination showed 100% (8/8) with severe axonal loss. The main autopsy findings included cerebellar vermal atrophy due to Purkinje cell loss with variable atrophy of the lateral hemispheres and preservation of the dentate nucleus. Selective neuronal loss was seen from ganglia of dorsal root and cranial nerves V, VII and VIII (specifically Scarpas not cochlear). Vestibular nerves were demyelinated and atrophic with preserved cochlear nerves. Posterior column secondary demyelination was seen.

CANVAS is a new progressive ataxic syndrome with pathology highlighted by cerebellar atrophy and loss of neurons from dorsal root and specific cranial nerve ganglia with secondary changes related to axonal loss seen in posterior columns, cranial and sural nerves. CANVAS should be considered in the autopsy differential of neuronal loss from dorsal root and specifically Scarpas ganglia with cerebellar vermal atrophy and in the in life differential of peripheral sural nerve biopsies with severe axonal loss.

P03. Behavioral

P03‐01

The insula cortex and experience of disgust

Da Silva WG¹, Caixeta LF¹, Silva LMBM², Leite MB³ and de Paula EC¹

¹ UFG; ² Anhanguera Educacional; ³ BIOCITO, Brazil

Introduction: The insula is located deep to the lateral sulcus. Its cortex is associated with the functions of dislike, disgust experience and inability to interpret signs of disgust/aversion and also the physiognomies instinctive behaviors related to survival and control of blood pressure (BP). Atrophy of insula was not described in Pick's disease, in the form of clinical symptoms related to disgust/aversion, vocal expressions of disgust, or correlated to BP observed in this disease. In cases of insular atrophy associated with other diseases, however, changes were observed in instinctive behavior: subjects were not disgusted with toxic potential of food and non‐edible biological material.

Materials and Methods: Case study in a patient with Pick's disease.

Discussion of Results: PC, 62‐year‐old male, was getting progressively placid, apathetic, emotionally indifferent to their families, egocentric, unable to abstract. Had constant fluctuations in blood pressure levels. Began to eat leftover food and even feces, indicating impaired disgust/aversion, in addition to dietary changes and indiscriminate dietary behavior (Kluver–Bucy syndrome). This type of syndrome is associated with limbic circuitry related to orbitofrontal–insular–temporal cortex. The autopsy showed marked atrophy of the left insular cortex.

Conclusion: Lesions of the insula cortex in Pick's disease may be associated with manifestations of placidity and lack of experience of disgust/aversion, as well as changes in blood pressure.

P04. Cell Biology and Pathology

P04‐01

Different vulnerability of cultured glioma cells (A172 and C6) to co‐administration of temozolomide and sulfasalazine

Ignarro RS, Facchini G, Avansini SH, Melo DR, Vieira AS, Athié MCP, Dogini DB, Rocha KJP, Lopes‐Cendes I, Ferreira CV, Castilho RF and Rogerio F

UNICAMP, Brazil

Temozolomide (TMZ) is an alkylating agent used to treat high‐grade gliomas. The antioxidant glutathione may be synthetized by glioma cells by importing cysteine through a cystine/glutamate antiporter, which is inhibited by sulfasalazine (SAS). We treated human glioblastoma (A172) and rat glioma (C6) cells for 3 days with 25 μM TMZ and/or 0.5 mM SAS and evaluated cellular viability, metalloproteinase‐2 (mmp2) gene expression/activity and apoptosis. MTT colorimetric assay showed that neither TMZ nor SAS alone significantly reduced viability of A172 cells. Co‐administration of TMZ and SAS led to a significantly reduction of 64% in A172 cellular viability compared with vehicle‐treated group. However, the percentage of apoptotic A172 cells, detected through flow cytometric TUNEL assay, did not differ from vehicle‐treated group after TMZ and/or SAS exposure. Zymography showed no modification of invasiveness of the remaining cells after all treatments. Regarding C6 lineage, TMZ showed a no significant trend for viability reduction. Surprisingly, SAS caused cell viability reduction of nearly 100%, compared with vehicle‐treated group. Co‐administration of TMZ and SAS induced a similar response. 48% and 5.3% of the cells were TUNEL‐positive after SAS and vehicle treatment, respectively. Quantitative PCR analysis showed a fivefold increase in mmp2 gene expression after TMZ and/or 0.25 mM SAS. Nevertheless, such increase was not followed by alteration in enzyme activity. Our data showed an additive cytotoxic effect of TMZ and SAS on A172 cells and C6 lineage vulnerability to SAS. The latter finding could be due to oxidative stress induced by SAS. Financial support: FAPESP (2013/02618‐1), FAEPEX and CAPES.

P04‐02

Effects of propentofylline in astrocytic response following gliotoxic injury

Bondan EF and Dossa PD

University Paulista, Brazil

Propentofylline (PPF) is a xanthine derivative that depresses activation of microglial cells and astrocytes, whose responses usually contribute to neural tissue damage during inflammation and hypoxia. It is known that ethidium bromide (EB) injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation, blood–brain barrier disruption and Schwann cell invasion. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). In such context, the aim of this study was to evaluate if PPF had the capacity of affecting astrocytic response following gliotoxic injury. Wistar rats were divided into groups: I – rats injected with 10 μL of 0.1% EB into the cisterna pontis and treated with PPF (12.5 mg/kg/day by intraperitoneal route during the experimental period); II – rats injected with EB but without receiving the xanthine. The rats were euthanized from 7 to 31 days after EB injection and brainstem sections were collected and processed for GFAP immunohistochemical staining astrocytic reactivity was determined in a computerized system for image analysis. In PPF‐treated rats, GFAP‐stained area around lesion was significantly smaller at 7 days after EB injection than in untreated animals. Similar observations were found at 15 and 21 days, but no difference was noted between groups at 31 days. Results demonstrate that PPF decreased astrocytic activation as estimated through GFAP expression from 7 to 21 days after neural damage, suggesting that this drug may have a role in preventing or reducing glial scar development following injury.

P04‐03

Evaluation of redox balance in glioblastoma cell lines after exposition to retinol or phytol

Facchini G, Ignarro RS, Castilho RF and Rogerio F

UNICAMP, Brazil

Glioblastoma is the most common primary brain tumor. Apoptosis in glioblastoma xenograft implants has been reported in nude mice treated with the antioxidant retinol (RET). In order to better understand the cytotoxic effects of RET on glioblastoma, we assessed the viability of two cell lines (U87‐MG and T98G) exposed to 10–60 μM RET or phytol (PHY), a retinoid receptor ligand, for 48 h. Redox status was evaluated through activity of superoxide dismutase (SOD) and catalase (CAT) and by treating the cells with Trolox^®, an antioxidant. MTT colorimetric assay showed cytotoxicity of RET and PHY (IC₅₀ for U87‐MG: 18,60 μM RET and 21,32 μM PHY; IC₅₀ for T98G: 6,25 μM RET and 2,78 μM PHY). Redox balance was supported by significant modulation of SOD and CAT activity by RET and PHY. Regarding U87‐MG, SOD activity was increased by 10 μM RET and reduced by 30 and 60 μM RET. Moreover, all concentrations of PHY inhibited SOD activity. All treatments with RET or PHY reduced CAT activity. Concerning T98G cells, RET decreased SOD activity only at 60 μM and PHY reduced SOD activity at all concentrations. RET decreased CAT activity at 15, 30 and 60 μM. PHY showed no significant changes in CAT activity. Furthermore, co‐treatment with Trolox^® (100 μM) intensified the cytotoxicity induced by 30 μM RET or 30 μM PHY alone, suggesting that redox balance may be paramount to T98G and U87‐MG cell demise after RET or PHY treatment. Financial support: FAPESP (2013/02618‐1), CAPES and FAEPEX.

P04‐04

Implications for lysophosphatidylcholine in periinfarct microglial activation

Inose Y, Yamamoto T, Kato Y, Kitagawa K, Uchiyama S and Shibata N

Department of Pathology, Tokyo Women's Medical University, Japan

Several studies have suggested a critical role for periinfarct microglial activation in determining lesion size in acute stage cerebral infarction. Given previous reports showing upregulation of phospholipases A2 in periinfarct zone, the enzyme reaction product lysophosphatidylcholine (LPC) is a candidate of proinflammatory mediator activating periinfarct microglia. To address this issue, we investigated the effects of LPC stimulation on the murine microglia‐derived BV‐2 cell line. BV‐2 cells were maintained. After 24 h serum starvation, cells were incubated for 12 h with or without 20 μM LPC in the presence or absence of preincubation for 1 h with inhibitors of Rho kinase, inhibitor of κB‐α kinase (IKK), phosphatidylinositol‐4,5‐bisphophate 3‐kinase, mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated kinase kinase, c‐Jun‐N‐ternminus kinase, and p38 MAPK. After harvested, total RNA was extracted from cell lysates and used for reverse transcription‐quantitative polymerase chain reaction analysis, using specific primer sets for mRNA of interkeukin‐1β (IL‐1β), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein‐1 (MCP‐1), and its receptor CCR2. The IL‐1β, iNOS, and MCP‐1 but not CCR2 transcription levels were significantly increased by the LPC stimulation group as compared to the vehicle group. The LPC‐driven increases of the IL‐1β, iNOS, and MCP‐1 but not CCR2 transcripts were significantly abrogated by Rho kinase or IKK inhibitor but not other inhibitors. The present results provide in vitro evidence that LPC activates microglia to upregulate the neuroxic IL‐1β, iNOS and MCP‐1 via the Rho kinase and IKK pathways.

P04‐05

Mitochondrial energy metabolism in proliferation of cultured U87 and T98G glioma cells

Ruas JS, Ignarro RS, Santos ES, Rossato FA, Melo DR, Rogério F and Castilho RF

Universidade Estadual de Campinas (UNICAMP), Brazil

Most cancer cells rely on glycolysis for ATP turnover during rapid proliferation despite the availability of saturating levels of oxygen for mitochondrial energy transduction. The aim of the present study was to evaluate the role of mitochondrial oxidative metabolism in the proliferation of human glioma cells U87 and T98G. When the cells were cultured in the presence of the either oligomycin (an ATP synthase inhibitor) or antimycin (a respiratory chain complex III inhibitor), only a partial inhibition of cell proliferation was observed. Remarkably, the incubation of these cells with both inhibitors caused an almost complete inhibition of cell proliferation. Oxygen consumption measurements indicated that glioma cells partially rely on oxidative phosphorylation for ATP turnover and exhibit a well‐coupled respiration. In fact, shutting down mitochondrial ADP phosphorylation in these glioma cells with either oligomycin or antimycin inhibitors slightly increased glucose consumption and lactate release. However, the treatment with both mitochondrial inhibitors promoted lower glucose consumption and lactate release as compared with the effects of antimycin alone, which indicates that ATP synthase is operating reversely and thus hydrolysing ATP and pumping H⁺ out when the respiratory chain is inhibited by antimycin. In agreement, a full collapse of mitochondrial membrane potential was only observed when the glioma cells were incubated in the presence of both antimycin and oligomycin, but not of only antimycin. Overall, our results suggest that rapid proliferation of glioma cells relies on mitochondrial membrane potential, but not on oxidative phosphorylation or electron transport in the respiratory chain.

P04‐06

Protective effect of valproic acid (VPA) on cultured motor neurons under glutamate excitotoxic conditions. Ultrastructural study

Naganska E, Matyja E, Taraszewska A, Grzywaczewska E, Gebarowska J and Janina R

Department of Experimental and Clinical Neuropathology, M. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

Valproic acid (VPA), a short‐chained fatty acid, is widely used for the treatment of seizures and bipolar mood disorder. The real mechanisms underlying numerous beneficial effects of VPA remain complex. It has been reported that VPA directly inhibits histone deacetylase (HDAC) and might play the role in the neuroprotection. The aim of this ultrastructural study was to evaluate the effect of VPA on the motor neurons (MNs) damages in a Glu excitotoxic model in vitro. It has been previously documented that chronic Glu excitotoxicity resulted in various MNs injury related with necrotic, apoptotic and autophagocytic mode of neuronal cell death. The present experiments were performed on 14‐day organotypic cultures of the rat lumbar spinal cord that were subjected to 100 μM glutamate for 3, 6 and 12 days. The cultures were pretreated with 10 μM VPA. Ultrastructural findings revealed time‐dependent neuroprotective effect of valproic acid against Glu neurotoxicity. The pretreatment with VPA effectively prevents Glu‐induced MNs apoptosis and autophagy. The typical apoptotic changes displaying peripheral condensation and aggregation of nuclear chromatin were seen only occasionally. The features of MNs autophagy and autophagosomes were rarely encountered. Some neurons exhibited cytoplasmic vacuolization and slightly damaged mitochondria but totally destructed cells appeared only sporadically. Numerous well‐preserved large motor neurons could be identified. These data documented the neuroprotective efficacy of VPA in experimental model of neurodegeneration related with GLU excitotoxicity in vitro. The prevention of neuronal excitotoxic changes was related with inhibition of apoptosis and autophagy. It could be suggested that this mechanism may underlay the VPA‐induced therapeutic effects in neurodegenerative processes.

P05. Cerebrospinal fluid

P05‐1

Cerebrospinal fluid cytology in a cohort of pediatric patients with metastatic medulloblastoma

Andreiuolo F^1,2, Valent A¹, Calderaro J³, Beaugrand A¹, Pages M², Varlet P², Grill J¹, Dufour C¹ and Vielh P¹

¹ Institut Gustave Roussy, Villejuif; ² Centre Hospitalier Sainte‐Anne, Paris; ³ Hôpital Henri Mondor, Créteil, France

Introduction: High‐risk criteria for medulloblastoma are age under 3 years, the presence of metastases and incomplete surgical resection. Studies on morphology of metastatic cells in CSF are scarce. The aim of this study was to evaluate morphological criteria for the diagnosis of metastatic medulloblastoma in CSF.

Patients and Methods: Retrospective analysis of CSF cytology was performed in specimens from a cohort of pediatric patients with metastatic medulloblastoma diagnosed by imaging and/or CSF cytology treated from 1989 to 2009.

Results: 129 CSF samples from 63 patients were reviewed. 45 «positive» results were rendered, defined as clusters of at least 2 tumor cells (n = 28 patients). Most common morphologic findings were irregular nuclear shape (45/45), high nuclear to cytoplasmic rate (45/45), and nuclear molding (43/45). Nucleoli were easily identified on 16/45 samples. 7 samples were considered «suspicious» of which 6 showing suspicious clusters and 1 only isolated suspect cells (n = 6 patients). Among the 45 positive cases 12 showed only one tumor cell cluster, 6 had 2 clusters, 7 had between 3 and 10 clusters and 20 had more than 10 cell clusters. 15 patients had at least one discordant result (a positive and a negative result) from different specimens.

Conclusion: Tumor cells were found in CSF in 28/63 patients with metastatic medulloblastoma. Cell clusters and irregularly shaped nuclei were the most characteristic findings. In 7 cases, the diagnosis of positive CSF was uncertain.

P05‐02

Importance of cytological examination of cerebrospinal fluid in the follow‐up of patients treated in a cancer hospital

de Albuquerque AKAC, Chimelli L, do Nascimento MF and Eisenberg ALA

INCA, Brazil

Introduction: The cytopathology of cerebrospinal fluid (CSF) is very important to investigate the occurrence of primary and secondary central nervous system (CNS) tumors disseminated in the subarachnoid space, especially when associated with clinical and radiographic findings.

Material and Methods: Cytopathology of the CSF in 276 patients with malignant neoplasms treated at the National Cancer Institute (2000–2010) considered as positive and suspicious for malignancy were reviewed by three pathologists. Clinical and radiographic findings were surveyed from medical records and radiology reports.

Results: After reviewing the slides, the 276 patients were reclassified into three categories consensually: 178 (64.5%) positive, 23 (8.3%) suspected and 75 (27.2%) negative for malignancy. Neurological findings were referred to 164 (88.6%) patients, of whom 114 (69.5%) were positive for malignancy. Headache was the most frequent neurological symptom (44 patients, 26.8%), followed by the involvement of cranial nerves (35 patients, 21.3%). Radiological abnormalities were identified in 151 (66.2%) patients, of whom 96 (63.6%) were positive for malignancy. The intraparenchymal brain lesion was observed in 83 (55.0%) patients and meningeal contrast enhancement, in 60 (39.7%) patients. Among patients with known primary tumors, 64 (23.2%) were primary and 207 (75.0%) were metastatic. Hematological diseases were the most common (107 patients, 38.8%), followed by carcinomas (74 patients, 26.8%) and the neuroepithelial neoplasms (53 patients, 19.2%).

Conclusion: An understanding of the indications and limitations of this diagnostic tool is important to ensure greater effectiveness of its use in clinical practice.

P06. Clinical

P06‐01

Application of predictive nursing intervention for patients with severe anti‐N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis

Gao L, Deng Q, Du H, Lin H and Li H

Department of Neurology, The First Hospital of Jilin University, Changchun, China

Introduction: Anti‐N‐methyl‐D‐aspartate receptor (anti‐NMDAR) encephalitis is newly identified immune‐related encephalitis with characteristics including psychosis, seizures, impaired consciousness, central hypoventilation and movement disorders. The need for critical care and duration of ICU stay is prolonged in severe anti‐NMDAR encephalitis patient. This is a report of 7 cases to observe the effect of predictive nursing intervention for patients with anti‐NMDAR encephalitis.

Patients and Methods: A total of 7 patients admitted to our hospital from December 2012 to May 2014 were diagnosed with anti‐NMDAR encephalitis. These patients were treated with predictive nursing intervention including predictive nursing for central hypoventilation, artificial airway, preventing rhabdomyolysis and acute renal failure, epileptic seizures, drug adverse reaction, plasmapheresis, deep vein thrombosis (DVT) and nutrition support.

Results: Five female patients ranged from 19 to 31 in age. Age of two male patients is 47 and 58. Six patients were positive for anti‐NMDAR antibody from both cerebrospinal fluid (CSF) and serum, one was positive only from CSF. Mental and behavior disorders are the first sign for all patients. Four had central hypoventilation and received mechanical ventilation for 7–21 days. All patients had seizure and orofacial, limbs and trunk movement disorders during the course of disease. Five patients’ condition improved and they were discharged with mental dysfunction, and with no other clinical complication. No unplanned extubation, VAP, rhabdomyolysis, DVT and malnutrition occurred during the hospitalization. One patient had poor prognosis, and the other one died.

Conclusions: Application of predictive nursing intervention is advantageous to the prognosis of patient with severe anti‐NMDAR encephalitis.

P06‐02

Effects of enteral plus parenteral nutrition on early nutrition parameters and immune function in neurocritical patients

Cao J, Zhang H, Sun L and Gao L

The First Hospital of Jilin University, Changchun, China

Introduction: Neurocritical patients often have nutrition risk or malnutrition due to intense catabolism, dysphagia, disturbance of consciousness, gastric dynamic obstacles. The purpose of this paper is to evaluate the effects of different methods of nutrition support on early nutrition and immune parameters in neurocritical patients.

Patients and Methods: Sixty‐one patients admitted to our NICU from June 2013 to February 2014 who need nasogastric tube enteral nutrition were randomly divided into three groups: EN group, EN + early parenteral nutrition (EN + EPN) group and EN+ supplemented parenteral nutrition (EN + SPN) group. Target calorie level is 25 kcal/(kg.d). We compared basic data, early (7 days after admission) serum protein and immune parameters between groups.

Results: Age, gender, admission GCS score, protein and immune parameter level of patients in groups has no differences. Comparison among groups and intra‐group after treatment, serum level of total protein, albumin, pre‐albumin and transferrin is obviously decreased 7 days after admission in EN group (P < 0.05). Pre‐albumin and retinol binding protein levels increased after 1 week nutrition support in EN + EPN group and EN + SPN group (P < 0.05). The level of total protein albumin, pre‐albumin and transferrin in EN + EPN and EN + SPN group is higher than in EN group (P < 0.05). Serum immunoglobulin A and G level in EN + EPN group is higher than in EN group (P < 0.05). Total lymphocyte count in EN + EPN and EN + SPN group is also higher than in EN group (P < 0.05).

Conclusion: Single EN at early stage may cause nutrition risk. EN + EPN or EN + SPN providing enough calories can reduce incidence of malnutrition and immune dysfunction in neurocritical patients.

P06‐03

Effects of modified intensive insulin therapy on neurocritical patients

Cao J, Zhang H, Sun L and Gao L

The First Hospital of Jilin University, Changchun, China

Introduction: Hyperglycemia is associated with increased mortality and poor outcomes in neurocritical patients. Intensive insulin therapy may reduce the mortality, but the optimal glycemic target is controversial.

Patients and Methods: We conducted a prospective, randomized and controlled study of 187 adult patients admitted to our NICU from March 2013 to January 2014. Patients were divided into two groups: in one group patients received modified intensive insulin therapy to maintain the blood glucose (BG) 6.1–8.3 mmol/L, in another group patients received conventional insulin therapy to keep BG 8.3–10.0 mmol/L. Serum albumin, duration of hospitalization, incidence of infection, tracheal intubation and tracheotomy rate, days of mechanical ventilation, mortality, 30 days MRS score and incidence of hypoglycemia were observed to evaluate the safety and effectiveness of modified insulin therapy on clinical outcome.

Results: Ninety‐five patients were retained for final analysis. Incidence of infection and artificial airway rate in modified intensive insulin therapy group is obviously lower than that in conventional insulin group (p < 0.05). Serum albumin reduction in conventional insulin group is lower than that in modified insulin therapy group (p < 0.05). Incidence of hypoglycemia, duration of hospitalization, mechanical ventilation, mortality, 30 days MRS were not significantly different between two groups.

Conclusion: It suggested that modified intensive insulin therapy is safe and effective, and the blood glycemic level 6.1–8.3 mmol/L is appropriate target in the neurocritical patients.

P06‐04

Gastric retention related risk factors and prognosis analysis in critically ill patients

Liu C¹, Gao L², Luan S¹, Jin L² and Zhang H ²

¹ Psychology Department, The First Hospital of Jilin University, Changchun, China; ² Neurology Department, The First Hospital of Jilin University, Changchun, China

It was reported that there is a widespread malnutrition in hospitalized patients, especially in the neurological ICU (NICU). By observing occurrence of the gastric retention (GR) in the enteral nutrition (EN), analyzing of the relevant factors, we may find a reasonable nutritional support. A total of 73 EN patients were encompassed in the NICU (2012.7–2013.2), energy supply: 20–25kcal/(kg.d), more than 1 week. Gastric residues were sucked every 6 h, and patients were divided into GR group and non‐GR group according to whether the gastric residues were more than 200 mL or not. General information, serum hemoglobin and albumin, serum immune parameters and treatment strategy were compared between groups on the day of admission and the end of EN. GCS scores were documented respectively. The data were statistically analyzed using SPSS 19.0 software, line count data were compared using chi‐square test and multivariate analysis using logistic regression analysis further stepwise regression analysis, measurement data ($\stackrel{\circledR }{x}\pm \mathrm{s}$), using the t‐test, non‐normal distribution of measurement data with the rank sum test. The general information, the serum hemoglobin and albumin were comparable between the two groups at admission. GR was negatively related to the GCS scores (p < 0.05). Serum immune parameters showed no difference at 1 week of EN between the 2 groups, but IgG and IgA were significantly decreased in GR than that in non‐GR at 3 weeks (p < 0.05). EN plus parenteral nutrition (PN) in the GR and total EN in the non‐ER had the same nutritional support efficacy. However, EN may improve neurological immune function in the long run.

Keywords: neurology, critical care, gastric retention, nutritional support, risk factors, prognosis.

P06‐05

Partial characterization of the population of patients with medulloblastoma diagnosed at the Brazilian National Cancer Institute (INCA) from 2002 to 2012

Santos Z and Chimelli L

National Cancer Institute, Brazil

This work is the result of the partial characterization of the study population of a project titled: “Meduloblatomas: prognostic accuracy and basis for new therapies,” in which will be described the distribution of the 4 subgroups of medulloblastomas according to Taylor et al. (2012), by immunohistochemistry according Northcott et al. (2011) and by fluorescence in situ hybridization (FISH), in a population of patients from the Instituto Nacional do Câncer (INCA) diagnosed between January 2002 and December 2012. Through medical records review and review of slides, initially 87 patients met the criteria for inclusion in the study, namely, the biopsy where from the primary lesion, cases containing paraffin blocks archived in the INCA and there is enough biological material to make all future tests immunohistochemistry and FISH. It was observed that there is a predominance of male patients, most patients were younger than 16 years old at diagnosis, histological subtype most frequently found was the classic subtype. In this population, there were 31 deaths. Among the patients who died during this period, all had partial surgical removal of the tumor and most patients survived less than 2 years after diagnostic. We hope to correlate the current results with the 4 subtypes of medulloblastomas in order to personalize the treatment of patients according to their prognosis prognosis and reduce their comorbidities.

P06‐06

Relative risk factors and prognosis analysis of DVT in neurocritical patients

Gao L, Zhang H, Sun L and Cao J

The First Hospital of Jilin University, Changchun, China

Introduction: We observed the incidence, clinical features and relative risk factors of deep vein thrombosis (DVT) in neurocritical patients in order to find strategies in early prevention, early diagnosis and avoid occurrence of adverse consequences.

Patients and Methods: Neurocritical patients without limb vein thrombosis in NICU were recruited in the First Hospital of Jilin University from June to November 2013. A total of 86 patients were divided into DVT group and non‐DVT group according to ultrasound examination on 5 d, 10 d and 15 d after admission. Basic data, clinical conditions, treatment and prognostic parameters were compared between two groups.

Results: History of diabetes mellitus and drinking, APACHE‐II score at admission, white cell count, thrombocytocrit, blood fat and homocysteine level were obviously different between two groups (P < 0.05). Higher APACHE‐II scores were correlated with the high incidence of DVT positively. Incidence of infection, hypoalbuminemia, hypotension, hypothermia therapy, sedative usage, endotracheal intubation is higher in DVT group (P < 0.05). Logistic regression analysis showed diabetes, drinking history, infection, dyslipidemia, hypotension, hypoalbuminemia, hyperhomocysteinemia, hypothermia, sedatives and endotracheal intubation are positively related to DVT (OR 2.267–7.452, CI 1.027–36.553, P < 0.05). Incidence of DVT during first 5 d after admission is 53.49%. The duration of hospitalization is longer and mortality is higher in DVT group significantly (P < 0.05).

Conclusion: Diabetes, drinking history, infection, dyslipidemia, hypotension, hypoalbuminemia, hyperhomocysteinemia, hypothermia, sedatives and endotracheal intubation are relative risk factors of DVT. Incidence rate of DVT is high during first 5 d in NICU, and early prevention should be done for neurocritical patients with risk factors.

P06‐07

Research on incidence and feature of right‐to‐left shunt: a 195 normal population study

Yan S, Zhang H, Gao L and Bai J

Department of Neurology, The First Hospital of Jilin University, Changchu, China

Introduction: The most common cause of right‐to‐left shunt (RLS) is patent foramen ovale. Recent study showed that it played a role in the occurrence of migraine. The incidence, shunt volume and shunt type of normal population and features of RLS were not known.

Patients and Methods: One hundred ninety‐five volunteers were enrolled; the clinical data were collected and contrast‐enhanced transcranial Doppler (cTCD) were performed. They are divided into two groups: young (18–45 years old) and middle‐aged group (older than 45 years old). The physiological saline activating via elbow intravenous injection as a contrast agent and combining with Valsalva maneuver, RLS was diagnosed by power M‐model TCD monitoring and the shunt volume was measured. The distribution features between volume and type (the inherent and potential) were also analyzed.

Result: RLS incidence rate was 28.7%. Thirty‐nine were defined as minor shunt (20%). RLS rate in young groups was 28.9% (39/135), and 28.3% (17/60) in middle‐aged group. The incidence rate and shunt volume distribution were not significantly different between two groups. Minor shunts took a predominant percentage in all shunts. Thirty cases were inherent type (60.7%), and 22 cases potential type (39.3%). In young group, 25 cases were inherent type (64.0%), and 14 cases potential type (35.9%). In middle‐aged group, 9 cases were inherent type (52.9%), and 8 cases potential type (47.1%). There was no statistical difference between the two groups in shunt type distribution.

Conclusion: The incidence of RLS in the selected sample was about 28.7%, and the minor shunt took a predominant percentage. There was no significant difference between young and middle‐aged groups in RLS incidence, shunt volume and type.

Keywords: contrast‐enhanced transcranial Doppler, right‐to‐left shunt, normal population.

P06‐08

The clinical features and prognosis of patients with hypernatremia in neurological ICU: a retrospective study

Liu C¹, Cao J², Luan S¹, Chen Z² and Zhang H²

¹ Psychology Department, The First Hospital of Jilin University, Changchun, China; ² Neurology Department, The First Hospital of Jilin University, Changchun, China

Hypernatremia is common in critically ill patients in neurological ICU (NICU). It's associated with duration of hospitalization, mortality and worse prognosis. However, it didn't attract much attention. This study was to investigate the incidence, clinical features, risk factors and prognosis of patients with hypernatremia in NICU. A total of 200 patients in NICU were recruited of our hospital (2011.9.1–2012.7.1), hypernatremia group (HN, n = 47) and non‐hypernatremia group (NHN, n = 153). χ² test, t‐test, rank‐sum test and multi‐factor non‐conditional logistic regression model were utilized to assess hypernatremia predisposing factors. General information of the two groups (age, agenda, variety of diseases, hypertension, diabetes) showed no significant difference (p > 0.05). The incidence of hypernatremia was 40.4% in cerebral hemorrhage, 31.9% in cerebral infarction, 21.3% in intracranial infection and 6.4% in others. The incidence rate was not significantly different between diseases (p > 0.05) and so the mortality rate was. The average hospitalization time was 10.09 ± 9.91 days in HN and 9.27 ± 9.28 days in the NHN; while not statistically significant different (p < 0.05). Mortality rate (70.2%) in the HN was significantly higher than that in the NHN (45.8%), the different prognosis of patients in the two groups was significant (p < 0.05). Mortality rate in HN was higher than that in NHN group; hypernatremia was an independent risk factor affecting prognosis of critically ill patients in NICU and had no relationship to the variety of diseases. The application of large dose of mannitol, mechanical ventilation and renal dysfunction are risk factors of the hypernatremia.

Keywords: neurological critically ill patients, hypernatremia, risk factors, clinical features.

P06‐09

Limbic encephalitis‐like syndrome with voltage‐gated potassium channel complex antibodies may be associated with colangiocarcinoma

Schmidt SL, Tolentino Jr JC, Alvarenga RP, Simões EN, Schmidt JJ, Schmidt GJ and Canedo NHS

State and Federal Universities of Rio de Janeiro, Brazil

Limbic encephalitis (LE) was originally described as a rare cliniconeuropathological entity, involving seizures and psychological disturbances. Most patients with typical LE and voltage‐gated potassium channel complex (VGKC) antibodies usually do not have a tumor. Here we described a 77‐year‐old male patient who 3 years before his death exhibited persistent cognitive changes involving executive deficits. At that time two positron emission tomography scans indicated absence of any abnormalities with the exception of low activity in the frontal lobes. Six months before his death he presented normal pressure hydrocephalus and autonomic seizures. LCR analysis showed the presence of VGKC antibodies. At that time a new PET scan indicated the presence of a tumor confirmed by MRI. Post‐mortem examination showed colangiocarcinoma with multiple metastases including lungs and lymph nodes. The brain weighed 1300 g, a catheter was well positioned in the lateral ventricle. There was mild cortical atrophy, ex‐vacuum dilatation of the ventricles and mild focal thickening of the cerebellar leptomeninges, which were infiltrated by neoplastic epithelial cells. There were no other changes in the cerebral cortex and white matter, except some thickened hyalinized microvessels in the deep white matter with adjacent gliosis and calcification of vessel walls in the basal ganglia. An axonal peripheral neuropathy associated with microangiopathy, possibly related to diabetes, was also seen. These data indicated a possible association between LE‐like syndrome with VGKC antibodies and colangiocarcinoma.

P07. Disease Model

P07‐01

PGW5, a novel sarcosinyl‐linked olanzapine, exhibits high efficacy in rat schizophrenia models, without metabolic side effects

Gil‐Ad I, Portnoy M², Weller A³ and Weizman A¹

¹ Biological Psychiatry Lab, Felsenstein Medical Research Center, Campus Rabin, Tel‐Aviv University; ² School of Chemistry, Sackler Faculty of Exact Sciences, Tel‐Aviv Univesity; ³ Dept. of Psychology and the Gonda Brain Research Center, Brain Sciences Program, Bar‐Ilan University, Israel

Background: Schizophrenia is a chronic disease associated with hypofunction of glutamate transmission. Atypical neuroleptics induce weight gain and metabolic syndrome associated with medical comorbidities. We have developed a novel antipsychotic drug, PGW5, possessing an olanzapine moiety linked to positive modulators of NMDA.

Objectives: 1. Evaluate the efficacy of PGW5 in rat models of schizophrenia. 2. To determine the metabolic profile of PGW5 compared to olanzapine in a rat model of weight gain.

Methods: Efficacy studies (open field and Morris water maze [MWM]) were performed in male SD rats following exposure to the anti‐NMDA agent; MK801. PGW5 (10, 20 and 40 mg/kg po) was administered 90 min before testing. Weight gain was tested in female rats exposed for 16 days to oral olanzapine or PGW5 at bio‐equivalent doses (based on efficacy+ Ki levels for D2 receptors).

Results: In the open field tests, PGW5 effectively antagonized MK801‐induced hyperactivity without sedation. PGW5 but not olanzapine improved spatial cognitive performance in the MWM. In adult female rats, PGW5 unlike olanzapine administered daily (16 days), did not induce weight gain or alteration in lipid profile. Olanzapine, but not PGW5, increased the hippocampal orexigenic NPY5 receptor, and decreased the anorexigenic peptide POMC expression in the hypothalamus.

Conclusions: PGW5 shows neuroleptic activity without the side effects characteristic of olanzapine. PGW5 is a potential candidate for treatment of positive and negative symptoms of schizophrenia. The possible mechanisms underlying the differential effects of PGW5 and olanzapine may be related to brain GAD67, NPY and POMC.

Acknowledgement: The development of PGW5 was licensed to Mental‐Heal. For more information please contact Dr. Santiago Ini at santiago@naiot.com.

P07‐02

Propentofylline provides antioxidant protection and increases myelin repair in AR at model of demyelination

Bondan EF, Baliellas DEM, Gimenez CFM, Martins MFM, Pereira AAF, Barros MP and Poppe SC

University Paulista and University Cruzeiro do Sul, São Paulo, Brazil

Propentofylline (PPF) is a methylxanthine that depresses activation of microglia and astrocytes. Pathological glial activation contributes to progressive neural damage in neurodegenerative and neuroinflammatory diseases and PPF has been proposed to provide antioxidant protection to nervous tissue. As ethidium bromide (EB) injection into the white matter induces local oligodendroglial and astrocytic death, demyelination, glia limitans and blood–brain barrier disruption and Schwann cell invasion; this study aimed to evaluate the effects of PPF on lipid peroxidation (measured through its byproducts, thiobarbituric acid reactive species or TBARS) and on remyelination following gliotoxic injury. Wistar rats were divided into four groups: rats injected with EB into the cisterna pontis and treated with PPF (12.5 mg/kg/day by intraperitonial route); rats injected with EB but without receiving the xanthine; PPF‐treated rats (no EB); control animals (no EB, no PPF). Brainstem sections were collected from 7 to 31 days for light and transmission electron microscopy studies, as well as blood plasma and brain samples for TBARS measurement. Results from EB‐injected groups were compared using a semi‐quantitative method developed for documenting the extent and nature of remyelination in semithin sections. By 7 and 15 days, results showed that PPF significantly reduced lipid peroxidation by 22% in blood plasma and brain, although by 31 days its antioxidant effects were no more observed. Both oligodendroglial and Schwann cell remyelination was increased compared to untreated animals by 31 days. These data suggest that PPF stimulates remyelination and prevents redox imbalances in blood plasma and brain following neural injury.

P07‐03

Retrograde axonal delivery of TDP‐43 and FUS genes using AAV9 and lentivirus vectors to adult mouse motoneurons

Ishii T^1,5, Akiyama K¹, Kawakami E¹, Yanagisawa H¹, Sango K¹, Okado H², Miwa A², Miyake K³, Kato S⁴, Kobayashi K⁴, Misawa H⁵ and Watabe K¹

¹ ALS/Neuropathy Project and ² Neuronal Development Project, Tokyo Metropolitan Institute of Medical Science; ³ Dept. of Biochemistry and Molecular Biology, Nippon Medical School; ⁴ Dept. of Molecular Genetics, Fukushima Medical University; ⁵ Dept. of Pharmacology, Keio University Faculty of Pharmacy, Japan

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding TAR DNA‐binding protein 43 (TDP‐43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. We have previously demonstrated that retrograde co‐infections of adenovirus encoding shRNA for protein degradation machineries with TDP‐43 or FUS adenovirus enhanced cytoplasmic aggregate formation in adult rat facial motoneurons in vivo, suggesting that impairment of protein degradation pathways accelerates formation of TDP‐43 and FUS‐positive aggregates in ALS. However, adenoviral transduction of foreign genes is transient and diminishes within 2 weeks in vivo. In the present study, we produced recombinant adeno‐associated virus type 9 (AAV9) and lentivirus (LxFuGB2) vectors encoding wild type and mutant TDP‐43 or FUS, and those encoding shRNAs for proteasome (PSMC1) and autophagy (ATG5) systems to examine their long‐term retrograde transduction efficiency of the foreign genes in adult mouse facial and spinal motoneurons. When these recombinant AAV9 or LxFuGB2 vectors were injected into the facial or sciatic nerves of adult mice, exogenous TDP‐43 and FUS proteins or shRNAs for PSMC1 or ATG5 were successfully expressed in facial or lumbar motoneurons by a retrograde axonal transport of the viruses at 2–4 weeks postoperation as examined. We are currently examining the aggregate formation of TDP‐43 and FUS using these AAV9 and LxFuGB2 vectors in mouse facial and spinal motoneurons.

P07‐04

The neuroprotective effect of lentivirus on choline acetyltransferase mRNA expression of hippocampal neurons and behavior in senile dementia rats

Yan S, Cao J, Zhang H and Bai J

Department of Neurology, The First Hospital of Jilin University, Changchu, China

Alzheimer disease (AD) is a neurodegenerative disease and its pathogenesis and etiology are undetermined. There is no effective treatment. Our pilot study proved the neuroprotective effect of lentivirus peptide (rLent/NT4‐NAP) in rats.

Methods: SD rats underwent injection of beta‐amyloid protein and intracerebral transforming growth factor beta (TGF)‐1 into the hippocampus to mimic the senile dementia model. Fifty rats were randomized divided in control, sham‐operated control, model, model+rLent/NT4‐NAP and virus control groups. The latter two groups were administered by dropping nose. The change of ethology and ChAT mRNA expression of hippocampal neurons in SD rats were determined by water maze and hybridization in situ technique.

Results: The ability of learning and memory in rLent/NT4‐NAP group were significantly higher than that in model groups (p < 0.05). The ChAT mRNA positive neuron extent density in model group (0.0698 ± 0.0182) was lower than that in control (0.1217 ± 0.0165), sham‐operated control (0.1130 ± 0.0154) , rLent/NT4‐NAP operated (0.1183 ± 0.0112) and virus control (0.1193 + 0.01118) respectively (p < 0.05).

Conclusion: The neuroprotective effect of lentivirus of improving the ability of learning and memory in senile dementia rats may relate to the recovery of ChAT mRNA expression in the hippocampus.

Keywords: lentivirus neuroprotective peptid, choline acetyltransferase mRNA, senile dementia, rats.

P08. Epilepsy

P08‐01

A case of focal cortical dysplasia IIB with extensive calcification and tumor‐like nodules

Bodi I¹, Selway R² and Honavar M¹

¹ Clinical Neuropathology, King's College Hospital NHS Foundation Trust, London, UK; ² Neurosurgery, King's College Hospital NHS Foundation Trust, London, UK

Introduction: Focal cortical dysplasia (FCD) IIB is a relatively common neuropathological lesion that is identified in neurosurgical specimens of patients with pharmacoresistant epilepsy. Calcification may be seen occasionally in lesions of FCD IIB; however, we report a case of FCD IIB with extensive calcification in which tumor‐like nodules were also detected.

Patients and Methods: 6‐year‐old boy presented with motor and gelastic seizures since 5 months of age and learning difficulties. MRI showed increased T2 signal in the deep white matter of the right frontal lobe extending towards the frontal horn and subcortical calcification.

Results: Histology from the right frontal resection showed cortical dyslamination, presence of dysmorphic neurons and numerous balloon cells. There was extensive calcification in the subcortical white matter. In addition, two tumor‐like nodules were observed in the subcortical white matter, close to the calcified areas. The nodules were composed of polygonal cells with oval nuclei with moderate to abundant eosinophilic cytoplasm. Immunohistochemistry confirmed FCD IIB. The cells of the nodules were focally positive parvalbumin, but they showed decreased immunoreactivity with GFAP, nestin, EMA, CD68 and HLA‐DR compared to the surrounding areas and they were negative for CD34 and neuronal markers.

Conclusion: Unusual tumor‐like nodules were associated with FCD IIB with extensive calcification. The differential diagnosis of tuberous sclerosis was raised, but the patient had no stigmata of tuberous sclerosis and CGH array DNA testing was negative. The nature of the tumor‐like nodules remains uncertain in this case.

P08‐02

Activity of GW‐542573X in chronic epilepsy model and in neocortical human tissue from pharmacoresistant epileptic patients

Raza ML and Heinemann U

Institute of Neurophysiology, Charite Universitätsmedizin, Berlin, Germany

Despite wide range of available anticonvulsant drugs, around 30% of patients are resistant to clinically used antiepileptic. The other issues of antiepileptic drug therapy include bothersome side effects and reduced compliance due to poly drug therapy. This creates a necessity to explore new potential single drug targets not only with reduced side effect but addressing the problem of pharmacoresistant in epilepsy patients.

In the present study we have studied activity of GW‐542573X, a specific agonist of small conductance Ca²⁺‐activated K channel‐1 (SK‐channel 1), it tends to reduce neuronal hyperexcitability by regulating medium after hyperpolarization. We have used slices from pilocarpine treated chronic epileptic rats and human neocortical tissue from pharmacoresistant patients.

Horizontal slices of 400 μM were prepared from pilo‐treated chronic epileptic rats and kept in aCSF at 36 C supplied with carbogen. After 2 hrs of recovery phase, local field potential recording were performed in an interphase chamber using glass microelectrode. For comparing effect of vehicle age‐matched control rats that only received the saline instead of pilocarpine were also included in the study. However, in case of neocortical human tissue 500 micron thicker slices were used to induce seizure‐like events (SLEs). For inducing the SLEs in pilo‐treated rats 100 μM 4‐AP was used. In human tissue two different protocols were used, i.e. wash in with 4‐AP 100 μM or by elevating potassium to 8 mM combining with bicuculline methiodide (50 μM). In slices from pilocarpine‐treated rats blocking of SLEs by GW‐542573X at 200 μM was achieved in 50% of slices (n = 8), at 100 μM blockade was found in 2 out 7 studied slices (n = 7). Duration of SLEs and seizure interval were altered at even 100 μM. In age‐matched control rats blockade of SLEs was observed only in 2 out of 6 slices at 200 μM and at 100 μM failed to block SLEs. In human tissue SLEs induced by 4‐AP were not blocked (n = 4). While at 200 μM it blocked SLEs induced by elevated K⁺ bicuculline in 2 out of 8 slices. Based on studied results we may suggest that SK‐channel agonist have potential SLEs blocking effect as we found in chronic epileptic rats. It is worth to mention those drugs which acts on CNS often alters the cognitive function; therefore, further it is important to test the effects of SK‐channel agonist on memory and cognition in order to make a good antiepileptic drug target.

P08‐03

Modulation of NMDA receptor by miR‐219 in the amygdalae and hippocampi of patients with mesial temporal lobe epilepsy

Hamamoto O, Assirati Jr JA, Saggioro FP, Neder L, Velasco T, Sakamoto AC, da Cunha Tirapelli DP and Carlotti Jr CG

School of Medicine Ribeirão Preto, University of São Paulo, Brazil

Introduction: Epilepsy is a common neurological disorder, and drug resistance is the most important problem in patients with temporal lobe epilepsy. Studies searching for new anticonvulsant drugs have indicated a need for better understanding the molecular and cellular bases of epilepsy. Small RNA molecules called microRNAs, which have an important role in the development and regulation of synaptic plasticity in the nervous system of vertebrates, may be potential targets for epilepsy treatment. Several studies have demonstrated the involvement of microRNAs in many brain diseases. However, there are few studies regarding the role of microRNAs in epilepsy.

Patients and Methods: Using real‐time PCR, we evaluated the involvement of miR‐219, miR‐181b, miR‐195, and the receptors of excitatory (NMDA: subunit NR1 and AMPA: subunit GluR2) and inhibitory neurotransmitters (GABA_A: subunits α2, β3, γ2) in the amygdalae and hippocampi of patients with epilepsy.

Results: We observed an inverse relationship between miR‐219 and NMDA‐NR1 expression in both the amygdala and hippocampus relative to their expression in the controls. NR1 and GluR2 were upregulated in the amygdalae of epileptic patients. No difference was observed in miR‐181b expression between patients with epilepsy and the controls. Low miR‐195 expression was observed in the amygdalae of patients with epilepsy.

P08‐04

Multinodular and vacuolating neuronal tumor – an emerging entity

Bodi I¹, Curran O¹, Laxton R¹, Al‐Sarraj S² and Honavar M³

¹ Clinical Neuropathology, King's College Hospital NHS Foundation Trust, London, UK; ² MRC London Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, King's College, London, UK; ³ Servico de Anatomia Patologica, Hospital Pedro Hispano, Matosinhos, Portugal

Introduction: An unusual multinodular and vacuolating neuronal tumor (MVNT) has been described in the cerebral hemisphere (Thom et al.). We report two cases with similar features, a surgical resection and the other an autopsy specimen.

Patients and Methods: Case 1, a 34‐year‐old female, underwent surgical resection for a multinodular non‐enhancing frontal white matter lesion causing intractable epilepsy. Case 2 presented with motor neuron disease (MND) at the age of 71 and MRI scanning revealed extensive multinodular non‐enhancing white matter lesions in the temporal lobe. There was no history of epilepsy and post mortem histology confirmed MND.

Results: Macroscopically multiple small grey well‐formed, discrete and coalescent nodules were seen in the deep cortex and subcortical white matter. On histology, mature‐looking neurons with large cytoplasmic vacuoles were distributed in a fibrillary background, where vacuoles were also noted. In the resected tumor scattered oligodendroglia‐like cells were present. No ganglion cells were seen. The vacuolated cells exhibited immunopositivity for synaptophysin, HuC/HuD and p62 but were negative for NeuN, neurofilament, GFAP, IDH1, nestin and CD34. Electron microscopy showed non‐membrane bound cytoplasmic vacuoles in the neurons and in some neuronal processes. The seizures recurred in Case 1. Some clinicopathological features of this lesion suggest a possible relationship with dysembryoplastic neuroepithelial tumor (DNT) although the morphological features are not typical of DNT. Case 2 demonstrates that MVNT may remain asymptomatic.

P09. Infection Non‐Viral

P09‐01

Cerebral aspergilloma in young immunocompetent patient

Prados SR¹, Gómez A¹, Fajre L², Golcman J³

¹ Dept of Pathology, ² Dept of Radiology, ³ Dept of Neurology, University of Tucuman, School of Medicine, Argentina

Aspergillosis of the central nervous system is an uncommon infection mainly occurring in immunocompromised patients with a high mortality. We report a case of cerebral aspergilloma in a previous healthy immunocompetent 20‐year‐old female patient who presented with a history suggestive of intracranial space occupying lesion. The MRI showed a 2 cm lesion in the left precentral gyrus with a rym of high signal intensity and other smaller lesions with similar characteristics. She underwent a craniotomy with excision of the lesions. Histopathological study showed granulomas with dense fibrosis. The multinucleate giant cells contained slender septate, acute angle branching hyphae of Aspergillus. The patient received antifungal therapy and postoperative course was uneventful. This report highlights the rare presentation of Aspergilloma in immunocompetent patient. Early diagnostic procedures with rapid confirmation of Aspergillus infection are pivotal for a benign clinical course.

P09‐02

Cerebral malaria in Uberlândia, Minas Gerais: a case report

Silva ACAL, Dornelas BC, Alcantara TM and Rocha A (in memoriam)

Federal University of Uberlândia, Brazil

Introduction: Considered one of the greatest public health issues worldwide, malaria affects 250,000 individuals per year in Brazil. One percent of symptomatic infections can lead to severe disease. According to World Health Organization, cerebral malaria is a disorder characterized by coma, parasites on peripheral blood smears and no other cause to explain coma. In this paper we will report a rare autochthonous case of cerebral malaria diagnosed postmortem and highlight this neglected disease.

Patients and Methods: We reviewed the autopsy and medical records of a 34 year‐old man who died of cerebral malaria at the Hospital de Clínicas, Federal University of Uberlândia, Brazil.

Results: The patient was in a state of coma upon admission. He presented febrile illness and jaundice for 5 days prior to admission. At the moment of the death, no etiological diagnosis had been made. Gross examination of the brain showed edema and petechial hemorrhages in the white matter of the cerebral hemispheres and cerebellum.

Histopathology showed the lumen of the vessels distended by sequestered infected red blood cells containing hemozoin. Some vessels were occluded by thrombus and Dürck's granulomas were found.

Conclusion: A brain injury is the most serious outcome of a parasitic infection with Plasmodium sp. as a consequence of brain hyperinflammation, vascular obstruction and reduced cerebral blood flow. Uberlândia is a non‐endemic area for malaria. Health care providers have little experience with this disease. The possibility of cerebral malaria should be considered to avoid this outcome in patients with the aforementioned symptoms.

P09‐03

Effect of systemic tuberculosis in the neuronal and blood‐brain barrier alterations in a murine experimental model

Tena Suck ML^1,6, Lara CS^1,4, Santos LR^1,5, Benavides NKS^1,5, Arvizu KPC^1,5, Soto JLH², Morales VA², Manzano PR⁹, Benavides OJC^1,7, Garibay CS^1,6, Pando RH⁸, Contreras JCL⁸, Valverde FF³, Zamudio LJ⁷, Campos MEH⁵, Maldonado ER⁷, López MAG^1,5 and Bojórquez DR¹

¹ Departamento de Neuropatología; ² Neuroinfectología y; ³ Patología experimental del I.N.N.N; ⁴ Facultad de Estudios Superiores de Iztacala U.N.A.M; ⁵ Maestría en Ciencias de la Salud y; ⁶ Doctorado en Investigación en Medicina, Escuela Superior de Medicina I.P.N; ⁷ Posgrado en Ciencias Quimicobiólogicas, Escuela Nacional de Ciencias Biológicas, I.P.N; ⁸ Sección de Patología Experimental INNN; ⁹ Laboratorio de Biología de la Reproducción Animal de la Facultad de Ciencias de la U.N.A.M., Mexico

Background: The injury mechanism of M. tuberculosis to the blood–brain barrier, choroid plexus and neurons in central nervous system tuberculosis is unknown.

Materials and Methods: Male Balb/c mice were infected intratracheally with M. tuberculosis H37Rv and LAM3 isolated from a patient with leptomeningitis. They were sacrificed post‐infection at different days (1, 3, 7, 14, 21, 28, 60, 120 ds). Brains were processed for hematoxylin‐eosin, silver stains, electron microscopy, Ziehl‐Neelsen, PCR (IS6110) and immunohistochemistry for cytokines IL1‐β, TNF‐α, IFN‐γ, TGF‐β, IL‐4.

Results: There were neuronal alterations in frontal cortex and hippocampus with both strains (death, disorganization, eosinophilic degeneration). There were no positive cultures for any strain. Positive PCR was detected for LAM3. Cytokines were positive in ependymal cells, endothelial cells and choroid plexus with both strains, IFN‐γ with more intensity in LAM3. Electron microscopy showed cytoplasmatic vacuolation, loss of intercellular junctions, detachment and progressive cellular necrosis.

Discussion: Changes described in choroid plexus may be related with inflammatory cytokines, in absence of local inflammation it could be effect of systemic infection or the process mediated in the blood–brain barrier; neuronal damage may be due to activated glia. Data described here, for the first time, indicate that with no (H37Rv) or minimal infection (LAM3) there is damage in these central nervous system structures.

Conclusion: There are neuronal alterations in frontal cortex and hippocampus and in blood–brain barrier posterior to systemic infection of M. tuberculosis, being more severe the LAM3 strain than H37Rv, possibly mediated by the presence of cytokines.

P09‐04

Multiple intracranial lesions; tuberculoma mimicking neurocysticercosis

Hajjazi E¹, Samaha M², Al‐Loh S³ and Al‐Hussaini M³

¹ Department of Pathology, Islamic Hospital, Amman, Jordan; ² Department of Neurosurgery, Ibn Al Haytham Hospital, Amman, Jordan; ³ Department of Pathology and Laboratory Medicine, King Hussein Cancer Center, Amman, Jordan

Multiple disseminated brain lesions form diagnostic challenge, with differential diagnosis including infections as well as metastatic malignancy. In young immune competent patients where metastatic tumors are less likely, infections rank top in this differential diagnosis.

Tuberculosis is one of the most common infectious diseases of the central nervous system (CNS). We report a case of multiple CNS lesions involving the cerebrum and cerebellum in a 32‐year‐old female, radiologically mimicking neurocysticercosis. Examination of one of the lesions excised revealed a well delineated granulomatous reaction with a large central area of caseating necrosis, exhibiting multiple acid‐fast bacilli when examined by ZN special stain. No larval cysts or scolices could be identified.

Recognizing this unusual form of CNS tuberculosis is important for proper management of the patients.

P09‐05

Brain abscess caused by mucormycosis infection – case report

Ussene EP¹, Carrilho C¹ and Ordi J²

¹ Pathology Department, Maputo Central Hospital, Mozambique; ² Pathology Department, Barcelona Clinic Hospital, Spain

Mucormycosis is a rare infection caused by fungi of the genera Mucorales, being the species Rhizopus most frequently associated with human infection. His presence and pathogenicity in human is constantly associated to a basic debilitating illness as tuberculosis, cancer and especially diabetes. In this case, the authors report an autopsy case of this disease entity, given its rarity, complexity diagnostic and clinical severity, which ended in death. The case refers to a 27‐year‐old patient, male, with diabetes mellitus. He was admitted in the surgery department with occipital cutaneous infection with exposure of occipital bone. The semiological nonspecificity and the difficulty to isolating the microorganism in body fluids are factors that delay the rapid diagnosis and treatment contributing to an unfavorable outcome.

P10. Infection Viral

P10‐01

Acute necrotizing encephalopathy

Raghavan R¹ and Achiriloiae AF²

¹ Dept of Pathology and ² Division of Neuroradiology, Loma Linda University Medical Center, Loma Linda, CA, USA

Introduction: We report a case of fatal atypical necrotizing encephalopathy (ANE) following influenza infection.

Patient: A healthy 8‐year‐old boy with recent respiratory illness was admitted for refractory seizures. Enzyme immunoassay was significant for possible infection by influenza A/B. Patient was placed on phenobarbital coma but later needed ECMO due to hemodynamic instability. MRI revealed bilateral cerebral and cerebellar white matter edema and microhemorrhages, occipital diffuse NAA decrease and lactate peaks on MR spectroscopy.

Result: Following his demise, the brain grossly revealed a large, mainly white matter centered area of necrosis in the right centrum semiovale with similar lesions in the temporal and cerebellar regions. Microscopically the coagulative necrosis had a peculiar homogeneous consistency with prominent central neovascularization and perivascular ‘rosette‐like’ macrophage infiltrates that was unique. In addition, there was neuronal loss in hippocampal CA1 and cerebellar Purkinje cells, likely representing superimposed global hypoxic‐ischemic effects.

Conclusion/Discussion: The extensive necrotic white matter changes were striking, and compatible with acute necrotizing encephalopathy. Originally described in the Japanese, ANE presents with acute and progressive neurologic deterioration, with high mortality (30%). They are commonly associated with influenza A, but have also been linked to other viruses. Typically, brain findings are characterized by bilateral thalamic necrosis and petechiae with variable cerebellar and cerebral white matter edema. The somewhat atypical distribution of lesions in the present case may relate to effects of superadded cardiovascular collapse. In the few familial ANE cases described, a genetic susceptibility link has been shown to the gene for RAN‐binding protein‐2.

P10‐02

Detection of JCV DNA variant (JCV‐CPN1) in central nervous system of patients without progressive multifocal leukoencephalopathy

Palecka C¹, Arakaki N², Riudavets MA², Arias E², Fiol M², Rojas E², Schultz M² and Sevlever G²

¹ Hospital de Clínicas; ² FLENI, Argentina

Human JC polyomavirus causes an asymptomatic infection in 50% to 80% of the population that is kept under control by the immune system. In immunocompromised individuals, JCV usually causes a demyelinating disease termed progressive multifocal leukoencephalopathy (PML) with well‐known histopathological features.

In rare cases, mutations in JCV can trigger a change in tropism, i.e. granule cell neuronopathy with a deletion in VP‐1 gene or JCV encephalopathy‐JCVE (JCVcpn1), characterized by JCV infection of cortical pyramidal neurons associated with a 143 base pair deletion in the Agno gene.

We describe two patients. The first case was a 47‐year‐old, HIV‐negative woman who developed progressive cognitive impairment, aphasia and pyramidalism. Her MRI showed hyperintense, non‐enhancing cortical images on T2 and Flair sequences. The second case was a 31‐year‐old HIV‐negative woman with neuromyelitis optica; treated with interferon, steroid, cyclophosphamide and plasmapheresis. MRI showed hyperintense periventricular images on T2 and Flair extending to the corpus callosum and thalamus, and also in the spinal cord. CSF was positive for JCV‐DNA.

On both cases, brain biopsies did not display typical features of PML. However, JCV‐DNA was detected on both samples. Molecular analysis detected the JCV variant cpn1 coexisting with wild type strain (JCV Mad‐1) in one case, and an additional 18‐base pair in frame deletion in the other.

P10‐03

Encephalomyelopathies associated to HIV infection after an introduction of combined antiretroviral therapy (TARV): thirteen years of experience in a university hospital

Madruga M¹, Vieira TM¹, da Silva Pereira RC², Canedo NHS² and Chimelli L²

¹ INCA; ² Federal University of Rio de Janeiro, Brazil

The introduction of combined antiretroviral therapy (TARV) benefited patients with HIV, to recover immunity, reducing opportunistic infections, but was also responsible by the immune reconstitution inflammatory syndrome (IRIS), resulting in a paradoxical worsening of lesions. We intend to evaluate the alterations of the central nervous system (CNS) in biopsied or autopsied patients after the introduction of combined TARV, and compare with literature and our previous experience without the TARV.

Material and Methods: We analyzed the CNS in biopsies and autopsies performed at a University Hospital between April/1998 and May/2011.

Results/Conclusion: In 60 necropsies (n) and 40 biopsies (b) the most frequent diagnoses were: nonspecific lesions (10n/7b), normal (10n/3b), toxoplasmosis (6n/6b), progressive multifocal leukoencephalopathy/PML (1n/10b), cryptococcosis (5n/4b), ischemic lesions/hemorrhagic (6n/1b), lymphocytic meningitis (5n/1b), lymphoma (1n/4b), tuberculosis (3n/2b), HIV encephalitis (1n/1b), candidiasis (1n), CMV (1n). Concomitant injuries occurred on two occasions. Healed lesions (toxoplasmosis?), Nonspecific encephalitis, Wernike's encephalopathy, vasculitis and necrotizing myelopathy were less frequent. Microcephaly (1), cortical atrophy (2) gliosis and microcalcifications were seen in children (7) of this series. Three patients developed IRIS (2 toxoplasmosis and 1 PML). We conclude that the introduction of TARV has reduced, but still not abolished, the occurrence of opportunistic infections, corresponding to half of the cases. The non‐adherence to the treatment, the fact that at the beginning not every patient had access to the drug, in addition to reduction in mortality and in demand for autopsies, which was not observed in the earlier decades of HIV infection, have contributed to these results.

P10‐04

Enterovirus 71 infection of the brainstem via cranial nerves can be ameliorated by passive immunization

Wong KT¹, Tan SH¹ and Ong KC²

¹ Department of Pathology, Faculty of Medicine, University of Malaya; ² Department of Biomedical Science, Faculty of Medicine, University of Malaya, Malaysia

Introduction: Enterovirus 71 (EV71) associated hand‐foot‐and‐mouth disease may be complicated by encephalomyelitis. Infection in the brainstem via cranial nerves is believed to be an important cause of death. As antiviral drugs and vaccines are currently unavailable, passive immunization may be useful but its effectiveness in established brainstem infection has not been demonstrated. We investigated passive immunization before and after brainstem infection in a mouse model.

Materials and Methods: EV71 was injected into unilateral jaw/facial muscles of groups of 2‐week‐old mice and given a single dose of hyperimmune sera at 4 hours pre‐infection, 12 and 24 hours post‐infection (hpi), respectively.

Results: In mice sacrificed at 72 hpi, the brainstem in pre‐infection immunized mice was negative for viral titer, antigen and RNA by microtitration, immunohistochemistry and in situ hybridization, respectively. Mice treated at 12 and 24 hpi showed significantly lower viral titers and antigen/RNA. Untreated control mice all developed signs of disease and hind limb paralysis at 60 hpi and died at 96 hpi. Viral antigen/RNA was mainly found in cranial nerves, motor trigeminal nucleus, reticular formation and facial nucleus in infected mice. In another group of animals observed over 3 weeks, all pre‐infection immunized mice remained healthy. Mice treated at 12 and 24 hpi showed survival rates of 50% and 25%, respectively. Furthermore, sick mice showed delayed onset of disease and death.

Conclusion: Optimized hyperimmune serum could prevent and ameliorate brainstem infection in a time‐dependent manner. These findings provide a basis for current empirical treatment of patients with IVIG.

P10‐05

Is involvement of visual and craniobulbar pathway in rabies encephalitis the anatomical pathway for hydrophobia?

Mahadevan A, Satishchandra P, Suja MS, Shankar SK and Madhusudana SN

National Institute of Mental Health & Neurosciences, Bangalore, India

Introduction: Rabies, a fatal rhabdovirus infection, is common in India. Occurrence of hydrophobia, a dramatic but enigmatic phenomenon associated with this disease, suggests involvement of the visuo‐motor pathway by the virus.

Objective: To determine visual pathway involvement, we traced the rabies viral antigen distribution from retina to visual pathway in autopsied human rabies and experimental rabies in permissive adult Swiss Albino mice by immunohistochemistry.

Methods: Rabies viral antigen in nine cases of rabies was mapped by immunohistochemistry using antibody to the nucleoprotein of rabies virus. Only one manifested hydrophobic features, the rest had paralytic rabies and phobic spasms terminally. Permissive adult Swiss Albino mice inoculated via intramuscular/intracerebral route with virulent rabies street and lab attenuated CVS strain was also evaluated similarly.

Results: The retina, optic nerve, lateral geniculate body, superior colliculus and the striate cortex demonstrated rabies viral antigen. Rabies viral antigen was also found in nuclei tractus solitarius and V, VII, IX and X, XII nuclei in brain stem, the central pattern generators controlling swallowing reflex connecting via with the superior colliculus with the visual pathway to trigger swallowing reflex/phobic spasms and hydrophobia. Variable involvement of excitatory (cingulate, insular, thalamus and hypothalamus) and inhibitory centers (pontine reticular formation and periaqueductal gray) could determine occurrence of hydrophobia.

Conclusion: Presence of rabies antigen was detected along the entire visual pathway to striate cortex with caudocranial gradient. Involvement of the visual pathway may trigger the stimulation of craniobulbar nuclei in brain stem modulating contraction of pharyngeal and bulbar muscles producing phobic spasms/hydrophobia.

P10‐06

Neuropathology of neuroaids in the post‐HAART era: preliminary results

Schiavotelo NL, Mendes ND, Alves C, Silvestre RN, Bollela VR, dos Santos AC, Pittella JEH and Neder L

School of Medicine of Ribeirão Preto, University of São Paulo, Brazil

The involvement of the central nervous system (CNS) is still high in patients with HIV infection, although the general mortality has declined in the post‐HAART era. We performed a systematic study in a series of 70 patients autopsied with aids in an academic hospital at southeast region of Brazil from 2003 to 2013. The frequency of opportunistic infections was compared to clinical presentation and laboratory data, such as CD4 counts and viral load as well as adherence to treatment and presence of cognitive impairment in these patients.

Results: The frequency of CNS lesions, opportunistic infections and/or cognitive impairment was significantly higher in patients who did not receive any HAART therapy (p = 0.01). Even though the incidence of those alterations was not significantly distinct in patients who regularly followed the anti‐HIV therapy, it was associated with longer patient overall survival. The presence of opportunistic infections were more often observed in those who did not use HAART regularly and toxoplasmosis was the most frequent CNS infection and frequently related to the patient's death; followed by cryptoccocosis, mainly in individuals who were drug abusers. These data are quite similar to previous Brazilian series of neuroaids in pre‐HAART period, except for observing a greater incidence in women and elders, multiple sequelar CNS lesions and fewer cases of HIV encephalitis. Additionally, the use of HAART had small impact in incidence of toxoplasmosis but, even when the antiviral therapy was used irregularly, it was associated with better patient survival.

Support: CNPq (Proc. No. 486740/2013‐7).

P10‐07

Association of human neurotropic JC virus with pediatric gangliogliomas & dysembryoplastic neuroepithelial tumors

Struckhoff AP¹, Mullinax J², Ripple M¹, Trillo‐Tinoco J¹, Craver R^3,4 and Del Valle L^1,4

¹ Stanley S. Scott Cancer Center, Louisiana State University Health; ² Department of Hematology/Oncology, Children's Hospital of New Orleans; ³ Department of Pathology, Children's Hospital of New Orleans, New Orleans, LA, USA; ⁴ Department of Pathology, Louisiana State University School of Medicine, New Orleans, LA, USA

The human neurotropic virus JCV has previously been associated with brain tumorigenesis. It is oncogenic when inoculated in the brain of experimental animals and it has been detected in medulloblastomas and adult glial tumors in humans. JCV exerts its oncogenic effects through the oncoprotein T‐Antigen, which binds and inactivates tumor suppressor proteins p53 and Rb. In addition, T‐Antigen can translocate IRS‐1 to the nucleus, where it impedes faithful DNA repair, gangliogliomas and dysembryoplastic neuroepithelial tumors (DNETs) are rare pediatric brain malignancies with both glial and neuronal phenotypes. Tissue samples of 15 cases of gangliogliomas and 5 cases of DNETs were obtained from the archives of the Pathology Department at Children's Hospital in New Orleans. PCR using specific primers for the T‐Antigen coding region was positive in 11/15 gangliogliomas (73.3%) and 4/5 DNETs (80%). T‐Antigen expression was found in the nuclei of tumor cells with both glial and neuronal phenotypes in 7/15 gangliogliomas (47%),and in exclusively in the glial component in 3/5 DNETs (60%). There is a direct correlation between expression of T‐antigen and p53 in positive cases. Nuclear IRS‐1 was observed in all the T‐antigen‐expressing cases, suggesting alterations in DNA repair plays a role in pathogenesis.

The presence of JCV genomic sequences in pediatric tumors suggests that primary infection occurs early in life, leading to transformation. The expression of T‐antigen in both the glial and neuronal components of gangliogliomas suggests that these mixed tumors most likely originate in a common primitive cell, which then differentiates into two different phenotypes.

This work was possible thanks to a NIH grant (RR021970) awarded to LDV.

P10‐08

Slowly progressive dementia in adult‐onset subacute sclerosing panencephalitis

Neto AS¹, Nóbrega PR¹, Castro LHM¹, Nitrini R¹, Duarte MIS², Lucato LT³

Department of Neurology, Faculty of Medicine, University of São Paulo; Department of Pathology, Faculty of Medicine, University of São Paulo; ³ Department of Radiology, Faculty of Medicine, University of São Paulo, Brazil

Introduction: Subacute sclerosing panencephalitis is a rare inflammatory and neurodegenerative disease secondary to a persistent infection of the brain by measles virus and affects primarily children. The diagnosis is established by characteristic clinical, high measles antibody titers in cerebrospinal fluid, typical electroencephalogram and histopathological findings in brain biopsy or autopsy. Adult onset is rare and often has symptomatology that diverges from the classic clinical description.

Patients and Methods: A Brazilian man presented with a progressive cognitive decline, with signs of diffuse demyelinating leukoencephalopathy in brain magnetic resonance imaging and a pleocytosis in cerebrospinal fluid with very high gammaglobulin fraction in protein electrophoresis.

Results: Histological examination of the brain showed diffuse parenchymal involvement by inflammatory and neurodegenerative processes with astrocytic and neuronal inclusion bodies. Some vessel walls presented with an inflammatory infiltrate and endothelial cell reactivity, configuring a vasculitis. Immunostaining for measles was positive with granular stain in nuclei of astrocytes and oligodendrocytes.

Conclusions: This case had a clinical presentation of this uncommon disease in an unusual age of onset and the diagnosis was supported by brain biopsy. Reports suggest that adult‐onset subacute sclerosing panencephalitis may have atypical features.

P11. Inflammation

P11‐01

Paraneoplastic limbic encephalitis & neuroleptic malignant syndrome in a case of mature (cystic) ovarian teratoma

Salazar Morales MF¹, Estrada Hernández MR², Torres Mejía IA², Parraguirre Martínez S² and Rivera Zetina DJ³

¹ Hospital General de México “Dr. Eduardo Liceaga”; ² Hospital General “Dr. Manuel Gea González”; ³ Centro Médico ISSEMYM, Mexico

We document the case of a 28‐year‐old woman with behavioral changes, seizures, visual hallucinations, language impairment and acute sleep–wake cycle reversal during ten days. Imaging scans revealed a right ovarian tumor as well as effacement of both hippocampi, while serum markers showed anti‐neuron (anti‐NMDA‐R) antibodies. Surgical excision of the tumor was not possible as the patient persisted with unmanageable seizures, superimposed rhabdomyolysis and fever (40°C to 42°C). She died 14 days after admission.

Postmortem findings confirmed a 13 × 12 × 5.5cm right ovarian tumor with a variegated appearance at cut, which was diagnosed as mature teratoma. Conversely, despite only mild cerebral edema, microscopic examination of both hippocampi and amygdalae showed neuronal loss, gliosis, activation of microglial cells and perivascular lymphocytic cuffs.

Non‐viral limbic encephalitis is a rare autoimmune disorder commonly associated with lung and breast cancer, Hodgkin's lymphoma, thymoma and gonadal neoplasms. Its pathogenesis involves synthesis of antibodies directed towards specific CNS antigens, either intracellular (e.g. Hu, Ma2, CV2/CRMP5, amphiphysin, etc) or membrane‐anchored (voltage‐gated potassium channels and the so‐called “hippocampal neuropil antigens”; e.g. NR1/NR2B). Encephalitides related to the latter predominantly affect young women who develop ovarian teratomas (mature or immature).

Though recovery was expected, our patient unfortunately coursed with neuroleptic malignant syndrome – an idiosyncratic reaction to antipsychotic drugs.

P11‐02

Age‐related memory impairment induced by recurrent low‐grade systemic inflammation is associated to a deregulation of inflammatory response

d'Avila JC¹, Siqueira LD¹, Bozza FA¹, Foguel D² and Azevedo E²

¹ Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brasil; ² Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Ageing is the major risk factor for the development of dementia and neurodegenerative diseases. Clinical and experimental evidence suggests that systemic inflammation can contribute to cognitive decline and dementia. The molecular mechanisms associated to the age‐dependent cognitive decline are not completely understood. Here we aimed to develop an animal model to study the effect of recurrent low‐grade systemic inflammation (RLSI) on cognitive functions of young and aged mice. To produce RLGSI we injected Swiss male mice, two months old (young) or fourteen months old (aged), with lipopolysaccharide (LPS 0,3 mg/kg i.p.) once a week for eight weeks. Memory was evaluated with the object recognition task and depressive behavior was evaluated with the forced swimming task. Microglial activation was evaluated morphologically by immunohistochemistry for Iba‐1 and cytokines were quantified by multiplex analysis. Systemic inflammation as measured by blood leukocytes was more pronounced in the aged mice acutely after RLSI (P < 0.0001, two‐way ANOVA) and returned to normal levels after one week. Basal plasma IL‐6 (P < 0.05) and brain MCP‐1 (P < 0.05) were significantly higher in the aged mice. However, after RLSI aged mice presented unexpected lower levels of almost all cytokines measured as opposed to young mice. Interestingly, only aged mice presented memory impairment after RLSI. Both young and aged mice presented depressive‐like behavior after RLSI. Our data show that age‐dependent memory impairment induced by RLSI is associated with a deregulation in the inflammatory response, suggesting that a microglial dysfunction might play a role in the memory impairment presented by aged mice.

P11‐03

Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS): autopsy findings

Taipa R, Moreira I, Cruto C, Alves JE, Correia C and Melo‐Pires M

Neuropathology Unit, Centro Hospitalar do Porto, Porto, Portugal

Introduction: CLIPPERS is a recently defined inflammatory entity, prominently involving the pons.

Case Report: A 76‐year‐old woman presented with headache, right VI and left VII cranial nerve paresis and cerebellar syndrome recovered partially in 3 weeks. One month later developed incoherent speech, visual hallucinations, ophthalmoplegia, left facial and palate paresis, pyramidal tetraparesis, cerebellar syndrome and depression of consciousness progressing to coma. MRI showed contrast enhancing linear lesions in brainstem, middle cerebellar peduncle and cerebellar deep white matter. Infections, autoimmune disorders (including sarcoidosis, Devic and Behçet diseases), neoplastic and paraneoplastic disorders were excluded. With high dose corticosteroids she regained consciousness, with continued limitation of left eye abduction, cerebellar syndrome and tetraparesis. Due to side‐effects prednisolone was reduced and immunoglobulins attempted. She remained stable and died 2 years later from pneumonia.

Neuropathological study revealed brainstem lesions with small perivascular lymphocytic infiltrates (CD4 and CD8+) accompanied by CD68 positive histiocytes and activated microglia, predominantly involving the pontine tegmentum but with extension to medulla and midbrain. There was also striking involvement of cerebellar peduncles decussation and red nucleus, and demyelination of intrapontine fascicules of V and VII nerves. The cerebellum showed perivenular demyelinating lesions.

Discussion: Clinical, imaging and pathological criteria for CLIPPERS were met. Partial therapeutic response could be attributed to clinical severity, late onset of treatment or low corticosteroid dose. The autopsy findings demonstrate that lesions are more widely distributed than thought, although limited to brainstem and cerebellum and include demyelination that has not previously been described.

P11‐04

Limbic encephalitis mimicking a hippocampal neoplasm: when MRI appearances may be deceptive

Gardiman MP and Pizzi M

General Pathology and Cytopathology Unit, Department of Medicine‐DIMED, Padua University Hospital, Padua, Italy

A 52‐year‐old Caucasian man with unremarkable past clinical history presented at the Neurosurgery Department of Padua University Hospital for seizures, mild confusion and memory loss. Brain MRI revealed an expansile lesion localized at the medial aspects of the left temporal lobe (main diameter: 3.5 cm), with a hyperintense pattern at T2‐weighted scans. The clinico‐radiological features were consistent with a primary brain neoplasm and the lesion was surgically excised. Histological examination revealed a meningeal and perivascular lymphocytic infiltrate, composed by mature B‐ and T‐cells (mixed CD20+ and CD3+ lymphoid population). The brain parenchyma surrounding such inflammatory infiltrate featured microglial nodules with no evidence of neoplastic proliferations (negative IDH1 immunohistochemical stain). Immunohistochemical and molecular tests for cytomegalovirus, herpes simplex virus and Toxoplasma gave negative results. The overall histological findings led to a diagnosis of limbic encephalitis of possible infectious (non‐herpetic) or autoimmune/paraneoplastic origin. Subsequent laboratory tests and total body CT excluded any extra‐brain primary tumor. At present (1 year after clinical presentation) the patient is alive and in good clinical conditions.

Limbic encephalitis (LE) is a rare inflammatory disorder of the hippocampal region, characterized by a hyperintense pattern at T2‐weighted MRI scans. In the present case, the unilateral location and the sharp margins of the lesion led to a clinico‐radiological misdiagnosis of primary brain tumor. To our knowledge, this is the first case of LE mimicking a brain neoplasm at MRI. The recognition of such atypical features is of capital importance for the correct diagnosis and management of patients.

P11‐05

The B cells and T cells changing of brain biopsy in infectious and un‐infectious inflammation patients

Gao J, Mao C, Guo Y, Cui Q, Cui L, Peng B, Feng F and bohou

Neurological Department; Pathological Department, MRI Centre, Peking Union Medical College Hospital, Beijing, China

Introduction: Brain biopsy is valuable and message limited, especially to differentiate infectious from non‐infectious inflammatory lesions.

Patients and Method: We selected patients whose brain biopsies were diagnosed as encephalitis (infectious and non‐infectious) and have been followed up for more than 6 months. All cases were submitted to immuno‐histochemical staining with CD3, CD8 and CD20 antibodies, to compare T cell and B cell infiltrates, between infectious and non‐infectious patients.

Results: Thirty patients were included, 10 defined as infectious, 13 patients as immuno‐mediated inflammation and 7 patients presented inflammatory lesions which could not be defined as infectious or not. From the 10 infectious diseases, 2 are probable nocardiosis, 2 are fungi infection, 1 is PML in HIV infected patient, 1 is Gram (+) bacterial infection with HIV, 2 are Sparganum mansoni, 1 is tuberculosis and 1 is probable Rhodococcus equi. All 13 patients with immunological diseases had good clinical evolution. T cells infiltrates were the most common changes in all cases, most of them with more CD3+ than CD8+ cells. Only 2 infectious cases had more CD8 than CD3 cells. CD20 cells were usually distributed around the blood vessels.

Conclusion: The T and B cells infiltrates could not help to differentiate infectious from non‐infectious inflammatory changes. Only a few infectious cases had more CD8 T cells. Clusters of neutrophils in brain specimens are more helpful to diagnose the cerebral infectious diseases.

P11‐06

Thoracic epidural inflammatory pseudotumor: a study of three cases

Moura HB, Souza FS, Araujo FI and La Roque‐Ferreira A

Sarah Network of Rehabilitation Hospitals, Brazil

Inflammatory pseudotumors are benign tumor‐like lesions of unknown etiology, which have paucity of reported cases at various locations throughout the body. They are important because of the difficulty in differentiating them from true neoplasms clinically, radiologically and intraoperatively. From the files of our institution we retrospectively located 3 patients with inflammatory pseudotumor diagnosed over a period of 11 years, from 2003 to 2014. Clinical records, images and pathology reports of all lesions were reviewed. There were 3 men with 28, 42 and 57 years old. Clinical symptoms included thoracic pain, difficulty with walking, sphincter disturbance and paraplegic with numbness and sensory disturbance. Laboratory values were nonspecific. The 3 lesions suggested a neoplasm both radiologically and clinically located in the region of the thoracic spine. All patients required surgical procedures with complete excision of the lesion and intraoperatory frozen biopsy. Histology examination of all cases revealed strips of dense fibro‐collagenous connective tissue showing extensive infiltration by a mixed cellular infiltrate dominated by plasma cells, lymphocytes and macrophages. There was no evidence of cytological atypia or clonality. ALK protein expression was negative. Gram, acid/alcohol‐fast bacilli and fungal stains proved negative. The 3 patients showed no tumor recurrence after surgical treatment. The shortest follow‐up was 4 months and the longest was 108 months. Spinal involvement due to inflammatory pseudotumor is rare and their clinical presentation, imaging findings may mimic a neoplasm. Histological studies and maintaining a broad differential diagnosis were essential for correct diagnosis and treatment.

P12. Metabolic

P12‐01

Secondary striolenticular ferrocalcinosis: a subtle permutation of Fahr's syndrome

Salazar Morales MF¹, Estrada Hernández MR¹, Omaña Hernández S², Estrada Moscoso I² and Parraguirre Martínez S²

¹ Hospital General de México “Dr. Eduardo Liceaga”; ² Hospital General “Dr. Manuel Gea González”, Mexico

We inform about two autopsy cases with bilateral striato‐lenticular ferrocalcinosis occurring under two different backgrounds: secondary hyperparathyroidism and toxoplasmosis.

Cases:

• 1) Forty six year‐old female with high blood pressure and increased calcium, phosphorus and PTH levels. Autopsy unveiled parathyroid hyperplasia associated with end‐stage renal disease.
• 2) Fifty‐two‐year‐old female with abrupt seizures, motor dysfunction and HIV infection detected by ELISA. Partial autopsy revealed multiple CNS necrotizing abscesses containing Toxoplasma tachyzoites.

Results: In both cases, sections taken from corpus striatum and lenticular nuclei showed mineralized small and medium sized vessels (Bottcher type 1 & 3/Yanai type B) with extraneuronal basophilic concretions (Bottcher type 2/Yanai type A) positive for Perls, PAS and alcian blue stains; only one case was reactive for von Kossa stain.

Conclusion: Triggered by a myriad of causes, striolenticular ferrocalcinosis is an uncommon phenomenon conceptualized as a variation of Fahr's syndrome. Intriguingly, mineral concretions denote a mucinous organic matrix component that is most likely a result of degenerative vascular changes.

P12‐02

Danon disease with early onset cardiomyopathy in females is not caused by skewed X‐chromosome inactivation

Hedberg C¹, Máthé G¹, Karason K¹, Oldfors A¹, Thomson K² and Östman‐Smith I³

¹ University of Gothenburg; ² Regional Molecular Genetics Laboratory, Churchill Hospital; ³ The Queen Silvia Children's Hospital, Sweden

Danon disease is caused by mutations in the lysosome‐associated membrane protein‐2 gene, LAMP2, located on the X chromosome. Although female carriers with LAMP2 mutations most often present with late onset cardiomyopathy and slow disease progress, there are unusual cases that emerge early and show a more severe disease course. The aim of this study was to investigate the clinical characteristics, morphological changes and pathogenesis of early onset cardiomyopathy in female LAMP2 mutated carriers. We investigated the explanted heart and skeletal muscle biopsies in two girls aged ten and thirteen years who underwent cardiac transplantation because of hypertrophic cardiomyopathy due to de novo heterozygous LAMP2 mutations and one 41‐year‐old female with late‐onset familial LAMP2 cardiomyopathy with more typical clinical phenotype. We found no evidence of skewed X‐chromosome inactivation in the two young girls since both alleles were expressed at apparently similar levels. In accordance with this finding skeletal muscle biopsy revealed no pathological changes. Immunohistochemistry in cardiac muscles showed a remarkable pattern with lack of LAMP2 protein in large regions including thousands of cardiomyocytes that also showed myocyte hypertrophy, lysosomal enlargement and disarray. In other equally large regions there was preserved LAMP2 expression and nearly normal histology. An uneven distribution of LAMP2 protein may cause deleterious effects depending on which regions of the myocardium that are lacking LAMP2 protein in spite of an overall moderate reduction of LAMP2 protein. This may be a more common mechanism behind early aggressive disease in females than skewed X‐chromosome inactivation.

P12‐03

Galactose consumption caused severe inflammation and amyloid fibril accumulation in brain especially in hypercholesterolemic state

Cho K‐H^1,2

¹ School of Biotechnology, Yeungnam University, Gyeongsan, Republic of Korea; ² Research Institute of Protein Sensor, Yeungnam University, Gyeongsan, Republic of Korea

Exposure of galactose is associated with brain aging, although there has been little information about consumption of monosaccharide (galactose, fructose, and glucose) and brain damage under hypercholesterolemic state. Treatment of galactose to apoA‐I (final 250 mM) resulted increase of advanced glycated end (AGE) product in lipid‐bound state as similar as fructose treatment, while lipid free apoA‐I showed less production of glycated end product. Treatment of galactose (final 250 mM) into human HDL₃ caused more smear band with retarded electromobility compared with treatment of fructose and glucose. Galactose treated HDL₃ did not prevent uptake of oxLDL into macrophages. Treatment of galactose into apoA‐I caused severe structural modification as similar as fructose treatment. Gal‐treated apoA‐I lost phospholipid binding ability and could not inhibit cupric ion mediated LDL oxidation, while native apoA‐I inhibited.

During 9‐week consumption of monosaccharide, galactose‐consumed zebrafish showed swimming defect with severe inflammation. Under hypercholesterolemic diet, galactose consumed group showed the lowest survival with dosage dependent manner. In normal diet group, total cholesterol and TG was not changed by the galactose consumption. However, the swimming defect was associated with loss of cell nucleus, increase of inflammation, accumulation of amyloid fibril in brain. Especially in galactose and high cholesterol diet, nucleus was more severely disappeared and accumulation of amyloid fibril was more detected with dosage dependent manner.

Conclusively, treatment of galactose caused modification of lipoproteins and consumption of galactose induced severe inflammation and amyloid accumulation with swimming defects.

P13. Methods and Techniques

P13‐01

Nanofibers mats – a new perspective for experimental studies of the nervous system

Dziewulska D^1,2, Gadamski R², Taraszewska A², Chrapusta S³, Sulejczak D³, Kowalczyk T⁴, Chrzanowska A², Ogonowska W², Wojda R², Wąsowska L² and Rafałowska J²

¹ Department of Neurology, Medical University of Warsaw; ² Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, PAS, Warsaw; ³ Department of Experimental Pharmacology, Mossakowski Medical Research Centre, PAS, Warsaw; ⁴ Institute of Fundamental Technological Research, PAS, Warsaw, Poland

Introduction: Introduction of nanotechnology into medicine has provided new therapeutic options. It has been demonstrated that implantation of nanofiber mats after nervous system injury allowed to diminish scar size and inflammatory reaction. It is also possible that, due to their ability to release active factors, nanofiber mats may replace intracerebral probes. To assess potential usefulness of nanofiber mats in releasing active substances we implanted them into the spinal cord subarachnoid space in adult Wistar rats.

Material and Method: The experimental animals were divided into four groups: group 1 – rats with implanted nanofiber mats, group 2 – rats with implanted nanofiber mats releasing glutamate, group 3 – rats with nanofiber mats releasing glutamate and treated orally by sodium valproate, and group 4 – control animals without nanofiber mats. The animals were killed 21 days after the mate implantation. Then, histopathological, immunohistochemical and ultrastructural evaluation of the spinal cords was performed.

Results: Morphological assessment revealed that implantation of nanofiber mats caused neither spinal cord damage nor inflammation (group 1). Also nanofiber mats releasing glutamate did not produce inflammatory reaction (group 2 and 3) although in group 2 morphological changes indicating toxic influence of glutamate were observed. These changes were less severe in group 3.

Conclusions: (1) Nanofiber mats are biocompatible and can be useful in long‐term animal experiments. (2) Nanofiber mats are able to release glutamate into the subarachnoid space. (3) Sodium valproate has a protective influence against glutamate toxicity.

P13‐02

Dermal nerve analysis in resin embedded skin biopsy is a reliable method to diagnose small fiber neuropathies, reproducing morphological changes seen in nerve biopsies

Chimelli J, Raposo M, Maceira JP and Chimelli L

University Hospital, Federal University of Rio de Janeiro, Brazil

Introduction: Over the last decades there has been increasing interest in skin biopsy to diagnose small‐fiber neuropathies. However, the density of unmyelinated intraepidermal fibers obtained by labeling with antibody against the pan‐axonal marker PGP 9.5, does not allow the morphological evaluation provided by the resin embedded section of a nerve biopsy. We propose that the dermal nerve changes seen in semi‐thin sections may reproduce those observed in nerve biopsies.

Material and Methods: We examined the dermal nerves in 1 μm resin‐embedded sections stained with Toluidin Blue of 10 skin biopsies, divided in 2 groups. Group I (6 patients) with clinical evidence of small fiber neuropathy, and group II (4 cases) of healthy volunteers. In 1 case of each group ultrastructural examination was also performed.

Results: Group I had changes previously described in neural trunks (small myelinated fiber loss, axonal degeneration, increase in endoneurial collagen). Two cases with the clinical hypothesis of leprosy, showed also lymphocytes around nerve fibers. Group II had normal morphology and density of myelinated and unmyelinated fibers.

Conclusion: We propose that semi‐thin sections of skin biopsies can replace nerve biopsies to the diagnosis of small fiber neuropathies since small nerve branches present the same changes observed in nerve biopsies, and are more reliable than quantitation of epidermal fibers.

P13‐03

Post‐mortem brain examination: does it still hold its role?

Zunarelli E, Mataca E, Nosseir S and Mengoli MC

Division of Surgical Pathology, University Hospital Policlinico di Modena, Modena, Italy

Introduction: The number and variety of investigations applicable during life and the improvements in diagnostic practice have led to downgrade the clinical importance of the autopsy.

To verify if this procedure is to hold its place as a helpful investigation and a means of medical audit, we reviewed our series of post‐mortem brain examinations.

Materials and Methods: Over the period 2008–2013, 680 macroscopic and microscopic brain examinations were performed as a routine part of the autoptic procedure in our institution. In 94 out of 680 cases, the presence of clinically, neuroradiologically and/or neurosurgically detected disorders was known.

Results: In 680 cases, incidental findings were 1% (meningiomas, 5 cases; tubers, 1 case).

In 94 cases of known brain lesions, post‐mortem examination provided supplementary information in 8 cases (8,5%), where the extension and severity of vascular and/or neurodegenerative events was better defined.

In 6 cases (6,4%), post‐mortem examination was diagnostic. Two brain lesions originally interpreted as metastases turned out to be primitive high grade neoplasms. On the contrary, a supposed primitive cerebellar tumor was a metastatic renal cell carcinoma. In 1 case, a bilateral fronto‐ethmoidal fibrous dysplasia turned out to be a bone‐invasive grade I meningioma. After two unsuccessful stereotactic brain biopsies, the post‐mortem brain examination enabled the diagnosis of choroid plexus papillary carcinoma. In another case, a definite diagnosis of anaplastic oligoastrocytoma was achieved after the partial resection of a huge fronto‐basal tumor.

Conclusions: In 14,9% of cases, autopsy still proves useful in a mutually rewarding partnership with neuroradiologists and neurosurgeons.

P13‐04

The essential role of Brain Bank Committee, Japanese Society of Neuropathology, in establishing Japan Brain Net

Murayama S¹, Saito Y², Akatsu Y³, Iritani S⁴, Oshima K⁵, Kakita A⁶, Takahashi H⁶, Kowa H⁷, Takao M⁸, Takanashi M⁹, Tanaka S¹⁰, Nishimura H¹¹, Niwa S¹², Miyata H¹³, Mochizuki H¹⁴, Yamada M¹⁵, Yokota O¹⁶ and Yoshida M¹⁷

¹ Tokyo Metropolitan Geriatric Hospital & Institute of Gerontology; ² National Center for Neurology and Psychiatry; ³ Fukushimura Brain Bank; ⁴ Nagoya University; ⁵ Matsuzawa Hospital; ⁶ Brain Research Institute, Niigata University; ⁷ Kobe University; ⁸ Mihara Memorial Hospital; ⁹ Juntendo University; ¹⁰ Hokkaido University; ¹¹ Kawasaki Medical University; ¹² Fukushima Medical University; ¹³ Research Institute for Brain and Blood Vessels, Akita; ¹⁴ Osaka University; ¹⁵ Saigata Hospital; ¹⁶ Okayama University; ¹⁷ Aichi Medical University, Japan

The Brain Bank Committee (BBC) of the Japanese Society of Neuropathology (JNS) is integrating two major brain bank networks in Japan, the Japanese Brain Bank Network for Neuroscience Research (JBBNNR) whose core is in Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology (TMGHIG) funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan and Research Resource Network for Neurology and Psychiatry (RRNNP) in National Center for Neurology and Psychiatry (NCNP), funded by the Ministry of Health, Labor and Welfare, Japan. TMGHIG and NCNP form ties for both networks. The members of the committee consist of neuropathologists who are responsible for each brain bank belonging to the two networks or their possible future members. The committee is trying to provide quality assurance of neuropathological diagnosis of each bank, in order to follow the quality assurance committee of NIH‐funded brain banks in USA. The committee is also trying to provide a teaching opportunity for young neuropathologists, who are interested in neurodegenerative diseases, the major target of Japan Brain Net. Thus, JNS is playing a key role in establishing an important bioresource network in Japan.

P13‐05

Tissue microarray technique: a valid method for immunohistochemical assessment of IDH1 mutation in gliomas

Canedo NHS¹, Corr R¹, Almeida B¹, Correa AHP¹, Chimelli L¹ and Pires ARC²

¹ Federal University of Rio de Janeiro; ² Fonte Medicina Diagnóstica, Niteroi, Brazil

Introduction: Immunohistochemistry has been widely performed in tissue microarrays (TMA) as a cost and time saving alternative for whole slide staining. Here we aim to validate the use of TMA for the assessment of IDH1 mutation in gliomas, since such validation has not been reported yet. IDH1 is one of the three isoforms of isocitrate dehydrogenase, metabolic enzymes involved in the citric acid cycle which acts at the conversion of isocitrate into alpha‐ketoglutarate. A recent genomic analysis identified somatic mutations at codon 132 of the IDH1 gene in approximately 12% of glioblastomas and 5/6 secondary glioblastomas. Since then, the R132H mutation of the IDH1 has been found in 70% of diffuse astrocytomas and oligodendrogliomas.

Material and Methods: 25 gliomas (1astrocitoma grade 2; 3 astrocytomas grade 3; 6 oligodendrogliomas grade 2 and 15 primary glioblastomas) were studied. From each case, one 1.0 mm cylinder was extracted from a representative paraffin block for the TMA construction. Immunohistochemistry was performed with IDH1 R132H clone H09 (Dianova) on both the TMA and corresponding whole slide from each case.

Results/Conclusion: In the TMA slides, all the 10 gliomas grades 2 and 3 were positive for the mutation, while the 10 primary glioblastomas were negative. The 5 glioblastomas studied in stereotactic biopsies were not represented in the TMA, because the cores slipped off the slide. There was a 100% correlation between TMA and whole slide analysis, and in both methods the positivity was diffuse, nuclear and cytoplasmic in the neoplastic cells.

P13‐06

Enhancing pathology diagnostic and prognostic reporting using digitized image analysis

Gokozan HN, Ahmad Fauzi MF, Elder B, Winter J, Puduvalli VK, Gurcan MN and Otero JJ

Division of Neuropathology, The Ohio State University College of Medicine, USA

Neuro‐oncology patient management requires objective interpretation of visual data in the form of histological and radiological images. The objective of our group is to implement diagnostic workflows that decrease subjectivity in pathological diagnosis by implementing digitized image analysis protocols. We focused our developments on two diagnostic time points: intraoperative neurosurgical consultation by pathology and prognostic reporting in the final diagnosis. Using a multidisciplinary approach that included image analysis scientists, translational neuro‐oncologists/surgeons, a chemical engineer, and neuropathologists, we generated a method capable of aiding intraoperative diagnoses and generating objective quantification of prognostic markers ki67 and p53. We also report image analysis workflows capable of objectively interpreting multiplex micelle‐quantum dot (QD) immunostaining of tumor cells. We compared standard immunohistochemical detection using polymer‐based secondary antibodies (DAKO Envision^TM) to micelle‐QDs. The following antibodies were tested in glioblastoma tissue sections: phospho‐Ser473‐AKT, AKT, PI4 kinase p110alpha, S6 ribosomal protein, phospho‐Tyr1173‐EGFR, and phospho‐S6 ribosomal protein. The approach provides a promising new approach that can increase efficiency, reduce inter‐observer variability and provide a new standard for pathology diagnosis.

P14. Mitochondrial

P14‐01

Clinicopathological study of MELAS with necrosis of basal ganglia and mtDNAG13513A mutation proceeding chronic renal failure

Kurashige T¹, Motoda A¹, Sugiura T¹, Nezu T¹, Nakamura T¹, Yamawaki T¹, Arihiro K² and Matsumoto M¹

¹ Department of Clinical Neuroscience and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University; ² Department of Anatomical Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS) is one of the most common respiratory chain disorders, characterized by sudden development of cerebral lesions reminiscent of large or small vessel strokes which cross vascular territories. Recently, some cases were reported to show a chronic renal failure before stroke like episodes. We reported 36 years old woman with MELAS and chronic kidney disease awaiting renal transplantation. At age 26, she was diagnosed as chronic renal failure. At age 35, she presented a generalized convulsive seizure. Muscle biopsy showed ragged red fibers and strongly SDH reactive blood vessels. mtDNA analysis revealed G13513A mtDNA mutation. Brain MRI revealed DWI/T2 hyper intensity area in right temporal lobe at first, and of basal ganglia at later. She died at age 36 because of chronic heart failure. Neuropathological examinations showed the infarct‐like regions in the cerebral cortex and subcortical white matter, and the necrosis of the basal ganglia with calcification. Cerebral microvessels showed high SDH immunoreactivities. In addition, our case showed mild changes in the substantia nigra with SSV. However, the patient had no necrotic regions in pons, medulla, and cerebellum. Her muscle biopsy specimen had ragged red fibers and strongly SDH reactive blood vessels, and her renal biopsy specimen showed granular swollen epithelial cells. The mtDNA G13513A mutation might cause the overlap syndrome of MELAS and Leigh syndrome.

P14‐02

Neuropathology of MELAS in the acute stage of stroke‐like episode

Kaneda D¹, Shintaku M¹, Kubota‐Sakashita M², Kato T² and Goto Y³

¹ Dept of Pathology, Osaka Red Cross Hospital; ² Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute; ³ Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Japan

Objective: To investigate the stroke‐like episode of MELAS in the acute phase.

Method: We performed immunohistochemistry analyses of neurovascular unit elements for two MELAS brains. One patient had died 16 days after the onset of acute stroke‐like episode.

Results: Both cases showed laminar cortical necrosis and white matter changes in old lesions. In acute lesions, moderate gliosis were noted in non‐necrotic cortical layer, associated with rarefaction and capillary proliferation. Immunoreactivity of AQP4 was reduced in the I–III layers of affected cerebral cortex beyond GFAP‐negative lesions. In choroidal plexus, the polarity of AQP4 immunoreactivity within the swollen ependymal cells was lost.

Conclusions: These findings indicate that the MRI findings of MELAS could reflect vascular rearrangement and hyperperfusion to compensate energy demand. The imbalance of water channel function due to mitochondrial dysfunction may also be involved in the pathophysiology of stroke‐like episode in MELAS.

P15. Motor Neuron Disease and Related

P15‐01

Familial neurodegenerative disease associated with a P497L mutation in the UBQLN2 gene

Newell KL^¹, Fahed AC^², McDonough B^², Gouvion CM^¹, Dure LS^³, Bebin M^³, Bick AG^², Seidman JG^², Harter DH⁴ and Seidman CE^²

¹University of Kansas Medical Center, Kansas City, KS; ²Harvard Medical School, Boston, MA; ³University of Alabama at Birmingham,Birmingham, AL; ⁴George Washington University, Washington, DC, USA

Mutations in the Ubiquilin‐2 (UBQLN2) gene, encoding a ubiquitin‐like protein involved in protein degradation, have been linked to X‐linked amyotrophic lateral sclerosis (ALS)/dementia. We have studied a family with a clinically heterogeneous neurodegenerative disorder ranging from early‐onset hyperkinesia, spastic paralysis, and dementia, to later‐onset ALS. DNA was isolated for genetic analysis. Brain autopsies were carried out in 4 affected family members. Neuropathology reports for 2 affected boys (18y, 19y) were reviewed, as the autopsy specimens were no longer available. Histological sections from brain and spinal cord were evaluated from a 67‐year‐old (67y) woman with clinically diagnosed ALS and her 37‐year‐old (37y) daughter with spastic paralysis, dysarthria, dysphagia, and behavioral dementia. Histochemical stains included hematoxylin and eosin‐Luxol fast blue and Thioflavin S. Immunohistochemical stains included β‐amyloid, tau, α‐synuclein, ubiquilin‐2, TDP‐43, and GFAP. Both brains had corticospinal tract degeneration. In the 67y brain, motor neuron loss was present in the cerebral cortex and brain stem. The 37y brain showed frontal lobe atrophy with neuronal loss and gliosis in cortex and caudate nucleus. Substantia nigra degeneration ranged from moderate (67y) to severe (37y). The 67y brain contained Lewy bodies, sparse‐moderate beta‐amyloid–immunopositive diffuse plaques, and sparse phospho‐tau. Both brains contained aggregates immunoreactive with antibodies to TDP‐43 and UBQLN2. Using exome sequence analysis, a novel mutation, c.1490C>T, in the UBQLN2 gene was associated with X‐linked inheritance in all studied affected individuals. The clinical and neuropathological findings associated with the UBQLN2P497L mutation expand the spectrum of neurodegenerative disease linked to UBQLN2mutations.

P15‐02

Pathological expressions of FALS‐related pathogenic molecules in the spinal cord of autopsied sporadic ALS patients

Ikemoto A, Tatsumi S, Urushitani M and Takahashi R

Department of Neurology, Kyoto University Graduate School of Medicine, Japan

Introduction: Since a certain pathogenic molecule of familial ALS might play a critical role in the process of TDP‐43 accumulation and motor neuron degeneration, it could also act similarly in sporadic ALS (SALS). Therefore, expressions of valosin‐containing protein (VCP), angiogenin (ANG), and charged multivesicular protein 2B (CHMP2B) in the spinal cord of SALS were examined immunohistochemically, in order to investigate involvement of these molecules in SALS pathogenic mechanisms.

Materials and Methods: Formalin‐fixed sections of spinal cords from 6 SALS and 6 control cases were immunostained with anti‐TDP‐43, VCP, ANG, and CHMP2B antibodies. Antibody binding was visualized by the ABC method.

Results: There was apparently no VCP‐immunoreactivity in the anterior horn neurons (AHNs) of controls, while in SALS numerous AHNs showed VCP‐immunoreactivity in the nucleus and some AHNs had cytoplasmic immunoreactivity. Regarding ANG, SALS exhibited AHNs with ANG‐immunoreactivity throughout the cytoplasm, while controls didn't show the immunoreactivity in the cell body. Some of SALS patients had ANG‐immunoreactive glias in the anterior horns. Immunostaining for CHMP2B revealed diffuse cytoplasmic immunoreactivity in AHNs and glias of SALS; however, no CHMP2B‐immunoreactivity in the cell body of controls.

Conclusions: These results indicate that each molecule might contribute to SALS pathogenesis differently. Histological features enable us to surmise that each might be associated with RNA metabolism in AHNs, with cytoprotection against TDP‐43 neurotoxicity, or with degradation of accumulated TDP‐43. Thus, SALS pathogenic mechanisms would be multi‐factorial from a viewpoint of molecular pathogenesis, so that further studies would be necessary to clarify the whole aspects of them.

P15‐03

Upregulation of xCT in spinal cords from sporadic ALS patients

Shibata N¹, Inose Y¹, Niida‐Kawaguchi M¹, Toi S², Kakita A³ and Takahashi H³

¹ Department of Pathology, Tokyo Women's Medical University; ² Department of Neurology, Tokyo Women's Medical University; ³ Department of Pathology, Brain Research Institute, Niigata University, Japan

A growing body of evidence suggests the involvement of oxidative stress and glutamate toxicity in amyotrophic lateral sclerosis (ALS). It is known that the cystine/glutamate exchange antiporter (xCT) mediates glutamate release associated with cystine transport in response to increased extracellular cystine levels as a consequence of increased oxidative stress. To determine the role for xCT in ALS, we compared the state of xCT expression in spinal cords from10 sporadic ALS patients and 10 age‐matched control subjects, using morphological and quantitative approaches. Immunohistochemical analysis revealed that the xCT determinants were localized in AChT‐identified lower motor neurons, GFAP‐identified astrocytes and Iba1‐identified microglia in the ventral horns and lateral funiculi, and that staining was more intense in the ALS cases than in the control cases. In the ALS cases, immunoreactivities for hydroxynonenal‐histidine adduct, hydroxydeoxyguanosine and nitrotyrosine as the major oxidative stress markers were clearly detectable in the remaining motor neurons, reactive astrocytes and activated microglia. By contrast, these immunoreactivities were less intense in the control cases. Immunoblot analysis disclosed a significant increase in the β‐actin‐normalized xCT signals in the ALS group as compared to the control group (P < 0.05). The present results provide in vivo evidence that upregulation of xCT in the motor neurons, astrocytes and microglia in ALS spinal cords, in cooperation with reduced activity of astrocytic glutamate transporter in the presence of oxidative stress described in previous reports, participates in glutamate neurotoxicity.

P16. Muscle

P16‐01

Bmi1 protects satellite cells against oxidative stress induced DNA damage

Marino S and Di Foggia V

Blizard Institute, Barts and The London School of Medicine and Dentistry, UK

Introduction: The Polycomb group (PcG) protein Bmi1 is an epigenetic chromatin modifier involved in heritable gene repression and maintenance of stem cell self‐renewal and progenitor proliferation. Our recent data show that the loss of Bmi1 in the mouse results in depletion of Pax7 positive satellite cells (SCs) with reciprocal increase in MyoD positive cells and consequent reduction of muscle fiber size. Conversely, conditional activation of Bmi1 in the SC population increases their proliferation and capacity to re‐enter the cell cycle upon stimulation in vitro without preventing their differentiation.

Methods: We analyzed the downstream mechanisms mediating the observed effects of loss and gain of function of Bmi1 in SCs by means of trascriptome wide analysis of gene expression followed by functional validation with cellular assays.

Results: The expression of p16^Ink4a, p19^Arf and p21^waf/cip1, cell cycle inhibitors known to be downstream effectors of Bmi1 was increased in the SCs isolated from Bmi1–/– mice. Interestingly, the expression of these genes was not downregulated in SCs overexpressing Bmi1 and significant upregulation of metallothionein‐1 (Mt‐1) gene was observed instead. Conversely, a significantly reduced expression level of Mt‐1 was found in SCs isolated from Bmi1–/– mice. Mt‐1 is a cysteine‐rich, low molecular weight protein, which is involved in mitochondrial function regulation and provides protection against oxidative stress. Further studies based on a series of oxidative stress assays suggest that SCs overexpressing Bmi1 suffer much less oxidative stress and consequent DNA damage, while Mt‐1 knock down leads to the opposite effects.

Conclusion: These data suggests that Bmi1 plays an important role in protecting SCs against oxidative stress induced DNA damage.

P16‐02

Endomysial capillaries in myopathy associated with anti‐signal recognition particle antibody show distinct features from those of dermatomyositis

Kanda T and Omoto M

Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan

Pathology of myopathy associated with anti‐SRP antibody and dermatomyositis, both of which are inflammatory myopathies, shared endomysial capillaries with MAC/C5b‐9 deposition. Dermatomyositis has been viewed as a humorally mediated autoimmune disease, in which the primary antigenic target in muscle is the endothelium of endomysial capillaries and muscle damage is hypothesized as the result of ischemia. Muscular pathology of dermatomyositis includes necrotic endothelial cells, endothelial cell cytoplasm with vacuoles, and endothelial hyperplasia with tubuloreticular profiles, which contribute to the reduction of capillary density. On the other hand, the role of anti‐SRP antibodies in the pathogenesis of myopathy has not been defined, and ultrastructural pathologies of endomysial capillaries in this disorder have not been reported in detail. We describe three anti‐SRP antibody positive Japanese patients with similar clinical features (two men and one women aged 40 to 55 years; mean, 45.3). Light microscopical pathologies of our patients showed distinct stereotyped features including an active necrotizing myopathy, no or little inflammation and MAC/C5b‐9 deposition in endomysial capillaries. Ultrastructurally, necrotic endothelial cells were rare and the density of endomysial capillary was not reduced. Neither endothelial cell cytoplasm with vacuoles nor endothelial hyperplasia with tubuloreticular profiles were found. In conclusion, unlike in dermatomyositis, it is suggested that endomysial capillary is not the major target in the pathogenesis of myopathy associated with anti‐SRP antibody.

P16‐03

Gait analysis, muscular force and protein expression after disuse and rehabilitation on weanling rats

Mattielo‐Sverzut AC¹, Noguti KL¹, Foresto CS¹, Bianchi E¹, Ferrari RJ² and Huard J²

¹ Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Brazil; ² McGowan Institute of Regenerative Medicine, University of Pittsburgh, USA

Comprehensive approach of muscle abnormalities after disuse allows establishing a safety rehabilitation protocol of exercise. The aim of this study was analyze the effects of these treatments on gait, muscular force and collagen expression in weanling rats after hindlimb immobilization. Right hindlimb of twenty‐four female Wistar rats were immobilized for ten days (weight average of 114.47 g). After removal the device they were treated for ten days by free movement (IFMG), intermittent stretching (IISG), or eccentric exercise (IEEG). Images of the footprint during the gait (right foot on pre‐swing phase) were obtained before the immobilization and after each protocol. Total spread of the toes (TS) and spread of intermediate toes – second and forth – (IT) were measured. Western blot of collagens type 1 and 3 was developed with soleus muscle. The leg frequency–force curve was obtained during the stimulation of sciatic nerve. The data was normalized by cross sectional area of the triceps surae. Gait analysis showed an increase of the TS and IT values of the IFMG (p < 0.05). There are no differences in force–frequency curves between all groups (p > 0.05). All groups presented collagen 1 reduction, whereas collagen 3 presented values similar to control. These results show that ten days of intermittent stretching or eccentric exercise can recover the function of the hindlimb. The reductions of collagen 1 may have affected the contractility of the soleus muscle.

P16‐04

Macrophagic myofasciitis: a case series of five children from Indian subcontinent

Nalwa A¹, Sharma MC¹, Suri V¹, Sarkar V¹ and Gulati S²

Department of ¹ Pathology and ² Pediatric Neurology, All India Institute of Medical Sciences, India

Background: Macrophagic myofasciitis (MMF), first described in 1998, is a rare form of muscle disease characterized by perifascicular accumulation of macrophages as a reaction to intramuscular injection of aluminum containing vaccines. Most of the cases have been reported in adults; however, sporadic cases in children have been described. Herein, we report a case series of five children with macrophagic myofasciitis from the Indian subcontinent. To the best of our knowledge this is the first such series from this part of the world.

Patients and Methods: We present five cases of MMF reported over a period of four years in our institute. All of them were children who presented with hypotonia and motor or psychomotor delay. The muscle biopsies were done with the clinical diagnosis of congenital myopathy. Electron microscopy was performed in only one case.

Results: The children belonged to the age group ranging from four months to two years with a male to female ratio of 4:1. All of them presented with muscle weakness; however, creatine kinase levels were not raised. All of them had a history of hepatitis B vaccination. Histopathological examination revealed sheets of histiocytes with granular cytoplasm infiltrating the perimysium. The granular material in the histiocytes was Periodic Acid Schiff (PAS) positive. Ultrastructural examination revealed crystalline material in the histiocytes.

Conclusion: MMF is a rare muscle disease, however it should be considered in the differential diagnoses of congenital myopathies in children.

P16‐05

Molecular mechanisms of immobilization inducing muscle atrophy in different stages of rat's development

Mattielo‐Sverzut AC¹, Foresto CS¹, Gomes SP², Graça FA², Kettelhut IC^2,3, Noguti KL¹ and Gonçalves DAP²

Departments of ¹ Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, ² Physiology and ³ Biochemistry/Immunology, Ribeirao Preto Medical School, University of Sao Paulo, Brazil

Immobilization used in prolonged periods of time can cause muscle atrophy with negative impact on the duration and intensity of rehabilitation. The aim of this work was to investigate the effects of immobilization on the ubiquitin‐proteasome (UPS) and lysosomal/autophagy proteolytic systems and intracellular signaling pathways during different stages of rat's development.

Hindlimbs of weanling (3wk‐old) and adult (∼12wk‐old) female Wistar rats were unilaterally immobilized (IM) for 10 days. Samples of soleus muscle were used to identify atrogin‐1 and MuRF1 for UPS and LC3 for autophagy. The activation status of IGF‐1/Akt signaling was analyzed by qPCR (IGF‐1 mRNA) and immunoblot (Akt phosphorylation/activation). (Ethical Committee of Animal Research – protocol number 146/ 2012.)

IM reduced soleus mass by 80% and 57%, in weanling and adult rats, respectively. In both age groups, the lipidated and activated form of LC3, LC3‐2, was increased (∼3‐fold) by IM. Despite no change in Atrogin‐1, the protein content of atrophy‐related Ub‐ligase MuRF1 was up‐regulated (∼2‐fold) only in muscles from adult IM rats. In agreement with this data, Akt phosphorylation was reduced by 55% only in adult IM rats. On the other hand, IGF‐1 mRNA was down‐regulated (∼60%) in both age groups.

These results suggest the molecular mechanisms of muscle atrophy induced by IM occur in an age‐dependent manner. In adult rats, IM hyperactivates autophagy and UPS by down‐regulating IGF‐1/Akt signaling. However, in young animals, IM stimulated only autophagy and reduced IGF‐1 expression, with no effect on Akt phosphorylation indicating that other intracellular factor also contribute to restrain autophagy.

P16‐06

Muscular dystrophies in Arabian gulf population

Al Homssi MF and Mohamad A

College of Medicine, University of Sharjah, Sharjah, United Arab Emirates

Arab population is characterized by large family size and high level of inbreeding with consanguinity rates in the range of 25–60%. A very high dystrophin deletion mutation frequency is documented in Arabs compared to other ethnic groups. Frame shift deletion mechanisms are detected in 51.3% of Egyptian Duchene and Becker muscular dystrophy patients. Duchene‐like muscular dystrophy (DLMD) was recognized in Sudan, Tunisia, Kuwait, Saudi Arabia, Libya, and Qatar, and mapped to chromosome 13q in Tunisian families, and same locus was also reported in Algerian and Moroccan families. Autosomal recessive limb‐girdle muscular dystrophies (LGMD) have a particular high frequency in Arab states especially Tunisia. LGMD2B, Miyoshi myopathy, was mapped to chromosome 2p12 in 12 families including a Tunisian family, and reported in Moroccan, Saudi, and Libyan Jewish patients. LGMD2C is the most frequent LGMD2 in Tunisia where patients share the same founder g‐SGCG mutation. LGMD2I was described and mapped in a Tunisian family in which the causing gene FKRP was identified. Large Bedouin kindred with X‐linked EDMD presented features suggestive of a neurogenic atrophy suggestive of a co‐morbid, mild congenital myasthenic syndrome. Congenital muscular dystrophy MDC1A caused by LAMA2 gene was described in Saudi, Tunisian, and Kuwaiti CMD patients. A new autosomal recessive CMD was described and linked to a new locus in a family from the United Arab Emirates. North African and Yemenite Jews and Kuwaiti individuals have significantly myotonin gene CTG repeats in the normal range, explaining the low frequency of myotonic dystrophies in these populations.

P16‐07

Myopathy as an isolated manifestation related to HTLV‐1 infection

Pelição GR¹, Lenzi MER², Macêdo PJOM¹, Leite ACCB³, da Costa Carvalho MG⁴, Canedo NHS⁴ and Vasconcellos LFR^1,2

¹ Instituto de Neurologia Deolindo Couto, Federal University of Rio de Janeiro, Brazil; ² Hospital Federal dos Servidores do Estado, Rio de Janeiro, Brazil; ³ FIOCRUZ; ⁴ Department of Pathology, Hospital Universitário Clementino Fraga Filho, Federal University of Rio de Janeiro, Brazil

A 52‐year‐old afrodescendant woman insidiously developed over 10 years proximal tetraparesis, first limited to lower limbs, without sensory or sphincter manifestation. After six years she suffered a myocardial infarction, reason why 20 mg of simvastatin was prescribed. Worsening of weakness was reported after this drug, becoming dependant of crutches in two years, and no improvement was observed after statin withdraw. She was previously diagnosed with hypertension and hypothyroidism and had no family history of neurological disease. On examination she exhibited xeroderma over the extensor surface of limbs and skin folds, an anserine gait with bilateral support, proximal muscle atrophy with proximal tetraparesis and bilateral achillian hyporeflexia. Blood analysis showed raised levels of ESR, CK and LDH and a positive immune reaction to HTLV 1 with elevated proviral load. FAN, anti‐Jo 1, anti‐HIV and viral hepatitis serology were negative, as well as genetic study for limb‐girdle muscular dystrophy and facioscapulohumeral muscular dystrophy. Electromyography showed mixed polyneuropathy and myopathy. The sole change in CSF exam was a positive HTLV 1 immune reaction. Right deltoid muscle biopsy showed preserved muscle fibers, noting occasional fibers in degeneration/regeneration with rare lymphocytes around. The polymerase chain reaction using general primers for HTLV I was positive. Given the clinical feature and other findings the diagnosis of myopathy related to HTLV‐I was established and treatment proceeded with 3 cycles of pulse therapy with methylprednisolone and followed by oral prednisone. Patient showed partial improvement in strength, especially in lower limbs, with increased ability to walk a few meters without support and longer standing.

P16‐08

Pericytes play a central role in the microvascular niche of muscle stem cells

Kostollari E, Lafuste P and Gherardi R

INSERM U955 & Paris Est University, France

Stem cells reside in specialized environments, termed stem cell niches, producing factors that regulate their behavior. Myogenic stem cells called satellite cells (mSCs) are involved in both post‐natal muscle growth and muscle regeneration following injury. We previously showed that adult mSCs residing beneath myofiber basement membrane are closely associated with capillaries and functionally interact with endothelial cells (Christov et al., Mol Biol Cell 2007), and self‐renew in response to local release of Angiopoietin‐1(AbouKhalil et al., Cell Stem Cell 2009). We show now that (i) muscle vascularization is rudimentary at birth; (ii) post‐natal angiogenesis is spatio‐temporally coordinated with myofiber growth and mSC entry into quiescence; (iii) mSCs become progressively associated with NG2+ Nestin + pericytes; (iv) muscle pericytes exert dual paracrine influence on myogenic cells in vitro, including differentiation/growth‐promoting effects of IGF‐1 and quiescence‐promoting effects of Angpt‐1; diphtheria toxin‐induced ablation of muscle pericytes of Tg:NG2‐Cre:iDTRmice caused growing myofiber hypotrophy, and released adult mSCs from quiescence as also observed after selective inhibition of pericyte production of IGF1 and Angiopoietin‐1, respectively, in ad hoc Cre‐lox mouse models; (v) these effects did not involve the mesenchymal stem cell potential of pericytes (Delavalle et al., Nat Commun 2011), indicating that “stem cells support other stem cells” in muscle explaining how functional benefits of cell therapies may occur despite poor differentiation of the grafted cells. In conclusion, pericytes play a central role in the microvascular niche of muscle stem cells.

P16‐09

Riboflavin‐responsive multiple acyl‐CoA dehydrogenase deficiency (MAD): light and ultrastructural findings in muscle biopsy

Laín AH¹, Casanueva MAM², Gonzalez OT¹, Encinar AS², Perez RS² and Gonzalez CD³

¹ Neuropathology Unit, Pathology Department, Hospital Universitario 12 de Octubre, Madrid, Spain; ² Biochemistry Department, Hospital Universitario 12 de Octubre, Madrid, Spain; ³ Neurology Department, Hospital Universitario 12 de Octubre, Madrid, Spain

Patient: A 33‐year‐old female who presented with progressive neck and proximal limb weakness. She had no data of bulbar, respiratory or heart involvement. One year before she presented a similar episode during the third trimester of pregnancy that recovered spontaneously six months after delivery. She had no family history of neurological diseases. CKP was slightly elevated 162 IU/L (normal up to 145). EMG was normal. Serum acylcarnitines and urine organic acids were elevated. Free serum carnitine was normal.

Results: Left quadriceps muscle biopsy showed preserved architecture. There was variability in fiber size. Numerous vacuoles in most muscle fibers were identified; some of them were of large size. They were distributed throughout the sarcoplasm although they were predominantly subsarcolemmal. Vacuolar content showed positivity with Oil‐Red‐O staining being negative with PAS. The vacuoles were present in both fiber types, although they are more frequent and larger in type I fibers. On SDH, a focal decrease in enzyme activity was found. Electron‐microscopic showed a marked increase in intracytoplasmic lipid droplets both in number and size. There was also an increase in the number of mitochondria. Lipid droplets were often present next to mitochondria. No autophagic vacuoles were seen. The pathological diagnosis was lipid storage myopathy.

Conclusion: The genetic study showed a heterozygous mutation (c.1180G>A) in the ETFDH gen (*).

After 48 hours treatment with vitamin B2 (Riboflavin) the patient showed progressive improvement, remaining virtually asymptomatic after 1–2 months of treatment.

(*) We did not find a second mutation yet. The search for the second mutation and pathogenicity of this one are in progress for the meeting in September 2014.

P16‐10

Sarcolemmal membrane alterations in autosomal recessive centronuclear myopathy due to BIN1 mutations

Malfatti E¹, Nennesmo I², Laporte J³, Romero NB² and Oldfors A¹

¹ Department of Pathology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; ² Unité de Morphologie Neuromusculaire, Institut de Myologie, Groupe Hospitalier Universitaire La Pitié‐Salpêtrière, Paris, France; ³ Department of Translational Medecine, IGBMC, Illkirch, France

Introduction:  BIN1‐related centronuclear myopathy is an autosomal recessive congenital myopathy morphologically characterized by type I predominance and centrally located nuclei in the majority of muscle fibers. To date only few patients have been reported, making this entity only partially characterized. BIN1 protein appears to be essential for T‐tubules positioning and remodeling. A possible role in autophagosome maturation has been speculated.

Patients and Methods: Here we present an extensive histoenzymological, immunohistochemical, and ultrastructural analysis of five muscle biopsies (two patients had two muscle biopsies at different ages) from three unrelated AR‐BIN1 patients.

Results: All muscle biopsies showed rounded hypotrophic type I fibres with very high percentage of central nuclei. Fibroadipose tissue replacement of variable degree without any ongoing regenerative process was noted in all biopsies. Around fifteen per cent of fibers harbored sarcolemmal membrane invaginations extending toward the centrally located nuclei. Some fibers also showed large vacuoles associated with the sarcolemma. Both membrane invaginations and vacuolization manifested high acetylcholinesterase and acid phosphatase activities, and immunoreactivity for LAMP2 and P62/SQSTM1, suggesting an authophagic component.Electron micrographs confirmed the presence of invaginations and membrane vacuolization containing cellular debris and autophagic material; clusters of centrally located myonuclei surrounded by abundant amorphous material containing vacuoles were also a constant feature.

Conclusions: We suggest that sarcolemmal invaginations and vacuolization are characteristic findings that suggest BIN1 as the responsible gene in the diagnostic workup of centronuclear myopathies. Histoenzymomological and immunohistochemical studies suggest a possible authopagy dysregulation in BIN1‐related centronuclear myopathy.

P16‐11

Atypical neuromuscular manifestation in infant with pulmonary tuberculosis

Madruga M¹, Vieira TM¹, da Silva Pereira RC², Canedo NHS², Feitosa LP³, de Vasconcelos LG³, Baptista KL³, Torbey AFM³ and Vasconcelos MM³

¹ INCA; ² Universidade Federal do Rio de Janeiro; ³ Universidade Federal Fluminense, Brazil

In children under 3 years the tuberculosis (TB) is often more severe than in adults: the symptoms are nonspecific, the samples for culture are difficult to be obtained, and the results, are usually negative. This leads to many undiagnosed cases, which increases the risk of severity. Another important reason for an earlier diagnosis in children is that, in them, TB is considered a sentinel event, reflecting recent infection by some community source. It's imperative that doctors pay attention to the possibility that any neuromuscular symptoms are atypical manifestations of pulmonary TB.

Case Description: Female patient, 14 months old, 4 weeks with weakness in the lower limbs that progressed to the upper limbs and to the face, started after two successive episodes of tonsillitis treated with benzathine penicillin. Also emerged laced erythematous lesions on the face and the four limbs as well as edema. On physical examination, asymmetric flaccid tetraparesis with are flexia and facial disparesia. VHS: 56 mm/h; CK: 893 U/L; electromyography in myopathic pattern. MRI and CT of the head and spine unchanged. Bilateral axillary lymphadenopathy. Muscle biopsy showed atrophy of large groups and fibers with signs of degeneration/regeneration, which are associated to significant vasculitis, mainly lymphocytic and focal leukocytoclasia. It was noted still some groups of fibers of the same type. Weeks after muscle biopsy result for BAAR in gastric lavage was positive.

Conclusion: The first presentation of tuberculosis with neuromuscular involvement is rare, but should be considered in pediatric patients with lymphadenopathy, due to tuberculosis prevalence in our population.

P17. Myelin Disease

P17‐01

An autopsy case of inflammatory demyelinating disease showing subacute progressive cognitive impairment

Nishihira Y¹, Hasegawa J¹, Nishihira J², Sueyoshi T³, Ihama Y⁴, Miyazaki T⁴, Endo K¹ and Takahashi H⁵

¹ Dept. of Neurol., Tomishiro Central Hosp.; Dept. of ² Neurol and ³ Radiol., Minei Daiichi Hosp.; ⁴ Dept. of Legal Medicin., School of Medicin, Ryukyuuniv.; ⁵ Dept. of Pathol., Brain Res. Inst., Niigata Univ. Japan

This is a 62‐year‐old man with a 1.5‐month history of progressive cognitive impairment. On examination, he showed frontal and parietal cerebral cortex symptoms and severe dementia. His serum β‐D‐glucan level was mildly increased. Cerebrospinal fluid examination showed pleocytosis, elevated protein level and oligoclonal IgG bands. MRI revealed foci of intramedullary abnormal signal, with contrast effect, in the lumbar cord. He was suspected of having fungal meningoencephalitis and granulomatous disease. He was treated with anti‐fungal agents without significant effect. However, his symptoms improved with administration of steroids. The patient died by accident, about 6 months after onset of the disease. The brain weighed 1,280 g after formalin fixation. There was traumatic subarachnoid hemorrhage, which was considered to be the cause of death. Lymphocytes‐predominant inflammatory cell infiltration was evident in the subarachnoid space and the perivascular areas in the parenchyma, the picture being that of meningoencephalitis. No bacteria, fungi or tubercle bacilli were detected. No demyelinating lesions were evident in the brain. The lumbar cord lesions, which extended from white matter to gray matter, were those of demyelinating disease; loss of myelin was evident, but axons were well preserved. There were perivascularly mophocytic infiltration and parenchymal microglia activation, but no histological findings suggestive of sarcoidosis or ADEM were evident. This is an unusual case of subacute progressive cognitive impairment with lumbar cord demyelinating (multiple sclerosis?) and meningoencephalitic pathologies. Progressive dementia as a cardinal symptom appeared to be extremely rare in demyelinating disease.

P17‐02

Demyelinating disease and oligodendroglioma: case report

Pereira RCS¹, Vieira TM², Madruga M², Silva MR³ and Canedo NHS¹

¹ Departamento de Patologia, Hospital Universitário Clementino Fraga Filho, UFRJ; ² Departamento de Patologia, Instituto Nacional de Câncer, INCA; ³ Departamento de Neurocirurgia, Hospital Universitário Clementino Fraga Filho – UFRJ, Brazil

There are very few reports on the occurrence of brain tumors in patients with demyelinating disease and vice versa: demyelinating lesions in patients with brain tumor. It has been suggested that either the reactive glial cells at the demyelinating site could undergo transformation, or that the tumor itself, from the very beginning, could induce an autoimmune response to myelin leading to the concomitant diseases. We report the case of a 23‐year‐old female patient, previously asymptomatic, who presented hemiparesis on the left side with gradual progression in weeks. Cerebral NMR showed a round 1,9 cm hypersignal in T2 and FLAIR area in the white matter and extending into the subcortical region at the right frontal lobe, with contrast enhancement. Other similar lesions were found at nucleo‐capsular region on the right hemisphere, adjacent to the thalamus. Lumbar puncture did not show abnormalities. The patient was submitted to two cycles of methylprednisolone with no satisfactory results and a stereotactic brain biopsy was performed for evaluation. Bielchowsky and Luxol Fast Blue stainings showed maintenance of the axons with multifocal loss of myelin. CD68 immunostaining revealed a substantial increase in phagocytic cells, and Olig‐2 antibody showed an increase in the number of oligodendrocytes, but proliferation index with Ki‐67 was very low, under 1%. IDH‐1 was negative. The diagnosis was demyelinating disease. Eight months after the first biopsy, there was a change in the NMR pattern, and the right frontal lobe lesion became heterogeneous. Additional neurological impairment developed, such as sensory symptoms, as well as VII and XII cranial nerve palsies. A new biopsy was performed and an Anaplastic Oligodendroglioma (WHO Grade III) was diagnosed. This case alerts to the need of tight vigilance of unexpectedly unfavorable outcome in patients with demyelinating disease, and adds to the still short list of concurrent occurrence of demyelinating and neoplastic lesions.

P17‐03

Transverse myelitis due to cyanocobalamin deficiency – case report

Wilson AHB, Guerra‐Peixe MCC, João RB and Hampshire‐Araújo FC

Faculdade de Medicina de Petrópolis, Hospital Alcides Carneiro, Brazil

Introduction: Cyanocobalamin is required for the initial development and myelination of the CNS as well as for the maintenance of its physiological function. Its deficiency can cause demyelination of various structures, among them lateral and dorsal columns of the cervical and thoracic cord.

Case Report: A 47‐year‐old female patient treated for symptomatic iron deficiency anemia for 2 years, started paraparesis 8 months later. Three months ago started with paraplegia, loss of sphincter control, hypoesthesia (LL), sacral ulcer.

Examination: Obese, restricted to bed, pale 2+/4+, hypochromic spots in the cheek, angular cheilitis, atrophic tongue. Cardiac, respiratory and abdominal devices without changes.

Neurologic: Mental function without changes, no meningeal signs, coordination preserved, paraplegia, hypotonia (LL), preserved tropism, deep tendon hyporeflexia bilaterally (LL), cutaneous‐plantar bilaterally absent, sensory level at T10, loss of proprioception (LL). Cyanocobalamin: 159 pg/ml; VCM: 120 fl; TSH: 26.7 μUI/ml; free T4: 0.2 ng/dl; Anti‐TPO: 245.3 U/ml; Anti‐parietal cell antibody: 1/40 (RV 1/20); EDU + Biopsy: Moderate enanthematous gastritis and intestinal metaplasia incomplete; Negative Urease Test; MRI Thoracic‐Lumbar: Unchanged. Treatment with intramuscular cyanocabalamin therapy was started and six months after showed significant improvement in anemia and progression to grade III force, partial sphincter control and recovery of sensory functions.

Results: We observed association between pernicious anemia, Hashimoto thyroiditis and B12 deficiency in this case. Sensory level is a rarely observed condition, associated with poor prognosis.

Conclusion: Cyanocobalamin deficiency's inclusion is required among the differential diagnosis of transverse myelitis, especially in patients with a history of chronic anemia where the etiology has not been clarified. It is up to the clinician to prevent clinical details go unnoticed, because the consequences of the misdiagnosed patient may be severe and sometimes irreparable.

P18. Neurodegeneration, Other and General

P18‐01

Clinical feature of 19 cases of neuronal intranuclear inclusion disease diagnosed by skin biopsy

Sone J¹, Kitagawa N², Sugawara E³, Iguchi M⁴, Inagaki T¹, Yoshimura H⁵, Ishii J⁵, Kawamoto M⁵, Mori K⁶, Araki K¹, Masuda M¹, Yoshida M⁷, Iwasaki Y⁷, Tanaka F⁸ and Sobue G¹

¹ Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; ² Department of Neurology, Kosei Chuo General Hospital, Tokyo, Japan; ³ Department of Neurology, National Hospital Organization Yokohama Medical Center, Yokohama, Kanagawa, Japan; ⁴ Department of Neurology, Sapporo Yamano‐ue Hospital, Sapporo, Hokkaido, Japan; ⁵ Department of Neurology, Kobe City Medical Center General Hospital, Kobe, Hyogo, Japan; ⁶ Department of Neurology, Oyamada Memorial Spa Hospital, Yokkaichi, Mie, Japan; ⁷ Department of Neuropathology, Institute for Medical Sciences of Aging, Aichi Medical University, Nagakute, Aichi, Japan; ⁸ Department of Neurology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan

Introduction: Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease characterized by widespread presence of eosinophilic hyaline intranuclear inclusions in neuronal cells and somatic cells. Recently we reported the capability of skin biopsy for antemortem diagnosis of familial and sporadic NIID. We studied clinical and pathological features of NIID cases diagnosed by skin biopsy.

Patients and Methods: We studied skin biopsy samples of 9 NIID patients from two familial NIID lineages presenting neuropathy symptoms in which some patients were diagnosed as NIID by autopsy, and 10 cases of sporadic NIID presenting dementia, leukoencephalopathy and characteristic MRI DWI high signal in cortico‐medullary junction diagnosed by skin biopsy. Skin biopsy samples from patients undergoing treatment at Nagoya University hospital and normal volunteers.

Results: Intranuclear inclusions were found in all skin biopsy samples of NIID patients. Inclusions have identical features with those of reported NIID inclusions in neuronal cells. Weakness and sensory disturbance are observed in almost all familial NIID cases, but rare in sporadic NIID cases. Besides, ataxia, abnormal behavior and epilepsy were observed only among sporadic cases.

Conclusion: NIID cases with leukoencephalopathy present peripheral nerve damage. To elucidate the pathophysiology of NIID, we will promote the antemortem diagnosis of NIID with skin biopsy.

P18‐02

Direct comparison between LM and immunoEM of Pick bodies and Lewy neurites based on dual visibility of quantum dots

Uchihara T¹, Uematsu M¹, Kanazawa T¹, Nakamura A^¹, Endo K^², Ichikawa M^², Tsuchiya K³, Adachi E⁴

¹Structural Neuropathology, Tokyo Metropolitan Inst Medical Sci; ²Histology Center, Tokyo Metropolitan Inst Medical Sci; ³Pathology, Tokyo Metropolitan Matsuzawa Hosp; ⁴Matrix Biology & Tissue Regeneration, Kitasato Univ, Japan

Direct comparison between light microscopy (LM) and immunoelectron microscopy (immuno EM) was achieved with “Three dimension (3D)‐oriented immunoEM,” where, we used quantum dots (QDs), fluorescent and electron‐dense nanocrystals of uniform size containing selenium (Se) and cadmium (Cd), as immunolabeling particles on thick floating sections including the entire target. This dual visibility allowed to choose the QD‐labeled target based on the fluorescent signal, which is reconstructed into 3D data on confocal microscopy. After landmarking by microdissection around the target, the same section was subjected to epon embedding for EM. Because fluorescent data are already available, it is possible to retrieve the LM plane exactly corresponding to the final EM section. Energy dispersive X‐ray spectrometry (EDX), which demonstrated pixel‐dependent parallel peaks corresponding to Se and Cd on the QD‐like spots, confirmed the identity of QD labeling. Advantages of this LM‐guided, target‐oriented EM method based on the dual visibility of QD include 1) intense immunolabeling, not attenuated by epon embedding, 2) trimming for EM after identification of desired targets on LM, 3) EDX confirmation for identity of QD labeling, 4) easy retrieval of LM plane exactly corresponding to the final EM section for precise comparison between LM and EM, 5) applicability to routinely handled human samples fixed in formalin 6) conventional confocal and EM (except EDX) are sufficient for this experiment. This LM‐guided immuno EM is a powerful tool to identify ultrastructures of desired target in relation to relevant molecules such as tau, alpha‐synuclein as we have demonstrated recently. For example, we were successful in identifying amorphous alpha‐synuclein deposits in axon, which were named “pale neurites” as earliest lesion of Parkinson disease.

P18‐03

High prevalence of TDP‐43 in Asians older adults: a clinicopathological study on a population‐based sample

Nascimento C¹, Rodriguez RD¹, Leite RP^1,2, Suemoto CK², Zatz M³, Pasqualucci CA⁴, Filho WJ⁵ and Grinberg LT⁶

¹ Experimental Pathophysiology Discipline, University of Sao Paulo Medical School and Brazilian Aging Brain Study Group/LIM22; ² Departments of Geriatrics, University of São Paulo Medical School and Brazilian Aging Brain Study Group/LIM22; ³ Human Genome Research Center, University of Sao Paulo, Brazil; ⁴ Departments of Pathology, University of São Paulo Medical School and Brazilian Aging Brain Study Group/LIM22; ⁵ Departments of Geriatrics, University of São Paulo Medical School and Brazilian Aging Brain Study Group/LIM22; ⁶ Departments of Pathology, University of São Paulo Medical School, Brazilian Aging Brain Study Group/LIM22 and Department of Neurology, University of California, San Francisco, USA

Aggregation of abnormal proteins is one of the hallmarks of neurodegenerative processes. TDP‐43abnormal deposition is central in the majority of frontotemporal lobar degeneration and amyotrophic lateral sclerosis cases. Several factors including educational level, language and cultural aspects influence the clinical expression of these pathological changes. Susceptibility to disease also depends on ethnicity. Our group previously demonstrated that Africans ancestry protects against the accumulation of β‐amyloid plaques. In this study, we examined non‐demented subjects from the Brain Bank of the Brazilian Aging Brain Study Group of the University of Sao Paulo Medical School for the presence of abnormal TDP‐43 deposition. Ethnicity was ascertained by DNA markers in most of the cases. We included 202 non‐demented subjects, older than 50 years: either Caucasians (n = 176) or Asians (n = 26). Presence of abnormal TDP‐43 was assessed in slides immunostained against phospho‐TDP (MAb409/410 Cosmo Bio, Tokyo, Japan, 1:1000). Positivity required identification of inclusions in at least, one of the regions of interest (inferior temporal gyrus, hippocampal formation and amygdala). Chi‐square test showed significant difference in TDP‐43 positivity between Caucasians and Asians (p = 0.04). Logistic regression analysis showed higher prevalence of TDP‐43 in Asians compared to Caucasians subjects, after adjusting for several confounding factors (OR = 0.34, 95% CI 0.12–0.94, p = 0.04). Our results suggest that Asians are more prone to exhibit TDP43 inclusions than Caucasians, although it is not clear if this deposition results in clinical decline. Studies using different ethnic group dwelling in the same city may help to identify genetic protective factors against the expression of clinical dementia.

P18‐04

Incidence and extent of TDP‐43 proteinopathy in aging human brain

Uchino A^1,2, Takao M¹, Saito Y³, Sumikura H¹, Nakano Y, Hatsuta H¹, Nishiyama K² and Murayama S^1,4

¹ Departments of Neuropathology (Brain Bank for Aging Research) and ⁴ Neurology, Tokyo Metropolitan Geriatric Hospital & Institute of Gerontology, Itabashi‐ku, Tokyo; ² Department of Neurology, Kitasato Medical School, Sagamihara‐shi, Tokyo; ³ Department of Laboratory Medicine, National Center Hospital for Neurology and Psychiatry, Kodaira, Tokyo, Japan

Background: TDP‐43 is pathogenic for amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD). TDP43 is also reported to accumulate in subgroup of Alzheimer's disease (AD), Lewy body disease (LBD), argyrophilic grain disease (AGD) and hippocampal sclerosis. However, very few studies focused on incidence and extent of TDP‐43 deposition, which is independent from these neurodegenerative process.

Methods: We immunochistochemically screened consecutive autopsy brains with anti‐ tau, Abeta and alpha‐synuclein antibodies and selected 136 brain with minimal senile changes (MSC), whose mean age at death was 78.5 ± 9.4. Brains from 29 AD, 11 LBD and 11 AGD patients were also examined for comparison. Sections of hippocampus, amygdala, medulla oblongata and lumbar spinal cord were immunostained with anti‐phosphorylated (p) TDP‐43 antibody (PSer409/ 410) and the immunoreactive structures were classified into neuronal or glial cytoplasmic inclusion and dystrophic neurite (DN).

Result: Immunoreactivity with pTDP‐43 was observed in 55 among 136 (40%) MSC, 21 among 29 AD (72.4%), 8 among 11 LBD (72.7%) and 6 among 11 AGD (54.5%) brains. pTDP‐43‐immunoreactive structures in the MSC brains mainly consisted of DNs and were preferentially present in uncinate gyrus or posterior ambient gyrus. The cases carrying the pTDP43 immunoreactivity was significantly older than those without the immunoreactivity (p < 0.05).

Conclusion: TDP 43 proteinopathy could occur independently from other neurodegenerative process with aging process, preferentially involve suncinate gyrus and takes a form of DN.

P18‐05

Intracellular processing of disease‐associated α‐synuclein in the human brain suggests prion‐like cell‐to‐cell spread

Kovacs GG¹, Breydo L², Green R², Kis V³, Puska G³, Lőrincz P³, Perju‐Dumbrava L¹, Giera R¹, Pirker W⁴, Lutz M¹, Lachmann I⁵, Budka H¹, Uversky VN^2,6,7, Molnár K³ and László L³

¹ Institute of Neurology, Medical University Vienna, Austria; ² Department of Molecular Medicine, University of South Florida, Tampa, FL, USA; ³ Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University of Sciences, Budapest, Hungary; ⁴ Department of Clinical Neurology, Medical University Vienna, Austria; ⁵ AJ Roboscreen GmbH, Leipzig, Germany; ⁶ USF Health Byrd Alzheimer's Research Institute, Tampa, FL, USA; ⁷ Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Russia

Dementia with Lewy bodies (DLB), Parkinson's disease (PD) and multiple system atrophy are characterized by the deposition of disease‐associated α‐synuclein. In the present study we examined the molecular specificity of the anti‐α‐synuclein 5G4 antibody and evaluated immunoreactivity patterns and their correlation in human brain tissue with micro‐ and astrogliosis in 57 cases with PD or DLB. In addition, we performed a systematic immunoelectron microscopical mapping of subcellular localizations. We show that 5G4 strongly binds to the high molecular weight fraction of β‐sheet rich oligomers. Disease‐associated α‐synuclein immunoreactivity was found in perivascular macrophages, ependyma and cranial nerves. α‐Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. We document ultrastructurally the pathway of processing of disease‐associated alfa‐synuclein within neurons and astroglial cells. Interaction of mitochondria and disease‐associated alfa‐synuclein plays a key role in the molecular‐structural cytopathogenesis of disorders with Lewy bodies. We conclude that Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning. Both neurons and astrocytes internalize disease‐associated α‐synuclein in the human brain, suggesting prion‐like cell‐to‐cell spread of alfa‐synuclein by uptake from surrounding structures, as shown previously in experimental observations.

Disclosure: Supported by European Commission's 7th Framework Programme under GA No 278486, “DEVELAGE.” Ingolf Lachmann is employed by diagnostic company AJ Roboscreen. Antibody 5G4 is patented by AJ Roboscreen, inventors are Gabor G. Kovacs, Ingolf Lachmann, Awad A. Osman and Uta Wagner.

P18‐06

Involvement of the sigma‐1 receptor in neuronal nuclear aggregate formation in mutant Huntingtin induced cells

Miki Y, Tanji M, Kon T, Mori F and Wakabayashi K

Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Japan

The sigma‐1 receptor (SIGMAR1) is now known to be one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins in cells via the ER‐related degradation machinery linked to the ubiquitin–proteasome pathway. Involvement of ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated accumulation of SIGMAR1 was common to neuronal nuclear inclusions in various neurodegenerative diseases. Our study also indicated SIGMAR1 might shuttle between the cytoplasm and nucleus.

We performed double‐immunocytofluorescence and Western blot analyses of Hela cells transfected with mutant Huntingtin to investigate the role of SIGMAR1 in neuronal nuclear aggregate formation, using a SIGMAR1 agonist (PRE‐084), SIGMAR1 antagonist (BD1063), SIGMAR1 siRNA, specific proteasome inhibitors for proteasome (epoxomicin), and exportin 1 (leptomycin B). Mutant Huntingtin induced cells caused neuronal nuclear aggregates, which were immunopositive for SIGMAR1. SIGMAR1 siRNA and epoxomicin significantly caused neuronal nuclear aggregates and cell death. Leptomycin B also caused cell death. Huntingtin became insolubilized in Western blot analysis after SIGMAR1 siRNA and epoxomicin treatment. Furthermore, proteasome activity was significantly reduced in cells transfected with SIGMAR1 siRNA compared with control. Although LC3‐I level was decreased, LC3‐II and p62 levels did not increase compared with controls. PRE084 and BD1063 affected no cellular viability and proteasome activity. These findings indicate ubiquitin–proteasome pathway might be more implicated in nuclear aggregate formation rather than autophagy–lysosome pathway. SIGMAR1 might be involved in transport of aberrant proteins from the nucleus to the ER‐related degradation machinery.

P18‐07

Neurodegenerative disease with widespread tauopathy and iron accumulation in childhood

Bonnin JM¹, Murrell JR¹, Oblak AL¹, Sokol DK², Walsh LE², Vidal RG¹, Goedert M³ and Ghetti B¹

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine; ² Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, USA; ³ Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

Severe accumulation of pathologic tau protein in neurons throughout the cerebral cortex and subcortical structures is uncommon in childhood. It has been observed in a number of unrelated conditions, including Niemann–Pick disease type C, subacute sclerosing panencephalitis, neuronal brain iron accumulation, SLC9A6‐related mental retardation and some cortical dysplasias. We report a case of neurodegeneration in a 14‐year‐old male. He had no developmental abnormalities until age 2 when speech and cognition started to deteriorate and was diagnosed as having autistic spectrum disorder. Multiple MRIs did not reveal brain abnormalities until age 11, when generalized cortical atrophy, ventriculomegaly, thinning of the corpus callosum and atrophy of the brainstem were documented. Since then, he developed behavioral problems, gait abnormalities, dysphagia, and slight ptosis. He also had some cog wheeling, marked rigidity in both lower extremities and tended to fall backward. Extensive work‐up did not reveal any genetic, mitochondrial, enzymatic, or storage disorders. EEG confirmed the presence of severe slowing in the background and bifrontal epileptiform discharges that became generalized at times. Later in his course, he developed severe spasticity and mutism. He died at age 14 and autopsy revealed marked cerebral and brainstem atrophy. There was no obvious atrophy of the cerebellum. Morphologically, numerous neurofibrillary tangles, tau‐positive neurons and neuropil threads were seen throughout the brain but were most severe in the brainstem. Prominent iron deposition was observed in the substantia nigra and in the globus pallidus. MAPT gene mutations were not found. Additional molecular studies to further classify this disorder are in progress.

P18‐08

Neuropathological study in a sample of cognitively normal elderly brains belonging to aging brain research group, University of São Paulo Medical School

Padrao IL and Grinberg LT

University of Sao Paulo Medical School, Brazil

Introduction: Started by Alois Alzheimer, Arnold Pick, Gaetano Perusini and Emil Kraepelin over 100 years, the neuropathological study of aging through the autopsy is widely used, providing morphological correlation in the study of dementia. The aging brain is characterized by cerebrovascular and stochastic degenerative processes, intracellular, such the accumulation of abnormal tau protein, and extracellular, by the accumulation of β‐amyloid protein in senile plaques, both constituting the pathological substrate of Alzheimer's disease.

Material and Methods: We evaluate the neuropathological findings in a sample of 57 cognitively normal older adults, whose average brain weight was 1147.7 g, mean age 77.8 ± 11.5 years, obtained by autopsy. The cognitive status was obtained from structured interviews with reliable caregivers. Fourteen brain areas, vulnerable to age related processes, were examined using H&E and immunohistochemistry for phospho‐tau, β‐amyloid, and α‐synuclein.

Results: Neuropathological abnormalities were found in 91.2 % of cases. AD‐type (AD‐t) pathological findings was more common from the eighth decade, representing 33.3% of the sample; 43% of the cases presented micro‐infarcts, 15.7% in subjects with AD‐t. Pathological findings of Parkinson's disease accounted for 8.7% (3.5% in association with AD‐t). Argyrophilic grains was found in 40.3% of the sample and one case had multiple system atrophy.

Conclusion: Neurodegenerative changes are frequently found in cognitively normal older adults. Protective factors and poor sensibility of clinical assessment may explain the lack of clinical symptoms in these subjects.

P18‐09

Neuropathological study in dementia‐free centenarians

Iwase T¹, Yoshida M² and Hashizume Y³

¹ Dept. of Neurol., Nagoya City Koseiin Medical Welfare Ctr.; ² Inst. for Med. Sci. of Aging, Aichi Medical Univ.; ³ Inst. for Neuropathol., Fukushimura Hosp. Japan

Introduction: Several studies have reported that some older persons are free of dementia despite the presence of extensive Alzheimer's disease pathology. We attempt to assess the prevalence of cognitively unimpaired centenarians, and to ascertain presence of dementia‐related neuropathological changes in dementia‐free centenarians.

Materials and Methods: The Mini‐Mental State Examination or Hasegawa dementia scale in the medical record of 46 centenarians were examined. The autopsied brains from dementia‐free centenarians were studied with standard hematoxylin‐eosin, Klüver–Barrera and Gallyas–Braak stainings, and immunostainings for α‐synuclein, phosphorylated tau, amyloid‐β (Aβ) and TDP‐43. CERAD criteria of neuritic amyloid plaques, Braak and Braak staging of neurofibrillary tangles and Saito's staging of argyrophilic grains were used to assess the brains.

Results: Thirteen of 46 (28%) centenarians were not demented. The average brain weight of dementia‐free centenarians (1098.1 g) was heavier than that of 46 centenarians (1050.8 g). While they were not clinically demented, they were neuropathologically diagnosed as Lewy body disease (5 cases), argyrophilic grain disease (3 cases), senile dementia of the neurofibrillary tangle type (3 cases), and Binswanger's disease (2 cases). Although none of the cases was diagnosed as Alzheimer's disease, substantial amount of senile plaques (CERAD B) and neurofibrillary tangles (Braak IV) were observed. Only 4 cases with subtle age‐related changes were diagnosed as “normal‐aged.”

Conclusion: Many dementia‐free centenarians had neuropathological evidence of dementia. These results may indicate that dementia‐free centenarians have cognitive reserve that provides greater brain capacity to compensate for the clinical expression of a particular degree of damage to the brain.

P18‐10

Oligodendroglial response in the spinal cord in TDP‐43 proteinopathy with motor neuron involvement

Rohan Z¹, Matej R¹, Rusina R² and Kovacs GG³

¹ Department of Pathology and Molecular Medicine, Thomayer Hospital, Prague, Czech Republic; ² Department of Neurology and Center of Clinical Neuroscience, First Faculty of Medicine, Charles University in Prague and General University Hospital, Prague, Czech Republic; ³ Institute of Neurology, Medical University of Vienna, Vienna, Austria

Background: TDP‐43 proteinopathies represent a spectrum of neurodegenerative disorders. Variable clinical presentations, including frontotemporal dementia, amyotrophic lateral sclerosis, and mixed forms are associated with the spatial heterogeneity of the TDP‐43 pathology. Recent studies emphasize the role of oligodendrocytes in the pathogenesis of amyotrophic lateral sclerosis. We evaluated whether TDP‐43 proteinopathies associate with an oligodendroglial response.

Methods: We performed a study on 7 control and 10 diseased cases with spinal cord involvement. Using the oligodendroglia‐specific antibody, TPPP/p25, we assessed oligodendrocyte density in the lateral corticospinal tracts along with the presence of perineuronal oligodendrocytes in the anterior horns. We performed densitometry of myelin basic protein (MBP) immunoreactivity. The numbers of TDP‐43 and p62 immunoreactive inclusions were counted in both lateral corticospinal tracts and anterior horns.

Results: Double immunolabeling confirmed that oligodendrocytes harbor TDP‐43 inclusions. In the lateral corticospinal tracts MBP density, but not the number of oligodendrocytes, was decreased in the diseased group. However, oligodendrocyte counts in the lateral corticospinal tract correlated positively, while the density of MBP inversely, with the number of neuronal inclusions in the anterior horn suggestive of a compensatory response of oligodendrocytes. The number of neurons with perineuronal oligodendrocytes correlated with the amount of inclusions.

Conclusion: Our study further emphasizes the importance of oligodendroglia in the pathogenesis of TDP‐43 proteinopathies with spinal cord involvement.

P19. Neurodevelopment

P19‐01

An autopsy case of syringobulbia and syringomyelia with severe antecollis

Yasui K¹, Hasegawa Y¹, Maeda N¹, Tsuzuki T¹, Tatsumi S², Mimuro M², Iwasaki Y² and Yoshida M²

¹ Department of Neurology, Nagoya Daini Red Cross Hospital; ² Institute for Medical Science of Aging, Aichi Medical University, Japan

Introduction: The exact pathogenesis of syringomyelia associated with Chiari type 1 malformation is unknown. Currently prevalent hydrodynamical theories assert that obstruction of the subarachnoid space at the foramen magnum interferes with flow of cerebrospinal fluid (CSF). As a result, spinal CSF is driven into the spinal cord through the central canal to form a syrinx. Some autopsy cases, however, showed that communication between the syrinx and the subarachnoid space at the entry zone of the posterior nerve roots was considered to play an important role in the pathogenesis of syringomyelia.

Patients (or Materials) and Methods: A healthy 12‐year‐old girl felt numbness in the right leg after coughing. Numbness then developed in the right upper limb after coughing. Her voice was hoarse and she had difficulty in swallowing. At 15 years old, she felt numbness and weakness in the left limbs. On her first visit to our hospital aged 22 years, she was diagnosed as syringomyelia between C2 and Th7 and Chiari malformation. At 32 years old, the syrinx was reduced with an S‐S shunt. Her symptoms, however, remained unchanged. After the shunt procedure, antecollis developed. At 35 years old, she suffered from sleeping apnea syndrome; at 40 years old, she experienced a shunt tube failure; and at 45 years old, she contracted spondylolisthesis and sciatica. At 54 years old, she died suddenly in her bed.

Results: She was autopsied. Neuropathological findings are as follows: Her brain weighed 1500 g. Macroscopically, the cervical spine was severely flattened. Microscopically, syrinx, extending from the medulla oblongata to L3 level, was localized in the central gray matter and the posterior horn. The syrinx surrounded by thick glial tissue. The syrinx was communicated with the subarachnoid space at the entry zone of the posterior nerve roots, but not with the central canal except at some levels.

Conclusion: In our case the entry zone of the posterior nerve roots is closely related to the pathogenesis of syringomyelia than the central canal.

P19‐02

Blockade of the potassium channel KCa3.1 as a potential target for the treatment of encephalopathy of prematurity

Lechpammer M, Huebner PA, Tran YP, Jin L‐W, Berman RF and Wulff H

Departments of Pathology and Laboratory Medicine, Pharmacology and Neurosurgery, University of California Davis, Medical Center and School of Medicine, Sacramento, CA, USA

Encephalopathy of prematurity (EOP) caused by neonatal hypoxic/ischemic (HI) brain injury is characterized by diffuse white matter (WM) damage and often leads to behavioral and motoric deficits in later life, including autism, hyperactivity and cerebral palsy. Previously we have demonstrated a neuroprotective efficacy of the selective small molecule KCa3.1 inhibitor TRAM‐34 in a rat model of EOP. In this study we have investigated the potential of TRAM‐34 to prevent or reverse EOP‐like behavioral deficits after selective WM injury produced in male Long Evans rats at P6 by unilateral carotid artery ligation (UCL) and severe hypoxia (UCL/HI).

The social behavior assessed in young rats (P31–33; n = 15) exposed to UCL/HI at P6 using a three‐chamber test showed a resistance to change and impaired preference for social novelty. TRAM‐34(40 mg/kg i.p. every 12 hours for 7 days) prevented this autism‐like behavior and restored normal preference for social novelty (p = 0.032) as observed in healthy controls. When the same animals were assessed by the open field assay, they exhibited hyperactive exploratory behavior and low anxiety levels. TRAM‐34 has reduced traveled distance, significantly decreased time in the field center (p = 0.01), and increased time in the field perimeter (p = 0.02) thus normalizing hyperactive behavior.

Our experiments have showed both behavioral hallmarks of EOP – autism‐like altered socialization and hyperactivity – in the rat model of EOP. Our results also demonstrate feasibility of prevention/reversal of cognitive consequences of EOP by in vivo pharmacological inhibition of KCa3.1 and offer a potential new target for treatment of autism and hyperactivity disorders.

P19‐03

Impact of cuprizone as a demyelinating agent on rat hippocampal neurogenesis

Abe H¹, Itahashi M¹, Tanaka T¹, Kimura M¹, Mizukami S¹, Saito F², Akahori Y², Imatanaka N², Yoshida T¹ and Shibutani M¹

¹ Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo, Japan; ² Chemicals Evaluation and Research Institute, Chemicals Assessment and Research Center, Tokyo, Japan

Introduction: Hippocampal dentate gyrus is a unique structure that can continue neurogenesis throughout the lifetime. Hilar interneuron populations regulate neurogenesis. We have shown that various kinds of neurotoxicants can affect hippocampal neurogenesis in rats and mice. In the present study, we examined a possibility for detection of neurotoxicity of cuprizone (CPZ), a demyelinating agent, targeting the hippocampal neurogenesis by postpubertal‐stage exposure in comparison with developmental exposure using rats.

Materials and Methods: For developmental exposure, CPZ was given to maternal SD rats at 0, 0.1 or 0.4% in diet from gestational day 6 until postnatal day (PND) 21 on weaning. For postpubertal stage exposure, 5‐week male SD rats were treated with 0, 120 or 600 mg/kg of CPZ by oral gavage for 4 weeks.

Results: Offspring after developmental exposure revealed decreases in Tbr2⁺ type 2b progenitor cells and PCNA⁺ proliferating cells in the subgranular zone (SGZ) and increases in reelin⁺ interneurons and NAChR7α⁺cholinergic interneurons in the hilus at 0.4% on PND 21. By postpubertal‐stage exposure, animals decreased Tbr2⁺ type 2b and double cortin⁺ type 3 progenitor cells and PCNA⁺ proliferating cells in the SGZ at 120 and 600 mg/kg, while the interneuron populations did not fluctuate the number.

Conclusion: Our results suggested that both developmental and postpubertal‐stage CPZ exposure affect neurogenesis targeting intermediate populations of granule cell lineages, while interneurons responded differently between the stages. In conclusion, CPZ may target hippocampal neurogenesis causing developmental neurotoxicity that can be detected even by exposure in the framework of regular toxicity study using young‐adult animals.

P19‐04

Maternal exposure effect of T2‐toxin on hippocampal neurogenesis in mouse offspring

Tanaka T^1,2, Abe H^1,2, Itahashi M^1,2, Kimura M^1,2, Mizukami S^1,2, Murakami T³, Yoshida T¹ and Shibutani M¹

¹ Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology; ² Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University; ³ Laboratory of Veterinary Toxicology, Tokyo University of Agriculture and Technology, Japan

Introduction: T‐2 toxin is a trichothecene mycotoxin produced by Fusarium species fungus and known to cause neurotoxicity as well as immunotoxicity and hematotoxicity through oxidative stress responses. There is a concern for human health risk from ingestion of T‐2 toxin contaminated in foods. In the present study, we examined the developmental exposure effect of T‐2 toxin on hippocampal neurogenesis in mice.

Materials and Methods: Oral doses of T‐2 toxin at 0, 1, 3, 9 ppm in diet were given to maternal mice from gestational day (GD) 6 to day 21 after delivery. The hippocampal dentate gyrus of offspring was immunohistochemically examined on postnatal day (PND) 21 and PND 77.

Results: The dams showed forestomach mucosal hyperkeratosis and hepatocyte hypertrophy at 9 ppm. Offspring showed lower absolute brain weight from 3 ppm, and lower relative weight of the liver, thymus and spleen at 9 ppm. Immunohistochemically, decreases in GFAP⁺ or BLBP⁺ type 1 stem cells and Tbr2⁺ type 2b progenitor cells were observed in the subgranular zone (SGZ) from 3 ppm on PND 21, but not on PND 77. In the hilus of the dentate gyrus, increase of reelin⁺ interneurons, suggestive of aberration of granule cell mismigration, was noted at 9 ppm on PND 21, but not on PND 77.

Conclusion: In conclusion, we revealed that maternal T‐2 toxin exposure reversibly affects hippocampal neurogenesis targeting early‐stage populations in mouse offspring at ≥ 3 ppm in diet, a level translating to 0.40 mg/kg body weight/day during gestation and 1.39 mg/kg during lactation.

P19‐05

Neuronal alterations in human brains with documented in utero alcohol exposure

Del Bigio MR, Basalah D and Jarmasz J

Department of Pathology, University of Manitoba, Winnipeg, Canada

Fetal alcohol spectrum disorder (FASD) is among the most common neurodevelopmental disorders. Fewer than 30 severe cases have been described at autopsy. The pathogenesis of the neurological disorder is not clear. A retrospective review of autopsies at the Health Sciences Centre (HSC) in Winnipeg, Canada from 1980–2013 revealed 153 cases with a history of gestational alcohol exposure or a clinical diagnosis of FASD. Autopsy reports and slides were reviewed. To investigate neuronal changes, 18 cases ranging from 2 months to 18 years were selected on the basis of history, tissue preservation, and absence of major malformations. Age and sex‐matched controls were identified. Antibody to excitatory amino acid transporter 3 (EAAT3) was used for glutamatergic neurons. Antibodies to parvalbumin and calbindin were used for inhibitory GABAergic neurons. Neurons were quantified in the hippocampus (CA1, CA3, and dentate gyrus regions), temporal lobe, and frontal lobe. Major abnormalities included hydrocephalus (4 cases), agenesis of corpus callosum (2), in utero cerebral infarct (2), periventricular white matter damage (3), microcephaly (5), anencephaly (2), and one case each of myelomeningocele, lissencephaly, holoprosencephaly, and Dandy–Walker malformation. Microscopic heterotopia were identified. Paired statistical analysis of neuron immunoreactivity showed no significant changes in excitatory neurons. Inhibitory neurons were significantly reduced in hippocampus and temporal cortex for the entire group of alcohol‐exposed vs. control and when analyzed separately by age (infants, children, and teenage) or by sex. This relative loss of inhibitory neurons might help explain some of the behavioral (attention deficit) and motor abnormalities (tremor) in FASD.

P19‐06

Rhombencephalosynapsis in oculo‐auriculo‐vertebral syndrome

Gener MAH¹, Slemp SN², Richey JD², Hawley DA² and Bonnin JM¹

¹ Divisions of Neuropathology and ² Anatomic Pathology, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

Oculo‐auriculo‐vertebral syndrome (OAVS) is a rare disorder characterized by congenital anomalies in the eyes, ears, and vertebral column. Central nervous system anomalies have been reported in approximately 50% of cases. They range from mild hydrocephalus to severe malformations, including Arnold–Chiari malformation, with or without syringomyelia or hydromyelia, encephalocele, holoprosencephaly, arhinencephaly, lissencephaly and others. Cerebellar abnormalities have also been observed in association with OAVS. We report a case of an 11‐year‐old female with this syndrome. Neuroimaging studies revealed ventriculomegaly and diffuse white matter loss, particularly in the peritrigonal region, atrophy of the corpus callosum, hypoplasia of the middle ear, fusion of the ossicles, hypoplasia of the right external auditory canal and absence of the right temporalis muscle. Fusion of the cerebellar hemispheres was also documented. A CT scan of the spine showed focal levoscoliosis in the lumbar region and multiple segmentation anomalies of the midthoracic vertebral bodies. At birth she had esophageal atresia with tracheoesophageal fistula for which multiple surgical procedures were performed. A horseshoe kidney was demonstrated radiologically. The patient was admitted because of severe abdominal distention following two days of diarrhea. She deteriorated rapidly and went into cardiac arrest. Resuscitation efforts were unsuccessful. An autopsy confirmed the presence of multiple anomalies in the spine, absence of right pinna, bicuspid aortic valve, horseshoe kidney and hydrocephaly. Examination of the hindbrain revealed absence of the cerebellar vermis and fusion of the cerebellar hemispheres and dentate nuclei. To our knowledge, rhombencephalosynapsis has not been previously reported in association with OAVS.

P19‐07

Tracing neuronal connections through genetic approaches

Gokozan H, Corcoran S, Czeisler C, Gygli P, Catacutan FP and Otero J

The Ohio State University, USA

Premature babies are at risk for life‐threatening autonomic dysfunction, including abnormal CNS control of breathing. These problems arise in part from immaturity of the autonomic nervous system in premature babies. The transcription factor PHOX2B is a master regulator of autonomic nervous system function as well as the specification of noradrenergic neurons. All autonomic afferent and efferent circuits require normal PHOX2B function, and therefore PHOX2B‐derived neurons must have attained developmental maturity at birth for normal physiological function. Incomplete maturity and/or absence of noradrenergic and serotonergic neuron networks are a proposed mechanism of infant breathing disorders including apnea of prematurity, sudden infant death syndrome, and congenital central hypoventilation syndrome (CCHS). PHOX2B mutations result in the “chemosensation” loss characteristic of CCHS, resulting in poor respiratory drive secondary to hypercarbia (high pCO2). The developmental processes occurring during late embryonic development that permit proper respiration at birth are unknown. So that we may ultimately determine the mechanisms of respiratory neuron maturation in mammals, we utilized a transgenic mouse model of neuronal circuitry development. This transgenic mouse reagent (Jackson Labs stock number 010590) shows upon cre‐mediated excision in neurons tdTomato expression in neuronal axons and somata and GFP expression at synapses. Using a conditional gene targeting approach we were able to conclude that neurons derived from PHOX2B progenitor domains innervate serotonergic neurons.

P20. Non‐Alzheimer Dementia

P20‐01

‘Posterior’ Pick's disease with posterior cortical atrophy phenocopy: a clinico‐pathological case study

Caixeta LF¹, Da Silva WG², Brasil R³, Leite MB⁴ and de Paula EC⁵

¹ Associate Professor of Neurology, Federal University of Goiás (UFG) School of Medicine, Goiânia, Goiás, Brazil. Coordinator of the UFG‐Brain Bank, Clinics Hospital – UFG, Goiânia, Goiás, Brazil; ² Professor of Pathology, State University of Goiás, UniEvangelica, FAMA, Anápolis‐Goiás, Brazil; ³ Student University of Goiás (UFG) Internal Medicine, Goiania, Goiás, Brazil; ⁴ Professor of Pathology, BIOCITO, Goiânia, Goiás, Brazil; ⁵ Professor of Pathology Federal University of Goiás (UFG) School of Medicine, CAPC, Goiânia, Goiás, Brazil

Introduction: Atypical presentations of Pick's disease do not include significant occipital atrophy. The present case was considered to represent a rare case of Pick's disease of the posterior type (at least in the beginning of the disease), presenting as a syndrome of progressive posterior cortical dysfunction (PPCD). After a very brief period of time, disease progressed to more anterior (frontotemporal).

Materials and Methods: Case report of posterior Pick's disease with pathological confirmation. MRI, SPECT, neuropsychological and autopsy evaluations are presented.

Results: With 56 years old patient D.C.A. initially presented with higher order visuospatial dysfunction, developed features of Balint syndrome, Gerstmann syndrome, environmental agnosia, transcortical sensory aphasia and a very rapid dementia. Soon after emerged severe behavior alterations. CT and MRI revealed atrophy of the posterior cortex with isolated posterior ventricular enlargement, in a fashion very classic for PPCD. The initial differential diagnosis included Creutzfeldt–Jakob disease, but CSF and EEG ruled out this possibility. Posterior (parietal‐occipital) fibrillary gliosis was the finding bearing a striking resemblance to “progressive subcortical gliosis.” Severe loss of nerve cells was observed in occipital as well as in frontal lobes, insula, gyrus cinguli, head of caudate nucleus and partial areas of temporal lobes, where Pick cells and Pick bodies were found. Moderate or mild loss was observed in the parietal lobes.

Conclusion: Our case expands the list of etiologic possibilities that may be associated with PPCD. Besides that, a posterior variant of Pick's disease is first reported.

P20‐02

Comparative study on TDP‐43‐related frontotemporal lobar degeneration and Alzheimer's disease with clinical frontotemporal dementia

Riku Y¹, Yoshida M², Watanabe H¹, Mimuro M², Iwasaki Y² and Sobue G¹

¹ Department of Neurology, Nagoya University Graduate School of Medicine; ² Institute for Medical Science of Aging, Aichi Medical University, Japan

Introduction: Behavioral variant frontotemporal dementia (bv‐FTD) is a clinical phenotype of frontotemporal lobar degeneration (FTLD) manifesting psychobehavioral deteriorations, and a primary immunohistochemical component is TAR DNA‐binding protein 43kDa‐related FTLD (FTLD‐TDP). However, Alzheimer's disease (AD) occasionally manifests early psychobehavioral symptoms, and comparative study between FTLD and AD patients presenting with clinical bv‐FTD has not been described. We compared clinicopathological features in FTLD‐TDP and AD patients clinically presenting with bv‐FTD using consecutively autopsied patients.

Patients and Methods: We included pathologically proven 53 and 77 subjects with sporadic FTLD‐TDP and AD, respectively. The 12 bv‐FTLD (FTLD‐TDP patients presenting with bv‐FTD syndrome) and 11 bv‐AD (AD patients presenting with bv‐FTD syndrome) groups were identified. For pathological analyses, the 26 regions of cerebrum were sampled from all patients, and the neurodegenerative changes were semi‐quantitatively compared.

Results: Clinically, the ages of onset were 66.3 ± 11.58 and 73.8 ± 12.8 in the bv‐FTLD and bv‐AD groups, respectively (p = 0.013). Upon semi‐quantitative evaluation of neurodegenerative changes, several regions in the temporal lobes and subcortical gray matters showed significant differences between the groups.

Conclusion: Regarding patients presenting with clinical bv‐FTD, these regions clearly characterizes FTLD‐TDP when compared to AD.

P20‐03

FTDP‐17 associated with the MAPT P301L mutation and severe brainstem pathology

Gener MAH, Murrell JR, Oblak AL and Ghetti B

Division of Neuropathology, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

Among mutations in the Tau gene, P301L isone of the most frequent. We studied a case with a long clinical course, extremely severe cortical and subcortical atrophy, and prominent involvement of the brainstem. A 44‐year‐old woman presented with sudden‐onset of delusions and hallucinations. Retrospectively, disease onset may have been insidious, beginning at age 40. She was first treated for a psychiatric disorder; however, initial symptoms were followed by a rapid cognitive decline. At age 46, imaging studies revealed bilateral frontotemporal hypometabolism and atrophy. She continued to deteriorate neurologically, was admitted to a long‐term nursing facility, and died at 53 years of age. At autopsy, the brain was extremely atrophic and weighed 652 grams. Atrophy was severe in the frontal and temporal lobes, and in the brainstem. Histological examination revealed marked neuronal loss and gliosis in the frontal and temporal cortices. In the midbrain, only a few neurons had survived in the substantia nigra. There was a significant loss of myelinated axons in the hemispheric white matter and in descending tracts. For immunohistochemistry, a panel of antibodies to tau (AT8, 4‐repeat tau, and 3‐repeat tau) was used. Tau and 4‐repeat tau were positive in neurons and glia. Tau deposits were most numerous in the frontotemporal cortices, in the amygdala and brainstem. MAPT sequencing revealed a C to T nucleotide substitution at codon 301 resulting in a leucine for proline change (P301L). The functional effects of 4‐repeat tau pathology in the brainstem, as it relates to autonomic functions, needs further investigation.

P21. Parkinson and Related

P21‐01

Differential expression of galanin in the basal forebrain of patients with Lewy body disorders

Gentleman S, Liu A, Pearce R and Alexandris A

Neuropathology Unit, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK

The cholinergic neurons of the nucleus basalis of Meynert (nbM) are severely depleted in Parkinson's disease (PD), PD dementia (PDD), dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). The resultant cholinergic deficits are considered as a key neuropathological substrate of cognitive deterioration. Galanin, a multi‐functional neuropeptide, is found in fibers that innervate the nbM and, in late stage AD, these galanin‐containing fibers are reported to enlarge and hyper‐innervate the surviving cholinergic neurons of the nbM. The aim of this study was to determine if a similar phenomenon could be observed in the basal forebrain of patients with Lewy body disorders (LBD). Galanin immunostaining was carried out on basal forebrain sections from 39 cases (15 PD, 16 PDD, 5 DLB, 3 mixed PD/AD) and 6 aged‐matched controls from the Parkinson's UK brain bank. Hyper‐innervation of the nbM neurons was evident only in a minority of cases and there were no differences in the extent of fiber innervation among LBD and age‐matched controls. However, the nbM neurons in many cases displayed intense somal galanin immunoreactivity. Semi‐quantitative analysis showed that somal staining was significantly increased in all LBD compared to controls and was correlated with Braakalpha‐synuclein staging. Upregulation of somal galanin in LBD is consistent with animal models of neuronal injury and may represent a reactive protective mechanism. By contrast, the cause of the hyper‐innervation observed in some cases remains elusive.

P21‐02

Immunohistochemical localization of neuron derived orphan receptor‐1 in neurodegenerative diseases

Kon T¹, Miki Y², Tanji K², Mori F, Toyoshima Y³, Kakita A⁴, Takahashi H⁵, Utsumi J⁶, Sasaki H⁶ and Wakabayashi K²

¹ Koichi Wakabayashi; ² Department of Neuropathology, Hirosaki University Graduate School of Medicine; ³ Department of Pathology, Brain Research Institute, University of Niigata; ⁴ Department of Pathological Neuroscience, Brain Research Institute, University of Niigata; ⁵ Department of Pathology, Brain Research Institute, University of Niigata; ⁶ Department of Neurology, Hokkaido University Graduate School of Medicine, Japan

Neuron derived orphan receptor‐1 (NOR‐1), also known as NR4A3, plays key roles in cell cycle, neuronal differentiation, apoptosis, inflammation and metabolism. These conditions may be involved in the pathogenesis of certain neurodegenerative diseases. We immunohistochemically examined the brain and spinal cord of patients with various neurodegenerative diseases and normal control subjects using anti‐NOR‐1 antibody. In controls, the cytoplasm and processes of neurons and glial cells were faintly immunostained with anti‐NOR‐1 antibody. In neurodegenerative diseases, immunoreactivity of NOR‐1 was observed in cortical and brainstem‐type Lewy bodies in Parkinson's disease and dementia with Lewy bodies, and glial and neuronal cytoplasmic inclusions in multiple system atrophy. Double‐labeled immunofluorescence study demonstrated co‐localization of NOR‐1 and phosphorylated α‐synuclein in these inclusions. No immunoreactivity was found in neuronal and glial inclusions in tauopathy (Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration and argyrophilic grain disease), TDP‐43 positive inclusions in TDP‐43 proteinopathy (sporadic amyotrophic lateral sclerosis and frontotemporal lobar degeneration), or neuronal nuclear inclusions in polyglutamine diseases (Huntington's disease, dentatorubral‐pallidoluysian atrophy, spinocerebellar ataxia type 1 and Machado–Joseph disease). These findings suggest that accumulation of NOR‐1 is specific to α‐synucleinopathy.

P21‐03

The effect of disaccharide, trehalose, on the formation of alpha‐synuclein‐positive inclusions

Tanji K, Miki Y, Kon T, Mori F and Wakabayashi K

Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

Accumulation of mis‐folded and/or abnormally modified proteins is a major characteristic of many neurodegenerative diseases. Abnormal alpha‐synuclein is widely deposited to the presynaptic terminals as well as the cytoplasm of neurons and glial cells in the distinct brain regions of Parkinson's disease (PD) and dementia with Lewy bodies. It is well known that autophagy‐lysosome system serves as an efficient degradation of abnormal proteins in cells. To test the possibility that activated autophagy can degrade abnormal molecules and leads to potential therapeutic strategy for diseases that characterized as protein aggregations, we here investigated the effect of trehalose on abnormal aggregation in model for PD. Trehalose is a disaccharide composed of two glucose units, and functions as a chemical chaperone. On cultured cells, trehalose increased levels of autophagosomal protein, LC3. Especially, since significant increased level of lipidated form (LC3‐II) was evident. Like the results of cell‐based assays, trehalose also increased the LC3‐II level in the brains of normal adult mice. However, there were no differences of abnormal alpha‐synuclein depositions between PD model mice treated with or without trehalose. These results suggest that autophagosomal numbers can be higher in trehalose‐treated cells and mice compared with control, but it would be not enough to degrade abnormal protein deposition in vivo.

P21‐04

The expression of acid alpha glucosidase in Lewy body and glial cytoplasmic inclusions

Kurashige T, Takahashi T, Maruyama H and Matsumoto M

Department of Clinical Neuroscience and Therapeutics, Institute of Biomedical and Health Sciences, Hiroshima University, Japan

Introduction: Gaucher disease is one of lysosomal storage diseases and caused by the deficiency of the lysosomal glucocerebrosidase (acid beta‐glucosidase, GBA). Recently, GBA loss of function variants were reported to be the most common genetic risk factor associated with parkinsonism. But, there were no reports about the association between other lysosomal enzymes and parkinsonism. In this study, we examined the involvement of acid alpha glucosidase (GAA), one of other lysosomal enzymes, in alpha synucleinopathy through immunohistochemical analysis of GAA.

Materials and Methods: Brains from five PD cases and four MSA cases were examined. In all cases, alpha synucleinopathy was ascertained by HE stain and/or Gallyas Braak silver staining. These specimens were subjected to immunohistochemistry and immunofluorescent technique, using anti‐phosphorylated alpha synuclein antibody and anti‐GAA antibody.

Results: A considerable number of halo of LBs in early PD cases and GCIs in MSA cases were immunopositive for GAA. Double immunofluorescence staining showed co‐localization of phosphorylated alpha synuclein and GAA in LBs and GCIs. In addition, the granular GAA immunopositivities were observed in the cytoplasm of neurons in DNMX of early PD cases.

Conclusion: This study suggests that the lysosomal dysfunction is implicated in the formation of LBs and GCIs.

P21‐05

Missense mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene can cause late‐onset Parkinson disease

Daher JPL

Department of Neurology – Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, EUA

Missense mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene can cause late‐onset Parkinson disease. Past studies have provided conflicting evidence for the protective effects of LRRK2 knockdown in models of Parkinson disease as well as other disorders. These discrepancies may be caused by uncertainty in the pathobiological mechanisms of LRRK2 action. Previously, we found that LRRK2 knockdown inhibited proinflammatory responses from cultured microglia cells. Here, we report LRRK2 knockout rats as resistant to dopaminergic neurodegeneration elicited by intracranial administration of LPS. Such resistance to dopaminergic neurodegeneration correlated with reduced proinflammatory myeloid cells recruited in the brain. Additionally, adeno‐associated virus‐mediated transduction of human α‐synuclein also resulted in dopaminergic neurodegeneration in wild‐type rats. In contrast, LRRK2 knockout animals had no significant loss of neurons and had reduced numbers of activated myeloid cells in the substantia nigra. Although LRRK2 expression in the wild‐type rat midbrain remained undetected under nonpathological conditions, LRRK2 became highly expressed in inducible nitric oxide synthase (iNOS)‐positive myeloid cells in the substantia nigra in response to α‐synuclein overexpression or LPS exposures. Our data suggest that knocking down LRRK2 may protect from overt cell loss by inhibiting the recruitment of chronically activated proinflammatory myeloid cells. These results may provide value in the translation of LRRK2‐targeting therapeutics to conditions where neuroinflammation may underlie aspects of neuronal dysfunction and degeneration.

P22. Pediatric Tumors

P22‐01

Chromosomal alterations as prognostic marker for risk stratification of pediatric ependymoma patients – validation in the German multicenter HIT2000 Trial

Pietsch T¹, Dörner E¹, zur Mühlen A¹, Mynarek M², von Bueren A², Friedrich C², von Hoff K² and Rutkowski S²

¹ Department of Neuropathology, University of Bonn, Germany; ² Department of Pediatric Hematology/Oncology, University of Hamburg, Germany

While histopathological grading varied in respect to its prognostic value in previous studies, the pattern of chromosomal alterations was identified to predict worse outcome.

To validate the impact of chromosomal alterations in a homogenously treated patient cohort, we analyzed the genome of 164 cases (median age 4.1 years, range 0.3–18.6) enrolled into the multicenter trial HIT2000 (irradiation and/or chemotherapy depending on age and extent of resection) with sufficient archival material available. Molecular inversion profiling (MIP) revealed 43 cases (26%) with gains or losses of whole chromosomes (numerical group), 65 (40%) without chromosomal alterations (balanced group) and 56 (34%) with structural alterations. The latter showed frequent gain of chromosome arm 1q. 109/164 ependymomas were located infratentorially. 26 cases represented WHO grade II ependymoma, 138 anaplastic ependymoma. 51 patients had postoperative residual tumor (4 not known).

At a median follow‐up of 4.4 years for survivors, patients with tumors showing structural alterations trended to worse overall survival without reaching significance. Multivariable analysis demonstrated that infratentorial vs. supratentorial localization [HR 3.31 (95% CI: 1.32–8.32; p = 0.001)] and the presence of structural vs. numerical chromosomal alterations [HR 2.67 (95% CI 1.08–6.63; p = 0.034)], but not residual tumor, were risk factors for OS. 5y‐OS in the most favorable prognostic group (supratentorial location, balanced group, no gain 1q; n = 14) was 100%, in the least favorable group (infratentorial location, structural chromosomal alterations, gain 1q; n = 14) 41+/–15%.

Genetic groups represent an independent marker for risk stratification of pediatric ependymoma patients, which can be evaluated by MIP representing a robust and reliable method.

P22‐02

Craniospinal metastasis of extracranial solid tumors: experience from a pediatric tertiary referral center in Argentina

Gargano PR¹, Alderete D², Baroni L², Zuccaro GN³ and Lubieniecki FJ¹

¹ Department of Pathology; ² Department of Neuro‐oncology; ³ Department of Neurosurgery, Hospital de Pediatria Prof. Dr. Juan P. Garrahan, Buenos Aires, Argentina

Introduction: Unlike in adults, in children the craniospinal metastases of solid tumors are very rare. They account for a small percentage of CNS tumors in this age group and usually correspond to an advanced stage of the primary tumor. The aim of the study was to describe our experience in these tumors.

Material and Methods: A retrospective analysis of cases of craniospinal metastases of extracranial solid tumors diagnosed at the department of pathology of Garrahan Hospital in the period 1988–2014 was conducted. Sex, age at time of resection, location, and histology of the lesions were evaluated.

Results: From a total of almost 2900 CNS tumors diagnosed over the period 1988–2014, 21 cases were metastases of solid extracranial tumors, accounting for 0.7 % of the total. Sex distribution: 13 males/8 females. Age range: 3–19 years. Location of the metastases: brain: 14, spine: 3, posterior fossa: 1, and skull: 3. The following neoplasms were diagnosed: Osteosarcoma: 6 (4 boys); neuroblastoma: 4; Wilms’ tumor 3 (boys); malignant germ cell tumor 2 (boys); paraganglioma/pheochromocytoma: 2; rhabdomyosarcoma: 1; synovial sarcoma: 1; renal clear cell sarcoma: 1; malignant odontogenic tumor: 1. Only in three of the cases the diagnoses of primary tumor and craniospinal metastases were made concomitantly.

Conclusions: Unlike in adults, there is a wide spectrum of histological subtypes of the primary tumor, however, they mostly correspond to sarcomas. Craniospinal metastases account for a small percentage of tumors that were neurosurgically treated in children.

P22‐03

Desmoplastic/nodular medulloblastoma and HIT‐SKK treatment. Clinicopathological data from a French series

Siegfried A¹, Bertozzi AI², Bourdeaut F³, Sevely A⁴, Loukh N⁵, Miquel C⁶, Sevenet N⁷, Pietsch T⁸ and Dufour C⁹

¹ Pathology and Genetic Department, CHU Toulouse, Toulouse, France; ² Hematology Oncology Department, Pediatrics, CHU Toulouse, Toulouse, France; ³ INSERM U830, Pediatric Oncology, Curie Institute, Paris, France; ⁴ Neuro‐radiology, CHU Toulouse, Toulouse, France; ⁵ Neuropathology Department, CHU Toulouse, Toulouse, France; ⁶ Neuropathology, CHU Sainte Anne, René Descartes University, Paris V, Paris, France; ⁷ Genetics, Bergonié Institute, Bordeaux, France; ⁸ Neuropathology Institute, University of Bonn Biomedical Center, Bonn, Germany; ⁹ Pediatry, Gustave Roussy Institute, Villejuif, France

Introduction: The aim of this study was to identify the clinical and bio‐pathologic features involved in the prognosis of patients with desmoplastic/nodular medulloblastoma (DNMB) treated with HIT‐SKK protocol.

Procedure: Between 2009 and 2012, 17 children (<5 y‐o), with initial DNMB were evaluated. Retrospective central radiological review, pathological and immuno‐histochemical study, array‐CGH and sequencing of SUFU and PTCH1 genes were performed.

Results: Median age at diagnosis was 26 months. 15/16 histologically reviewed cases were confirmed as DNMB/MBEN. Nodularity and desmoplasia were observed in varying degrees. All tumors showed a SHH immunoprofile. Mutation of PTCH1 was found in 3 tumors. One case was re‐diagnosed as a classic MB. It lacked reticulin lined nodules, showed a non‐SHH profile and a non‐specific array‐CGH profile. Radiological features were heterogeneous but the only 2 cases with T2 hypersignals presented a relapse. Treatment was quite homogeneous. Surgery was complete for 13 children, 4 patients had postoperative residual tumor. Chemotherapy began with a median delay of 23 days (14–119) after initial surgery. At 2 years, the event free survival was 64 +/– 16% and the overall survival 92 +/– 8%. Recurrence occurred in 4 patients (one with a classic MB, the only case with MYCN and MYCL amplifications). MIB1 index did not impact the outcome.

Conclusions: The survival rate with HIT SKK protocol was quite similar to the previously published results. Older age (>3), a longer delay before treatment, T2 hyperintensity and biopathological features might explain the worse evolution observed in some of our patients.

P22‐04

Dysembryoplastic neuroepithelial tumor (DNET) and Noonan syndrome. A case report

Delisle MB¹, Siegfried A², Tauber M³, Cave H⁴, Loukh N⁵, Boetto S⁷, Bertozzi AI⁶, Uro‐Coste E¹ and Cances C⁸

¹ INSERM UMR 1037, CHU Toulouse, University of Toulouse III, Neuropathology Department, Toulouse, France; ² Pathology Department, Genetic Department, CHU Toulouse, Toulouse, France; ³ INSERM UMR 5282, Endocrinology Department, Pediatrics, CHU Toulouse, Toulouse, France; ⁴ INSERM UMR 718, Genetics, Paris, France; ⁵ Neuropathology Department, CHU Toulouse, Toulouse, France; ⁶ Hematology Oncology Department, Pediatrics, CHU Toulouse, Toulouse, France; ⁷ Neurosurgery Department, CHU Toulouse, Toulouse, France; ⁸ Pediatric Neurology Department, Pediatrics, CHU Toulouse, Toulouse, France

Noonan syndrome (NS), an autosomal dominant disorder, is characterized by short stature, congenital heart defect, developmental delay and facial dysmorphism. PTPN11 mutations are the most common cause, PTPN11 encoding a non‐receptor protein tyrosine phosphatase, SHP‐2. Hematopoietic malignancies and solid tumors are associated with NS. Among these, brain tumors, described in children and young adults remain rare (less than 10 published cases).

We report here a 16 year‐old patient with PTPN11 related NS who, at the age of 12, was incidentally found to have a left temporal lobe brain tumor, a cystic lesion in the right thalamus. The main tumor showed hypointensity on T1 images, no contrast‐enhancement. Epilepsy occurred 2 years later. Worsening of crisis and increase of radiological images led to surgical resection of the temporal tumor. Microscopy showed nodules with characteristics of specific glioneuronal elements of dysembryo plastic neuroepithelial tumor (small round cells in columns, floating neurons) or of glial elements. Immunohistochemistry revealed positive staining with olig2, negative staining with IDH1R132H and BRAFV600E in small cells. MIB was less than 5%. 10 months after surgery, the patient is well.

SHP‐2 plays a key role in RAS‐MAPK‐ERK pathway signaling which controls several developmental cell processes and oncogenesis. An amino‐acid substitution in N‐terminal SHP2 domain disrupts the self‐locking conformation and leads to an ERK activation. Glioneuronal tumors and pilocytic astrocytomas were both described in NS. This report reinforces the relation of glioneuronal tumors with “RASopathies” even if these tumors are heterogeneous at the genomic level with BRAF, NF1, FGFR1 or PTPN11 mutations.

P22‐05

Fusocellular brain neoplasm as the presenting form of pleuropulmonary synovial sarcoma in a child

Lubieniecki FJ, López Marti JM, Moretti L, Díaz DD, Vazquez V and de Dávila MTG

Department of Pathology, Hospital de Pediatría J.P. Garrahan, Buenos Aires, Argentina

Introduction: Synovial sarcoma is an aggressive neoplasm frequently arising from deep soft tissues of the limbs, accounting for 8% of soft tissue sarcomas and occurring mainly in adolescents. Intracranial metastasis of sarcoma is rare in children and generally happens late in the course of tumor progression. We report an unusual pediatric case of CNS synovial sarcoma as the first manifestation of a concomitant mediastinal tumor.

Patient and Methods: A 10‐year‐old boy, diagnosed with anaplastic oligodendroglioma, was referred. MRI showed a right heterogeneous contrast‐enhancing parieto‐occipital lesion with peripheral edema. The patient developed respiratory distress and pain. CT scan showed a large left solid pleuropulmonary/mediastinal tumor. The child died 12 days after the lung surgery. Immunohistochemistry, FISH, and RT‐PCR were performed in paraffin blocks and frozen tissue of both samples.

Results: Histological sections disclosed a fusocellular proliferation organized in sheets with alternating hyper‐ and hypocellular areas of loose stroma resembling a monophasic synovial sarcoma. Immunohistochemistry showed positivity for vimentin, CKAE1‐AE3, EMA, and BCL2 and negativity for desmin, CK19, CK18, CK7, and S100. Both samples revealed an 18q11 rearrangement by FISH and a SYT/SSX2 fusion transcript by RT‐PCR.

Conclusion: This is an unusual case of a primary mediastinal synovial sarcoma initially presenting with CNS involvement in a child. The discovery of the specific translocation (X;18) led to the identification of this neoplasm in a location other than limbs, such as CNS. Morphological, immunohistochemical, and molecular studies should be performed early to make an accurate diagnosis and exclude other primary or secondary spindle‐cell neoplasms.

P22‐06

High‐resolution genomic analysis of choroid plexus tumors separates two genetic entities and identifies recurrent focal alterations

Japp AS¹, Gessi M¹, Denkhaus D¹, zur Mühlen A¹, Pietsch T¹, Hartung S², Kordes U², Messing‐Juenger M³, Wolff JE⁴ and Klein‐Hitpass L⁵

¹ Department of Neuropathology, University of Bonn, Germany; ² Department of Pediatric Hematology/Oncology, University of Hamburg, Germany; ³ Department of Pediatric Neurosurgery, Asklepios Children's Clinic Sankt Augustin, Germany; ⁴ Department of Pediatric Hematology/Oncology, Cleveland Clinic, OH, USA; ⁵ Department of Cell Biology (Tumor Research), University of Duisburg‐Essen, Germany

Choroid plexus tumors are rare brain neoplasms mainly occurring in children. They include papillomas, atypical plexus papillomas and carcinomas. Detailed genetic studies of large series are rare and our knowledge on their molecular pathogenesis is limited.

In order to identify common genomic alterations and molecular features specific for each subgroup, we performed a molecular inversion probe (MIP) and RNA expression analysis in 66 CPT.

MIP analysis showed the frequent occurrence of hyperdiploidy in papillomas and atypical plexus papillomas, which appeared very similar in their cytogenetic profiles. Papillomas showed frequent copy number gains of chr. 7, 8, 9, 12, 15, 18 and 20. Atypical plexus papilloma predominantly displayed gains similar to CPP except chr. 15 and 18. In contrast to papillomas and atypical plexus papillomas, carcinomas showed a large number of chromosomal aberrations on many chromosomes. Besides recurrent focal chromosomal gains common for all choroid plexus tumors, including chr. 14q21‐q22 (harboring OTX2), chr. 7q22 (LAMB1) and chr. 9q21.12 (TRPM3), GISTIC analysis uncovered focal alterations specific for papillomas and atypical plexus papillomas (e.g. 7p21.3 (ARL4A) as well as for carcinomas (6p13.3 (RBFOX1) and 6p21 (POLH, GTPB2, RSPH9 and VGFRA). RNA expression profiling and gene set enrichment analysis revealed a significantly increased expression of cell cycle related genes in atypical plexus papilloma in comparison to papillomas.

According to these findings, it can be hypothesized that atypical plexus papilloma may represent an infantile variant of papillomas, characterized by increased proliferative activity. Plexus carcinomas represent instead a genetically distinct tumor group.

P22‐07

Histopathological grading according to the WHO classification of CNS tumors and H3.3K27 mutational status represent independent prognostic markers in pediatric high grade gliomas

Pietsch T¹, Gielen G¹, zur Mühlen A¹, Wolff J², Kwiecien R³ and Kramm C⁴

¹ Department of Neuropathology, University of Bonn, Germany; ² Department of Pediatric Hematology/Oncology, Cleveland Clinic, OH, USA; ³ Department of Biometrics and Clinical Research, University of Münster, Germany; ⁴ Department of Pediatric Hematology/Oncology, University of Goettingen, Germany

While the prognostic impact of histological grading is well established in adult high grade gliomas, this has not been shown in large series of pediatric HGG patients. We analyzed samples from 159 HGG patients (age range, 0–18 years, with 31 patients <3 years) treated with an age adapted postoperative (radio)chemotherapy according to the German HIT‐HGG/GBM or HIT‐SKK (infant) protocols for the presence of H3.3K27 mutations by pyrosequencing. Histological analysis led to the diagnosis of 44 anaplastic astrocytoma (WHO grade III, AAIII) and 115 glioblastomas (WHO grade IV; GBMIV), 27.7% of these HGGs carried H3.3K27 mutations. These mutated cases were mostly located in the midline and occurred in non‐infants.

A significant superior overall survival in diffuse pediatric HGG patients graded as AA III in contrast to GBM IV was detected (for all patients, p = 0.002, for non‐infants only, p = 0.025, log‐rank‐test). H3.3K27 mutation was a significant adverse prognostic marker. Multivariable analysis showed that WHO grading and H3.3K27 mutational status were independent prognostic markers. By combining these two features in Kaplan–Meier analysis for grouping non‐infant patients, overall survival was best for patients with H3.3K27 wildtype (wt) AAIII (n = 19, 5y‐OS, 30.5 +/– 13.6 %, followed by H3.3wt GBM IV (n = 40, 5y‐OS, 10.6 +/– 5.7%), and H3.3K27 mutated cases did worse with lowest survival rates, irrespective to WHO grading (AAIII H3.3K27 mutated, n = 11, 5y‐OS, 9.1 +/– 8.7%; GBMIV H3.3 mutated, n = 30, 5y‐OS, 0%).

These data suggest that WHO histological grading and H3.3 mutational analysis allows a three‐tier prognostic stratification of pediatric HGG patients and risk‐adapted therapeutic design.

P22‐08

Metastatic glioblastoma in child: a case report

Silva ACAL, Neder L, Vilela TM and Caputo S

Hospital of Clinics, Federal University of Uberlandia, Brazil

Introduction: Glioblastoma (GBM) is a grade IV astrocytoma that seldom occurs in children. Extraneural metastases from GBM or other high‐grade gliomas are rare, because the overall short‐term survival. Herein we describe a rare case of GBM in a child with metastases to cervical lymph nodes.

Patient and Results: The patient is a 9‐year‐old male who was referred to Hospital of Clinics, Federal University of Uberlandia for investigation of a short history of focal neurologic deficits and signs of intracranial hypertension. An MR revealed a large intra‐axial mass in the right fronto‐temporal region extending into deep white matter. There was a midline shift and the tumor showed irregular contrast enhancement. At frontotemporoparietal craniotomy the tumor was soft, yellow‐brown and involved leptomeninges. The diagnosis was glioblastoma. The patient underwent to radiotherapy and chemotherapy, but five months later, she presented cervical lymph node enlargement on the right side. A lymph node biopsy revealed a metastatic GBM with similar immunohistochemical profile of the previous brain biopsy. Three months later, the child exhibited spinal and gum metastases. She expired shortly after acute respiratory failure, but autopsy was not performed.

Conclusion: Spreading of extra cranial metastases from primary gliomas is extremely rare. In this case, the GBM spread by lymphatics or hematogenous routes may be occurred, due to tumoral growth to skull base/dura‐mater and/or tumor surgical manipulation. Although rare, extra cranial metastases of high‐grade primary brain tumors should be investigated in patients with high‐grade gliomas, particularly when complicated by extra cerebral manifestations.

P22‐09

Molecular characterization of FGFR1 expression in pilocytic astrocytomas: a clinicopathological study in Brazilian patients

Becker AP¹, Scapulatempo‐Neto C^1,2, Clara C³, Machado HR⁴, Oliveira RS⁴, Musselwhite E⁵, Varella‐Garcia M⁵, Neder L⁴ and Reis RM^1,6

¹ Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; ² Department of Pathology, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; ³ Department of Neurosurgery, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; ⁴ Ribeirão Preto School of Medicine, University of São Paulo (FMRP‐USP), São Paulo, Brazil; ⁵ University of Colorado School of Medicine, Aurora, CO, USA; ⁶ Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal

Introduction: Pilocytic astrocytoma (PA) is a WHO grade I glioma, in which key molecular event is a constitutive activation of MAPK pathway. Point mutation of FGFR1 was described as an alternative trigger for MAPK activation. Our aims were to assess the immunohistochemical (IHC) expression of FGFR1 and characterize its underlying molecular mechanisms in a series of Brazilian patients with PAs.

Patients and Methods: The IHC expression of FGFR1 and gene amplification was assessed by FISH in FFPE tumor samples from 69 patients (1.2 M/F, median 11.6 years‐old) on tissue microarray platforms. Hotspot mutations of FGFR1 (exons 12 and 14) were evaluated by PCR followed by direct sequencing. The findings were correlated with clinicopathological data.

Results: Overall, 73.9% of the cases (51/69) showed cytoplasmic expression of FGFR1 in neoplastic cells, without significant differences between clinical subgroups. FGFR1 amplification was not observed in 61 tested samples. Seven cases (11.5%) exhibited low level of copy number gain. FGFR1 activating mutation lys656glu was observed in 6.7% of cases (3 cerebellar lesions), with negative impact in the OS (p = 0.047). No correlation was observed between FGFR1 overexpression and gene alterations.

Conclusions: Overexpression of FGFR1 in PAs was not related to clinicopathological data, butin contrast to breast cancer, gene amplification was not the underlying mechanism in PAs. A subset of patients that harbored FGFR1 point mutation had a shorter OS, which highlights the importance of this alteration in PA progression and raises the question whether these patients could be candidates for FGFR1 target therapies.

Support: This project was financially supported by the BHC internal research funds (PAIP), FAPESP and NCI CCSG P30CA046934 grant.

P22‐10

Pilomyxoid astrocytoma: experience in a pediatric hospital

Gargano PR¹, Camarero S¹, Baroni L², Alderete D², Buznick J³, Zuccaro G³ and Lubieniecki FJ¹

¹ Department of Pathology; ² Department of Neuro‐oncology; ³ Department of Neurosurgery, Hospital de Pediatria Prof. Dr. Juan P. Garrahan, Buenos Aires, Argentina

Introduction: Pilocytic astrocytoma (PA) is the most frequent neoplasm of the central nervous system (CNS) in pediatrics. The defined pilomyxoid variant (PMA) incorporated in the WHO 2007 classification is considered to have a worse prognosis because of its tendency to recur and spread via CSF. The aim of this study was to present our experience.

Materials and Methods: We retrospectively evaluated 17 cases of PMA diagnosed over the last 16 years at our institution accounting for 0.5% of all CNS tumors and 6.3% of pilocytic tumors. Clinical and pathological parameters were evaluated. Routine techniques, GFAP, synaptophysin, neurofilament, and Ki67 were performed.

Results: Age range at diagnosis: 4–186 months (mean 95 months). Location: Posterior fossa 3/16, hypothalamus/chiasm region 9/16, and temporal region 4/16. Extent of resection: 3/15 total, 12/15 partial. Histologic features: Pure pilomyxoid pattern: 8/17; necrosis: 8/17; cellular pleomorphism: 6/17; infiltration of adjacent nervous parenchyma: 6/17. Proliferation index: 0.5% to 15%. Regrowth was seen in three mixed cases (two hypothalamus‐chiasmatic tumors): two in both samples and one with a pilocytic pattern in the first surgery and pilomyxoid features in the recurrence. Leptomeningeal dissemination was found in 1/15 (6.66%). Three patients died within the first year of diagnosis (17.64%), another was receiving palliative care.

Conclusion: Our experience shows a broad spectrum of morphologically pure or mixed patterns. Moreover, our cases reflect a more aggressive clinical behavior, shorter disease‐free survival, and higher mortality than pilocytic astrocytoma compared with the literature.

P22‐11

Posterior fossa and spinal gangliogliomas form two distinct clinicopathologic and molecular subgroups

Gupta K¹, Orisme W¹, Dalton JD¹, Punchihewa C¹, Collins‐Underwood R¹, Tatevossian RG¹, Ellison DW¹, Qaddoumi I² and Robertson T³

¹ Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA; ² Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA; ³ Department of Pathology, Royal Brisbane and Women's Hospital, Herston, Qld, Australia

Gangliogliomas are low‐grade glioneuronal tumors of the central nervous system and the commonest cause of chronic intractable epilepsy. Most gangliogliomas (>70%) arise in the temporal lobe, and infratentorial tumors account for less than 10%. Posterior fossa gangliogliomas can have the features of a classic supratentorial tumor or a pilocytic astrocytoma with focal gangliocytic differentiation, and this observation led to the hypothesis tested in this study – gangliogliomas of the posterior fossa and spinal cord consist of two morphologic types that can be distinguished by specific genetic alterations. Histological review of 27 pediatric gangliogliomas from the posterior fossa and spinal cord indicated that they could be readily placed into two groups: classic gangliogliomas (group I; n = 16) and tumors that appeared largely as a pilocytic astrocytoma, but with foci of gangliocytic differentiation (group II; n = 11). Detailed radiological review, which was blind to morphologic assignment, identified a triad of features, hemorrhage, midline location, and the presence of cysts or necrosis, that distinguished the two morphological groups with a sensitivity of 91% and specificity of 100%. Molecular genetic analysis revealed BRAF duplication and a KIAA1549‐BRAF fusion gene in 82% of group II tumors, but in none of the group I tumors, and a BRAF: p.V600E mutation in 43% of group I tumors, but in none of the group II tumors. Our study provides support for a classification that would divide infratentorial gangliogliomas into two categories, (classic) gangliogliomas and pilocytic astrocytomas with gangliocytic differentiation, which have distinct morphological, radiological, and molecular characteristics.

P22‐12

Stratification of infant patients with medulloblastoma: genomics and biology complement histological classification

Kapitza E¹, Denkhaus D¹, zur Mühlen A¹, Rutkowski S² and Pietsch T²

¹ Department of Neuropathology, University of Bonn, Germany; ² Department of Pediatric Hematology/Oncology, University of Hamburg, Germany

Desmoplastic/nodular histology is a favorable prognostic marker in medulloblastoma (MB) of infancy that allows the reduction of therapy to decrease side‐effects. In a cohort of 168 infant MB (<5 years) we analyzed the histology, hedgehog pathway activation (immunostaining for YAP‐1 and p75‐NGFR) and genomic landscape by molecular inversion profiling. Desmoplastic/nodular histology (MB with extensive nodularity (MBEN) and desmoplastic MB (DMB)) showed a high concordance (>98%) with hedgehog activation. The frequency of chromosomal copy number aberrations was significantly lower in the desmoplastic/nodular subtypes. MBEN and DMB were characterized by LOH of chromosomal arms 9q or 10q (MBEN, 77%; DMB, 76%), whereas classic MB (CMB) and large cell or anaplastic MB (LCA‐MB) showed 17q gains (CMB, 67%; LCA‐MB, 79%) or an isochromosome 17q (CMB, 30%; LCA‐MB, 57%), and gains of OTX2 (CMB, 73%; LCA‐MB, 79%). Moreover, 71% of LCA‐MB carried MYCC or MYCN amplifications. In summary, hedgehog activation and specific genetic features are strongly associated with distinct histological subtypes of infant MB and therefore represent useful diagnostic tools to correctly classify these tumors.

P22‐13

Useful diagnostic features in a challenging case of intraventricular dysembryoplastic plastic neuroepithelial tumor at frozen section

Kan ANC¹ and Wong S²

¹ Department of Clinical Pathology, TuenMun Hospital, Hong Kong; ² Department of Neurosurgery, TuenMun Hospital, Hong Kong, China

Dysembryoplastic neuroepithelial tumor (DNT) is an uncommon benign glioneuronal tumor usually in the cortex with or without associated cortical dysplasia. The histologic diagnosis of DNTs is known to be difficult. Clinical and radiological criteria are suggested in the latest WHO classification as diagnostic criteria which may not be helpful in case of extracortical DNTs and renders diagnosis challenging especially at frozen section. We report a case of intraventricular DNT in a 13‐year‐old boy who presented with convulsion. Imaging revealed a tumor in the left lateral ventricle with features suggestive of central neurocytoma. The patients underwent two operations. Frozen section material showed predominantly oligodendroglial‐like cells and rare neurons in focal myxoid background. The latter feature is absent in the frozen section biopsy from the second operation. Subsequent permanent sections revealed focal diagnostic myxoid areas containing scanty floating neurons and the presence of glioneuronal element which comprise a minor proportion of the tumor. Clinical information and frozen section biopsy material from the other 2 DNTs in our centre in 2001 to 2014 were also reviewed. Recent onset of seizure, cystic appearance of the lesion in imaging, myxoid area and the identification of “floating” neurons are the important features to be identified irrespective of location of the tumor.

P22‐14

V600E BRAF mutation in pilocytic astrocytoma is associated with a more diffuse growth pattern but does not confer a more aggressive clinical behavior

Bannykh S and Kandala G

Cedars‐Sinai Medical Center, Los Angeles, CA, USA

The V600E missense mutation is the most common tumor‐associated somatic alteration in the BRAF gene, recently observed in pilocytic astrocytoma (PA). Alternative BRAF activating mechanism involves BRAF‐KIAA1549 gene fusion with corresponding duplication at chromosome band 7q34. Here, we present our experience with a combined use of these two techniques to assess the utility of the BRAF alterations as an adjunct diagnostic test. 46 cases of IDH‐1 mutation negative non‐syndromic PAs were evaluated. We observed that 25 (54%) of tumors were positive for either V600E mutation or 7q34 duplication. We detected that all 12 of V600E+ tumors showed more “diffuse” infiltrative growth, whereas only 3 of 15 PAs with 7q34 duplication displayed such pattern. Correlation between “diffuse” growth and V600E mutation was statistically significant. Clinical follow up however revealed no adverse prognostic significance of BRAF V600E mutation. We conclude that combined molecular study for V600E mutation and 7q34 duplication can have a diagnostic utility, especially in cases of so‐called “diffuse variant” of PA.

P22‐15

Molecular markers in pediatric ependymomas

Araki A, Chocholous M, Gojo J, Dorfer C, Czech T, Dieckmann K, Slavc I and Haberler C

Institute of Neurology, Medical University of Vienna, Vienna, Austria

Introduction: Ependymomas account for approximately 10% of brain tumors in children. Whereas patients with subependymomas and myxopapillary ependymomas have in general a very good prognosis, ependymomas grade II and anaplastic ependymomas have a relatively poor outcome. The pathological criteria for differentiation between grade II and III are difficult to apply and the prognostic significance of grading is of uncertain clinical utility. Thus, molecular markers are needed for therapeutic stratification. In the present series we have analyzed the previously described prognostic markers 1q gain and Tenascin‐C expression in a series of ependymomas.

Patients and Methods: A total of 82 ependymomas were included in the study. 52.4% were male, 47.6% female. Age at diagnosis ranged between 1 month and 21 years (median 4.4 yrs). 31. Tumor location was infratentorial in 67%, supratentorial in 31.7%, and unknown in 1.2%. Analyses were performed on FFPE sections using commercially available 1q25/1p36 FISH probes (Vysis) and an antibody against Tenascin‐C.

Results: 28/82 tumors were classified as grade II and 54 as grade III ependymomas. Chromosome 1q gain was found in 12 tumors, 1 grade II and 11 grade III tumors. Tenascin‐C expression was detectable in 61/82 tumors, 15 grade II and 46 grade III ependymomas. Both, gain of 1q and Tenascin‐C expression were significantly associated with a shorter overall survival (p = 0.027 and p = 0.04).

Conclusion: We could validate the negative prognostic impact of 1q gain and Tenascin‐C expression on patient outcome in our series, thereby providing further evidence that both are useful prognostic markers in ependymomas.

P22‐16

Pediatric primary pachymeningeal fibroxanthoma–cholesterol granuloma with benign fibrous histiocytoma foci displaying cannibalistic features & focal melanin synthesis

Rodríguez Álvarez CA¹, Salazar Morales MF², Gómez Apo E², Estrada Hernández MR², Estrada Moscoso I² and Parraguirre Martínez S³

¹ Hospital Pediátrico Legaria; ² Hospital General de México “Dr. Eduardo Liceaga”; ³ Hospital General “Dr. Manuel Gea González,” Mexico

We put forth the unique case of a 4‐year‐and‐7‐months‐old girl who experienced a slow, painless and progressive occipital swelling that eventually caused aesthetical impairment. Magnetic resonance imaging showed a dural tumor of variegated intensity compressing the left occipital pole and apparently extending towards the superior sagittal sinus.

Grossly, the cut surface of the surgical piece submitted was pale yellow and soft with reddish brown kernel‐like crusts giving a whole appearance similar to a «raspberry bread‐and‐butter slice». Histologically the yellow areas resembled cholesterol granuloma – widespread coagulative necrosis, cholesterol clefts, powdery calcification, foreign body‐type giant cells and foamy macrophages – while the scattered red spots were constituted by spindle cells and plentiful multinucleated giant cells of Touton and foreign body types, the latter with amphophilic cytoplasm and occasional empty vacuoles; intriguingly, they also featured phagocytosis of cellular elements – cannibalism. The immunohistochemistry panel was positive for CD68, a1‐antitrypsin, a1‐antichemotrypsin and vimentin. Also, scarce macrophages contained dark brown granules non‐reactive for Perls Prussian blue but positive with the Fontana‐Masson silver stain.

Dural fibroxanthomas (also designated as benign fibrous histiocytomas) are extremely rare lesions usually found in young persons. On the other hand, intracranial cholesterol granulomas are also rare and usually comprise the frontal, zygomatic and petrous bones. As far as we know, no tumor displaying these hybrid features has yet been described.

P22‐17

Cerebral lipidized ependymoma in a child

Inenaga C¹, Otsuki Y², Tanaka T¹, Namba H³ and Takahashi H⁴

¹ Department of Neurosurgery, Seirei Hamamatsu General Hospital; ² Department of Pathology, Seirei Hamamatsu General Hospital; ³ Department of Neurosurgery, Hamamatsu University School of Medicine; ⁴ Department of Pathology, Brain Research Institute, University of Niigata, Japan

We report an unusual cerebral tumor in a 5‐yar‐old girl. She visited our hospital because of simple head trauma. Brain CT revealed a cystic tumor in the parietal lobe, showing high density with focal calcification. MRI revealed that the tumor was well enhanced and showed no attachment to the wall of the lateral ventricle. The tumor was removed totally. Histologically, the tumor consisted mainly of foamy (xhantic) cells without apparent nuclear atypia or cellular pleomorphism. Perivascular pseudorosettes or ependymal rosettes were not evident. There were no mitotic figures or necrotic foci. Immunohistochemically, the foamy cells were strongly positive for GFAP and S‐100 protein and negative for olig2, CD68, neurofilament protein orsynaptophysin. EMA‐positive granular and microvesicular structures were occasionally seen; the feature appeared to be in consistent with ependymomas. The MIB‐1 labeling index was <1%. Interestingly, strong expression of fatty acid synthase (IBL) was evident in the foamy tumor cells. In conclusion, we considered the present case to be an unusual example of ependymoma containing many lipidized tumor cells (a lipidized variant of ependymoma). The postoperative course was uneventful, and there was no evidence of recurrence 2 years and 8 months after excision.

P22‐18

Frequency of mismatch repair gene deficiency among pediatric pleomorphic xanthoastrocytoma

Amayiri N, Tabori U, Bakry D, Musharbash A, Swaidan M, Bouffet E, Hawkins C and Al‐Hussaini M

Department of Pathology and Laboratory Medicine, King Hussein Cancer Center, Amman, Jordan

Background: Biallelic mismatch repair deficiency (BMMRD) is a cancer predisposition syndrome affecting individuals from consanguineous families with multiple childhood cancers; most commonly malignant brain tumors. The prevalence of clinical findings suggestive of BMMRD among pediatric patients diagnosed with pleomorphic xanthoastrocytoma (PXA) was determined.

Patients and Methods: All patients <18 years old diagnosed at King Hussein Cancer Center/Jordan with PXA between 2003 and 2013 were included. Clinical data regarding presence of café au lait spots (CAL), family history of cancer (FHC), consanguinity, diagnosis and treatment were collected. Tumors were centrally reviewed and tested for evidence of BMMRD by immunohistochemistry of the MMR proteins.

Results: Seven patients (5 females) fulfilled the inclusion criteria. Median age at presentation was 14.1 year (range, 4.6–16.2yr). One patient had CAL, one had history of consanguinity and one had FHC. Five of seven patients had PXA with anaplastic features. A single case was product of consanguineous marriage however lacked further information on presence of CAL or FHC. This patient (14%) had lost PMS2 and MLH1 stains in her tumor as well as in the surrounding normal endothelium suggesting BMMRD. She survived only 13 months. At a median follow up of 28 months (ranges, 8–71 months), three patients were alive, two were dead and two were lost for follow up.

Conclusions: We reported the first PXA pediatric patient with likely underlying BMMRD. Germline mutation analysis is needed to confirm the diagnosis. Alterations suggestive of BMMRD in pediatric PXA can be seen in this WHO grade II brain tumor.

P22‐19

Oncocytic variant of choroid plexus papilloma in a three‐year‐old child

da Fonseca TC, Eisenberg ALA, Bandeira CL, de Oliveira JA and Grabois MF

INCA, Brazil

Introduction: Oncocytic differentiation occurs as a result of cytoplasmic accumulation of mitochondria, probably caused by defects of the oxidative metabolism. They may occur in numerous different types of neoplasias, but are more frequent in renal, thyroid, and salivary gland neoplasms. Oncocytic tumors are uncommon in the intracranial region and only 5 cases of oncocytic choroid plexus papilloma were documented to date in the international literature.

Patient and Methods: A clinicopathological correlation of a left lateral ventricular oncocytic variant of choroid plexus papilloma is described. The patient was a 3‐year‐old boy with a history of seizures and “café au lait” spots. Computed tomographic scan and magnetic resonance imaging revealed a mass in the left lateral ventricle. Surgical resection was performed, with intraoperative consultation. Cytologic smears showed solid and papillary areas represented by numerous large polygonal cells with ample eosinophilic granular cytoplasm with central nuclei and prominent nucleoli, consistent with choroid plexus papilloma. Paraffin embedded sections revealed a lesion with solid and papillary architectural pattern, containing or lined by large and polygonal cells, with distinct cytoplasmic borders and finely granular eosinophilic cytoplasm. Multinucleated pleomorphic cells were observed and mitotic figures were exceedingly rare. Necrosis and vascular hyperplasia were not seen. The diagnosis was oncocytic variant of choroid plexus papilloma.

Conclusion: To our knowledge, this is the sixth report of an oncocytic variant of choroid plexus papilloma.

P22‐20

Oncogene amplification in a child with medulloblastoma

Sakai Jr N and de Souza FS

Sarah Network of Rehabilitation Hospitals, Brasilia, Brazil

Medulloblastoma (MB) or primitive neuroectodermal tumor (PNET) represent 15%–30% of pediatric brain tumors and are the most common brain tumors in children; they are rare in adults. We describe amplification of the MYCN oncogene in a medulloblastoma with aggressive clinical behavior. The patient was an eleven year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy (Carboplatin, Ifosfamide, Etoposide and Vincristin) and total neuraxis radiation, the clinical course was one of relentless progression, with extensive spread and death within fourteen months of presentation. Cytogenetic studies from tumor were performed. The pathological features were consistent with the recently described, “large cell variant” of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin, CD‐99, GFAP, vimentin and KI‐67. Fluorescence in situ hybridization (FISH) assay revealed evidence of MYCN amplification. Conventional karyotypic analysis of tumor revealed a complex chromosome rearrangement with several double minutes (dmin), extensive MYCN amplification and absence of MYCC amplification. The MYCN oncogene encodes a transcription factor belonging to the MYC family. It is primarily expressed in normal developing embryos and is thought to be critical in brain and other neural development. High copy number amplifications found in MYCN oncogene suggesting a “pathogenetic” role of this gene in PNETs. Patients with high copy number amplifications of MYCN not responded to therapy. In the future, analysis of a greater number of cases will permit recognition of previously unknown genetic aberrations in medulloblastoma and its aggressive variants.

P22‐21

Pattern of SOX₂ expression in medulloblastomas – an immunohistochemical analysis of 56 cases

Pal L¹, Mehrotra A² and Behari S²

Departments of ¹ Pathology and ² Neurosurgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences, RaeBareli Road, Lucknow, India

Background: Medulloblastomas are highly aggressive tumor of childhood. Identification of cancer stem cells in medulloblastomas may provide therapeutic target in high risk patients who remain refractory to current treatment. Overexpression of SOX₂ has been reported with poor prognosis. It has also been shown that knockdown of SOX₂ in vitro impairs the growth and tumorigenicity of brain tumor cells. Hence this study was conceptualized to identify the morphological subgroups expressing SOX₂.

Material and Methods: Fifty‐six medulloblastomas were included in this study and immunohistochemistry was performed on paraffin embedded sections using standard technique for SOX₂ antigen. Strong nuclear expression of more than 10% of tumor area was considered as positive.

Results: The majority of medulloblastomas were in the pediatric age group and the age ranged from 6 to 49 years. There was male preponderance with male : female ratio of 3:1. Vermis was the most frequent site (n = 38) of involvement. 55.4% of the medulloblastomas had classic histology followed by desmoplastic (n = 14) and medulloblastoma with extensive nodularity (n = 4). Anaplastic/large cell morphology was present in 7 cases. Diffuse/patchy/focal strong SOX₂ was positivity was seen in 34% of cases and 78.6% of the desmoplastic medulloblastomas (11/14) showed strong internodular as well as diffuse positivity. On the contrary only 3 out of 31 classic medulloblastomas showed focal strong positivity. Diffuse strong positivity was noted in 5/7 medulloblastomas with anaplastic/large cell morphology. All 4 medulloblastoma with extensive nodularity were negative. Normal cerebellar tissue included in some of the cases showed SOX₂ positive nuclei at the junction of outer molecular and inner granule cell zone.

Conclusion: SOX₂ expression was more frequent in desmoplastic and anaplastic variant compared to classic ones, which may have prognostic significance, and larger study is required.

P23. Peripheral Nerve

P23‐01

Association of CMV in HIV related peripheral neuropathy – is it a manifestation of immune reconstitution in peripheral nervous system?

Mahadevan A, Satishchandra P, Shankar SK, Taly AB and Ravi V

National Institute of Mental Health & Neurosciences, Bangalore, India

Background: Incidence of peripheral neuropathy in patients with HIV/AIDS varies from 15 to 50% in Western literature. At our Centre, the incidence of HIV related peripheral neuropathy appears uncommon (2.9%). Pattern of peripheral neuropathy in India and its etiopathogenetic mechanisms, particularly the role of CMV has not been documented.

Objectives: The pathological spectrum of peripheral neuropathy in patients with HIV/AIDS in last decade was evaluated. The incidence of CMV in these cases was assessed by immunohistochemistry and in situ hybridization.

Methods: Sural nerve biopsies from 21 cases were processed for histological evaluation and immunostained for CMV, T cell, B cell, CD68 and neurofilament. Details of clinical features, electrophysiology and relevant serological tests were reviewed.

Results: Clinically, 7 presented with features of CIDP, 6 with mononeuritis multiplex, 3 with DSPN, and 5 with suspected toxic neuropathy (following HAART). Vasculitis was a common finding (mononeuritis multiplex: 5/6, CIDP: 4/7, and DSPN: 1/3 and 0/5: toxic neuropathy). The inflammation was T cell rich (majority CD8 phenotype) with variable demyelination and axonal loss. CMV positive mononuclear cells were seen 15/21 cases including cases of toxic neuropathies which had no vasculitis.

Conclusion: CIDP and mononeuritis multiplex were common modes of presentation unlike the West where DSPN predominates. Vasculitis was a common finding in majority except in drug induced forms. High prevalence of CMV antigen was seen (71%) irrespective of type of presentation. Finding of CMV antigen in patients receiving HAART raises speculation that peripheral immune reconstitution may occur. Use of steroid/immunosuppressive therapy needs to be evaluated with care.

Acknowledgement: This publication was partly supported by a subaward from The Johns Hopkins University, with funds provided from National Institute of Neurological Disorders and Stroke (NINDS) [Grant No: 1RO1NS055628‐01A2]. Its contents of the study are solely the responsibility of the authors and do not represent the official view of NINDS or JHU.

P23‐02

Patterns of amyloid deposition in salivary gland biopsies of familial amyloid polyneuropathy (FAP) patients

Domingos J, Coelho T, Moreira R, Melo‐Pires M and Taipa R

Department of Neurology, Centro Hospitalar do Porto, Porto, Portugal

Introduction: Salivary gland biopsy (SGB) is a very sensitive method to demonstrate amyloid in FAP patients. Our aim was to determine whether there were different patterns of amyloid deposition in SGB between FAP patients and those who received a domino liver transplant (DLT) from FAP patients.

Materials and Methods: We selected 79 SGB from FAP patients (32 early‐onset; 22 late‐onset) and from patients submitted to DLT (25). The observers were blind for the diagnosis. Amyloid deposition was characterized accordingly to its deposition around the acini/ducts, blood vessels and fibrous tissue in the submucosa.

Results: From the 32 early‐onset FAP patients 26 (81,2%) had amyloid deposits around the acini/ducts, 4 (12,5%) around the blood vessels and 13 (40,6%) in the submucosa's fibrous tissue (SFT). In late‐onset FAP 21 (95,4%) had deposits around the acini/ducts, 3 (13,6%) around the blood vessels and 12 (54,5%) in SFT. In DLT 22 (88%) had deposits around the acini/ducts, 7 (28%) around the blood vessels and 19 (76%) in SFT. Comparing the different groups we found statistically significant differences between the early‐onset FAP and DLT regarding amyloid deposition in SFT, being more frequent in DLT patients (p = 0.01). Analyzing the samples according to the presence or absence of amyloid in SFT we found that the age at biopsy was statistically different between the two groups (p = 0.03), amyloid being more common in the oldest.

Conclusion: Our data suggest that in an early stage of disease, amyloid deposition in SGB might be different in FAP patients and DLT recipients and that age might be a contributing factor.

P23‐03

Preoperative biopsies in peripheral nerve tumors: clinical consequences

de Sousa ACS¹, Guedes‐Correa JF², Costa JP³, Amorim RMP², Santos LL² and Pereira MRC²

¹ Pathology Service, Resident Physician, Hospital Universitario Gaffree e Guinle (HUGG), Escola de Medicina e Cirurgia da Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Rio de Janeiro, Brazil; ² Division of Neurosurgery, HUGG, UNIRIO, Rio de Janeiro, Brazil; ³ Undergraduate Medicine Student, Escola de Medicina e Cirurgia da UNIRIO, Rio de Janeiro, Brazil

Introduction: Peripheral nerve tumors (PNT) form a heterogeneous group of complex lesions of difficult treatment. Preoperative biopsy (POB) in patients with PNT is controversial. There are few references in the literature about both its indications and clinical consequences. Our objective is to evaluate consequences of POB in patients with PNT.

Patients and Methods: Seven adult patients (five females and two males) who underwent POB in other surgical services were evaluated. The initial diagnosis of each patient, after POB, was: schwannoma (28.6%), malignant peripheral nerve sheath tumor (MPNST – 14.3%) and inconclusive (57.1%). The location of the tumors were: four (57.1%) brachial plexus lesions, one (14.3%) right radial nerve, one (14.3%) right arm and one in right thigh (14.3%). All patients were evaluated by neurological examination, imaging exams and neurophysiological tests and were operated by the senior author (JFGC), with microsurgical technique.

Results: All patients had worsening of the pain after POB (100%). Five developed a new motor deficit (71.4%), three (42.8%) had a different final histological diagnostic after surgery and four (57.1%), who had obtained inconclusive results by POB, had the histological type of the tumor eventually clarified (two schwannomas, one neurofibroma and one lipoma).

Conclusions: POB must not be performed in PNT, except when there is a strong possibility of a MPNST. After POB patients have a high probability of developing or worsening neuropathic pain or even motor/sensory deficit. POB in PNT makes microsurgical resection of the tumor difficult by development of intraneural fibrosis, hemorrhage and lesion of functional fascicles.

P23‐04

Vasculitic neuropathy: leprosy as a possible diagnosis

Pinto TS, Antunes SLG, Sousa FS, Vital RT, Gomes ML, Freitas ABSB, Sano EN and Jardim MR

Fundação Oswaldo Cruz (FIOCRUZ), Ambulatório Souza Araújo, Hospital Universitário Pedro Ernesto (HUPE), Services of Neurology and Rheumatology, Brazil

Introduction: Peripheral nerve (PN) vasculitis are pathological conditions with pleomorphic clinical features, whose diagnosis frequently depends on the histopathological study of nerve biopsy samples.

Methodology: Retrospective study of patients who underwent histopathology of nerve samples for diagnosis of peripheral neuropathy in a referral center for leprosy in Rio de Janeiro/Brazil 2001 to 2014. Twenty nerve biopsy specimens showing histological alteration consistent with vasculitic neuropathy were selected. Patients underwent clinical, neurological, rheumatological and neurophysiological evaluation. The samples were routinely studied by hematoxylin‐eosin, Gomori's trichrome, Wade staining as well as semithin sections stained by toluidine blue.

Results: Out of 20 nerve patients (mean age 55 years, 55% men) selected, 6 were diagnosed as leprosy; 3 Wegener's granulomatosis; 2 systemic lupus erythematosus; 5 isolated peripheral nervous system vasculitis; 1 rheumatoid arthritis; 1 Sjögren's syndrome; 1 livedoid vasculopathy and 1 monoclonal Ig A hypergammaglobulinemia. The most common clinical presentation was asymmetric sensory neuropathy (63% of cases) followed by asymmetric sensory‐motor neuropathy (37% of cases). Histopathology of the nerve samples revealed alterations in all cases consistent with axonal neuropathy. In addition, inflammatory infiltrate surrounding vessel wall was seen in 64% of cases; fibrinoid necrosis (4%); acute thrombosis (4%); chronic thrombosis (8%); hyperplasia of the muscle layer (8%); thickening of adventitious layer (4%) and endoneurial and perineurial increased extracellular matrix (8%).

Conclusion: Vasculitis of the PN is a clinical entity difficult to diagnose given that laboratory tests are not specific for vasculitis, making biopsy an important tool to define diagnosis. Attention should be paid in the differential diagnosis to the possibility of leprosy as an etiology considering the high endemicity of this disease in Brazil.

P24. Prion

P24‐01

Comparison of brushes and swabs for olfactory mucosa sampling and RT‐QuIC analysis of patients with Creutzfeldt‐Jakob disease

Bongianni M^1,2, Orrú CD¹, Tonoli G³, Hughson AG¹, Ferrari S², Groveman BR¹, Sacchetto L⁴, Fiorini M², Pocchiari M⁵, Monaco S², Caughey B¹ and Zanusso G²

¹ Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; ² Department of Neurological and Movement Sciences, University of Verona, Verona, Italy; ³ S.O.C. di Otorinolaringoiatria, Ospedale “Santa Maria della Misericordia,” Rovigo, Italy; ⁴ Clinica Otorinolaringoiatrica, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; ⁵ Department of Cell Biology and Neurosciences, Istituto Superioredi Sanità, Rome, Italy

Introduction: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) in living patients remains a challenge. In a recent study, we identified prion seeding activity in olfactory mucosa (OM) of CJD patients by nasal brushing coupled with Real Time Quaking induced Conversion (RT‐QuIC), obtaining 100% specificity and >97% sensitivity for the detection of CJD. OM samples were collected with a sterile disposable Cyto‐brush (Kito‐Brush, Kaltek) that might cause mild discomfort for the patients. Therefore, we used less abrasive short nylon fiber Flocked swabs (Flocked swab, Copan Diagnostics) for sampling.

Materials and Methods: We collected OM samples in 18 CJD patients. To ensure efficient OM sample collection, each patient underwent to 1 sampling of OM with Flocked swabs for each nostril, followed by 1 with Cyto‐brush. OM samples were processed and analyzed by RT‐QuIC.

Results: Using Cyto‐brushes 17 out of 18 CJD cases were positive by RT‐QuIC analysis, while Flocked swabs were positive for 16 out of 18 CJD samples. In one patient, OM samples were negative for both Cyto‐brush and Flocked swab samplings. Neither OM sampling technique produced false positives.

Conclusion: Overall, Cyto‐brushes and Flocked swabs gave comparable sensitivity. However, for one patient the Cyto‐brush was positive by RT‐QuIC but the two consecutive Flocked swabs were negative. Although the efficacy of OM sampling with Flocked swabs needs further evaluation, this collection method shows promise as a gentle, patient friendly alternative to sampling by Cyto‐brush. Our current recommendation is to adopt a combination of Flocked swabs and Cyto‐brushes to maximize the collection of OM material and minimize patient discomfort.

The authors acknowledge Copan Diagnostics for their collaboration; Steven Medeiros CJD Memorial Research Grant and the Donna Millard CJD Memorial Research Grant.

P24‐02

Definite sporadic Creutzfeldt–Jakob disease: neuropathology and differential diagnosis

Caixeta LF¹, da Silva WG², da Silva MM³ and Daher MC⁴

¹ Associate Professor of Neurology, Federal University of Goiás (UFG) School of Medicine, Goiânia‐Goiás, Brazil. Coordinator of the UFG‐Brain Bank, Clinics Hospital – UFG, Goiânia, Goiás, Brazil; ² Professor of Pathology, State University of Goiás, Unievangelica, FAMA, Anápolis, Goiás, Brazil; ³ Pathologist, The Coroner Service, Unievangelica, Anápolis, Goiás, Brazil; ⁴ Infectologist, Hospital Evangélico Goiano, Anápolis, Goiás, Brazil

Introduction: The transmissible spongiform encephalopathies constitute an unusual and inexorably fatal group of neurodegenerative disorders caused by a protein called prion infection. It was first described in 1920 by Hans Creutzfeldt and Alfons Jakob, so called Creutzfeldt–Jakob disease (CJD). According to the etiology, CJD is divided into: a) sporadic CJD is the most cases; b) acquired by CJD prion‐contaminated food, surgical or biopharmaceutical products; c) hereditary CJD forms.

Materials and Methods: Case study in a patient with sporadic CJD in Central Brazil.

Discussion and Results: The patient had the disease for 71 years old, with rapid course of progressive dementia, with tic signs, ataxia, myoclonus. Western blot of CSF was positive for 14‐3‐3 protein. Death occurred two and a half years after disease onset. The neuropathological study at autopsy showed cerebral cortical atrophy and subcortical widespread, especially in the frontal, parietal and occipital lobes, with asymmetric distribution, involving the left hippocampus and basal ganglia. The cerebellar cortex was also atrophic. The histology of these regions revealed a spongiform degeneration, astrogliosis with, Amyloid plaques and loss of neurons of the cerebellum in the molecular and granular layers. Main differential diagnoses to be considered were: Lewy body dementia, Alzheimer's disease, frontotemporal dementia and corticobasal degeneration.

Conclusion: The neuropathological procedures are crucial to exclude other etiologies and to better analyze these disorders that often present with clinical heterogeneity.

P24‐03

Different patterns of microglial cell activation in distinct sporadic Creutzfedl–Jakob disease subtypes

Pasini G*, Ferrari S, Chilosi M, Bongianni M, Baldo M, Monaco S and Zanusso G

Department of Neurological and Movement Sciences and Department of Pathology and Diagnostics, University of Verona, Verona, Italy

*Presenting author; Email: jules21486@hotmail.com

Introduction: Microglial activation has been described in sporadic and variant Creutzfeldt–Jakob disease (CJD) and related to disease‐specific forms of the prion protein (PrP^Sc). These PrP^Sc forms act as prion strains showing specific tropisms towards neural or lymphatic tissue. In the present study, we characterized microglia phenotypes in definite cases of sCJD using specific markers and we investigated whether distinct sCJD subtypes might be distinguished based on microglial phenotype.

Methods: We carried out a neuropathologic and biochemical study on 24 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. We obtained from each subject formalin fixed sections from frontal and occipital cortexes, cerebellum, thalamus and basal ganglia. Serial sections were immunostained with monoclonal antibodies directed to PrP, GFAP and to different monocyte/macrophage markers such as CD68, CD11c, CD163. Quantitative analysis of microglial expression was also determined by Western blot analysis.

Results: In sCJD cases, MM and MV at codon 129 with a PrP^Sc type 1, immunohistochemistry showed a severe degree of microglia activation while in sCJD with a PrP^Sc type 2 was moderate. In particular, in sCJD with a PrP^Sc type 1 microglial cells were characterized by rounded cell bodies, short and swollen cell processes. In contrast, in sCJD cases with PrP^Sc type 2, microglia was mainly ramified and formed small clusters of activated cells in subcortical white matter and in perivascular spaces. The highest extent of microglial proliferation and activation was observed in cases with status spongiosus, in contrast in cortical areas with a coarse pattern of PrP deposition only rare microglial cells were observed.

Conclusion: These findings support the view that the phenotypic heterogeneity of microglia in different subtypes of sporadic CJD is influenced by PrP^Sc conformers. Moreover, the variable extent of microglia activation in distinct sCJD subtypes indicates specific prion strain tropisms and might provide clues on their potential properties of prion infectivity spreading.

P24‐04

E211K bovine spongiform encephalopathy: animal model for human E200K Creutzfeldt–Jakob disease

Greenlee JJ¹, Smith JD¹, Nicholson EM¹ and Greenlee MHW²

¹ Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA; ² Department of Biomedical Sciences, Iowa State University, Ames, IA, USA

Prion diseases can be acquired through exposure to infectious material, inherited as germline polymorphisms, or occur spontaneously. The majority of cases of bovine spongiform encephalopathy (BSE) have been ascribed to the classical, feedborne form of the disease that is transmissible to humans in the form of new variant Creutzfeldt‐Jakob disease (CJD). Since 2004, atypical forms of BSE have been described based on molecular profiles: H‐ or L‐type, for high and low, respectively. Most cases of atypical BSE arise spontaneously, but one case of H‐type BSE identified in the U.S. in 2006 was associated with a heritable amino acid substitution in the prion protein gene (prnp) called E211K, which is homologous to human E200K, the most common hereditary form of CJD. This work compares the features of transmission of E211K H‐type BSE and classical BSE including clinical signs, antemortem test results, histopathology, and tissue distribution of PrP^Sc. Calves were intracranially inoculated with either classical BSE or H‐type E211K BSE. E211K calves inoculated with E211K H‐type BSE had rapid incubation times (10 mo) when compared to wild type calves inoculated with the same inoculum (18 mo) or either genotype of calf inoculated with classical BSE (26 mo). In all instances, western blot demonstrated BSE with the same molecular profile as the inoculum used and PrP^Sc was widely distributed throughout neural tissues. Preclinical deficits in retinal function and thickness were demonstrated by electroretinography and optical coherence tomography, respectively suggesting these techniques can be a useful screening tool for both classical and H‐type BSE.

P24‐05

Expression of proteinase‐activated receptor 2 (PAR‐2) in human prion diseases and other neurodegenerations

Rohan Z, Smetakova M, Olejar T and Matej R

Department of Pathology and Molecular Medicine, National Reference Laboratory for Diagnostics of Human Prion Disorders, Thomayer Hospital, Prague, Czech Republic

Introduction: PAR‐2 was shown to mediate both neurodegenerative and neuroprotective effects in various neurodegenerative diseases. Previously we have shown that PAR‐2 deletion prolongs survival in murine model of prion disease. Aim of our study was to evaluate PAR‐2 expression in human brain and cerebrospinal fluid (CSF) of patients affected by human prion diseases.

Material and Methods: Both, fresh frozen and formalin‐fixed and paraffin‐embedded brain samples were collected according to standardized protocol during autopsy as well as we collected in some cases ante mortem cerebrospinal fluid. Neuropathological examination revealed diagnoses of sporadic and genetic forms of human prion diseases and compared with other neurodegenerations. We determined PAR‐2 brain expression by western blotting and immunohistochemistry. PAR‐2 expression in the CSF was evaluated using ELISA method.

Results: We did not find any significant difference in the brain and CSF expression of PAR‐2 between neither prion diseases subtypes nor between prion diseases and other neurodegenerations.

Conclusion: Our results did not show significant difference in PAR‐2 expression in prion diseases. Even though PAR‐2 role in prion diseases is unknown, its role as disease‐modulating factor is possible. Determining whether and how PAR‐2 contributes to the pathogenesis of prion and other neurodegenerative diseases may thus provide better understanding of the pathogenesis of these fatal disorders with potential future therapeutical possibilities.

The study was supported by project GACRP303/12/1791 and grant no. NT12094‐5 from Grant Agency of Ministry of Health.

P24‐06

Genetic Creutzfeldt–Jakob disease associated with the P105S‐129V mutation in the PRNP gene

Bonnin JM¹, Cracco L², Murrell JR¹, Richardson RM¹, Bonnin DA¹, Gambetti P² and Ghetti B¹

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine, USA; ² Department of Pathology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA

A man developed a shuffling gait at age 84. Clinical records revealed a history of coronary artery disease, coronary artery bypass surgery, aortic aneurysm and intermittent claudication, pain in left lower extremity, which improved with resting, and diabetes mellitus. At age 85, he was hospitalized because of rapidly progressing cognitive decline. Brain imaging and CSF analyses supported the diagnosis of Creutzfeldt–Jakob disease (CJD). He deteriorated rapidly with worsening confusion, frequent falls, and speech impairment. Death occurred two months after hospitalization. At autopsy, moderate to severe atrophy of the frontal, temporal and parietal lobes, and severe atherosclerosis in the circle of Willis were noted. Two lacunar infarcts were present in the left putamen. Neuronal loss, gliosis and spongiform changes were most severe in the neocortex, entorhinal cortex, subiculum, thalamus and basal forebrain. Immunohistochemistry revealed PrP immunoreactivity in a synaptic‐like pattern as well as focal coarse deposits in the neocortex, entorhinal cortex, hippocampus and cerebellum. Molecular analyses of the PRNP gene revealed a P105S mutation coupled with valine at residue 129. Western blot analyses carried out on proteinase K‐treated prion protein (PrP^res) from frontal cortex and cerebellum showed the presence of PrP^res type 1 with electrophoretic mobility of ∼20 kDa. All three PrP^res glycoforms were present. In a previously reported case, clinically and neuropathologically suggestive of Gerstmann‐Sträussler‐Scheinker disease variant, the P105S‐129V mutation was associated with PrP^res type 1 lacking the diglycosylated PrP^res isoform. These two cases highlight the phenotypic heterogeneity in this mutation. (CDC UR8/CCU515004 and Charles S. Britton Fund).

P24‐07

Human prion diseases in Brazil

Landemberger MC, Machado CF, Smid J, Gomes HR, Chimelli L, Rosemberg S, Nitrini R and Martins VR

AC Camargo Cancer Center, University of São Paulo, Federal University of Rio de Janeiro, Brazil

Global surveillance of vCJD and other forms of CJD was recommended from the WHO for a better understanding of potential causes of iatrogenic CJD, as well as the distribution of various hereditary forms. Prion diseases have been under compulsory notification in Brazil since 2005. From 2005 to 2013, we received 320 blood samples from notified cases of suspected CJD. Blood samples were analyzed by direct genomic sequencing to identify mutations and polymorphisms in the PRNP gene. Cases with mutation in direct sequencing were cloned to confirm results. The presence of 14.3.3 protein in cerebrospinal fluid (CSF) was evaluated using immunoblotting and brain tissue obtained by autopsy or biopsy was analyzed by imunohistochemistry for the presence of spongiosis and proteinase K resistant PrP. The average age of our patients was 56.8 years with a median of 60 years (range 13–90 years), males representing 48% of the cases. PRNP polymorphisms analysis showed that 53% of the cases were homozygous for methionine at codon 129 (M129M), 29% were heterozygous (M129V) and 18% were homozygous for valine (V129V). The silent polymorphism at codon 117 was detected in 9% of the patients and 4% had deletion at the octarepeat. E200K mutation at PRNP was found in seven unrelated patients and all of them presented methionine at codon 129 in the mutated allele. We found two patients with GSS syndrome with a P102L mutation. We also described the first case in America of Insomnia Familial Fatal with a punctual mutation in 178 codon (N178+M129). The mutation in 178 codon were found in other two unrelated patients, one of them associated to Creutzfeldt–Jakob disease. Brain tissue of 32 patients was available, 28 (87%) of them had spongiosis and were positive for proteinase K resistant PrP. After clinical evaluation, imaging exams, 14.3.3 protein presence, genetic and immunohistochemical analysis, notified cases were classified according to the WHO criteria. This study provides the first epidemiologic data about human prion diseases in Brazil. Similar to any other country the availability of brain tissue from these patients is a limiting factor to confirm the diagnosis of prion diseases. This study also represents an important tool for prion‐prevention policies and is of great importance for future implementation of clinical trials.

P24‐08

Intracytoplasmic prion protein in familial Creutzfeldt–Jakob disease associated with the PRNP E200K‐129V haplotype

Oblak A¹, Murrell J¹, Richardson R¹ and Epperson F¹, Chen W³, Bonnin D¹, Farlow M², Bonnin J¹, Gambetti P² and Ghetti B¹

¹ Division of Neuropathology, Department of Pathology and Laboratory and ² Medicine and Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, USA; ³ National Prion Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA

Familial prionopathies are neurodegenerative diseases caused by mutations of the Prion Protein (PRNP) gene. In familial Creutzfeldt–Jakob disease (fCJD), approximately 20 mutations have been reported. They are transmitted in an autosomal dominant pattern. Clinical and neuropathologic features of fCJD are comparable to those of sporadic CJD. We report a family in which four members have the PRNP E200K‐129V haplotype and died of the disease. All four subjects carried methionine in the normal PRNP allele. The oldest subject, a female, died at 85 years. Her mother, three of her sisters, and a brother also died of the same disease. Three of her daughters died of fCJD at ages 56, 55 and 51 years of age. Neuropathologic examination revealed mild to moderate cerebral atrophy with preferential involvement of the frontal and temporal cortices and the hippocampus. There was extensive prion protein (PrP) immunopositivity with a synaptic pattern throughout the brain. Intraneuronal PrP‐immunopositive inclusions were prominentin the neocortex, basal ganglia, entorhinal cortex and the dentate nucleus. Neuronal loss, gliosis and spongiform changes were observed in the same regions. Alzheimer disease pathology was present in two members of the family. The E200K‐129M haplotype is the most common form of fCJD while there are only five reported cases of E200K‐129V haplotype. The presence of intraneuronal PrP‐immunopositive inclusions appears to be a consistent finding in subjects with the E200K mutation; however, their presence needs to be further investigated in other prionopathies. (Funding sources: P30AG010133; NIH P01AG‐14359, Charles S. Britton Fund and CDC UR8/CCU515004).

P24‐09

Neuropathological investigation of the olfactory bulb and olfactory tract in sporadic Creutzfeldt–Jakob disease

Iwasaki Y, Mimuro M and Yoshida M

Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Japan

Introduction: Although it is well known that the cerebral cortex, including the limbic system, is pathologically involved in sporadic Creutzfeldt–Jakob disease (sCJD) from the early disease stage, there are no detailed investigations in the literature on pathological findings in the olfactory bulb and olfactory tract. Thus, we investigated the neuropathological findings in these regions in sCJD cases.

Materials and Methods: We examined pathologic involvement and prion protein (PrP) deposition in the olfactory bulb and olfactory tract of five autopsied cases of MM1‐type sCJD. The average age at disease onset of the cases was 75.2 years (range: 70–83 years) with an average total disease duration of 24.8 months (range: 5–51 months). In each case, formalin‐fixed paraffin‐embedded olfactory bulb and olfactory tract samples were obtained.

Results: Under hematoxylin and eosin staining, gliosis and numerous corpora amylacea were observed in the olfactory bulb and olfactory tract, but spongiform change was not so apparent. Mitral cell and granule cell numbers were preserved in all patients. The number of neurons in the anterior olfactory nucleus was also well preserved. The stainability of the myelin under of the olfactory tract was well preserved with Klüver–Barrera staining. PrP immunostaining revealed diffuse synaptic‐type PrP deposition, particularly in the glomerulus, mitral cell layer and anterior olfactory nucleus. Some small plaque‐type PrP deposits were also observed. The corpora amylacea did not show PrP immunoreactivity.

Discussion: These pathologic findings were similar in each case and their severity tended not to be associated with total disease duration. In the present study, the olfactory bulb and olfactory tract showed PrP deposition from the early disease stage to the prolonged disease stage. Furthermore, the findings that neuron numbers were preserved in number, and that apparent myelin loss was not observed in the olfactory bulb or olfactory tract indicated the resistance of these regions to the progression of prion disease pathology. These findings might also indicate that the olfactory bulb and olfactory tract are a possible route of PrP infection in the sCJD cases from the outside of the brain.

P24‐10

Prevalent abnormal prion protein in human appendices after cattle BSE epizootic in the UK

O Noel Gill¹, Yvonne Spencer², Angela Richard‐Loendt³, Carole Kelly¹, Reza Dabaghian⁴, Lynnette Boyes³, Jacqueline Linehan⁵, Marion Simmons², Paul Webb², Peter Bellerby², Nick Andrews¹, David A Hilton⁶, James W Ironside⁷, Jon Beck⁵, Mark Poulter⁵, Simon Mead⁵ and Sebastian Brandner^3,8

¹ Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, United Kingdom; ² HIV & STI Department, and CJD Section, National Centre for Infectious Disease Surveillance and Control, Public Health England, 61 Colindale Avenue, London, NW9 5EQ, United Kingdom; ³ Pathology Unit Science Directorate, Animal Health and Veterinary Laboratories Agency, AHVLA Weybridge, New Haw, Addlestone, Surrey, KT15 3NB United Kingdom; ⁴ Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, WC1N 3BG United Kingdom; ⁵ Virus Reference Department, Public Health England, 61 Colindale Avenue, London, NW9 5HT United Kingdom; ⁶ MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square London, WC1N 3BG United Kingdom; ⁷ Department of Cellular and Anatomical Pathology, Derriford Hospital, Plymouth, PL6 8DH United Kingdom; ⁸ National Creutzfeldt‐Jakob Disease Research & Surveillance Unit, School of Molecular and Clinical MedicineUniversity of Edinburgh, Western General Infirmary Edinburgh, EH4 2XU United Kingdom

Background and objectives: Widespread dietary exposure of the population of the United Kingdom to bovine spongiform encephalopathy (BSE) prions in the late 1980s and early 1990s led to the emergence of variant Creutzfeldt‐Jakob Disease (vCJD). A distinct feature of this form of prion disease is the extensive involvement of the lymphoreticular system, which does not occur after transmission through contaminated human growth hormone or in kuru. Accumulation of vCJD prions in spleen, tonsils or lymphoid tissue of the gastrointestinal tract, precedes invasion of the central nervous system. Thus, tonsil biopsies proved a specific and sensitive preclinical test for the detection of vCJD prions in patients, and the prevalence of vCJD carrier status in the UK population was estimated through screening archival appendicectomy and tonsillectomy specimens. In view of differences between the findings in previous studies a further large scale survey of appendix tissue was undertaken.

Design: Designed as an irreversibly unlinked and anonymised study prior to commencing laboratory testing, paraffin blocks were collected from Pathology departments in the UK and tested using immunohistochemistry for the presence of abnormal prion protein.

Results: In 32441 formalin fixed paraffin embedded (FFPE) appendix samples, we found 16 positive specimens, indicating a prevalence of 370 per million population, with a 95% confidence interval of 191 to 646 per million. The prevalence in those born in 1941–60 (733 per million) was not significantly different from those born between 1961 and 1985 (412 per million), and it was similar in both genders and across the three broad geographic areas sampled. Genetic testing for the PRNP codon 129 genotype revealed a high proportion of 129VV compared to the frequency in the normal population, and in stark contrast to confirmed clinical cases of vCJD who have all carried the PRNP 129MM genotype.

Conclusions: This study corroborates previous studies and suggests a remarkably high prevalence of subclinical vCJD prion infection in the population compared with the 176 vCJD cases to date. These findings have important implications for the surveillance and control of blood and blood products and for the handling of surgical instruments.

P25. Stem Cells and Repair

P25‐01

Intracranial pancreatic islet transplantation attenuates behavioral dysfunction and promote brain islets hormones reserve in rats with traumatic brain injury combined with hypoglutamatergic injury

Gil‐Ad I¹, Tarasenko I¹, Taler M¹, Weizman A¹, Vanichkin A², Vardi P³ and Bloch K³

¹ Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University; ² Laboratory of Transplantation, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University; ³ Laboratory of Diabetes and Obesity Research, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Israel

Background: Traumatic brain injury (TBI) is a major cause of morbidity leading to cognitive and neuropsychiatric disorders. Pancreatic islet hormones were reported to reduce neuronal injury and improve cognitive functions. Brain insulin alleviates symptoms of Alzheimer's disease. MK801 is an NMDA receptor antagonist that induces hyperlocomotion and cognitive deficits.

Methods: TBI was produced by dura and arachnoid perforation. The perforation in the frontal skull bone was used also for islet transplantation onto the olfactory bulb and prefrontal cortex. Animals were tested following acute or sub‐acute administration of MK801 (0.15mg/kg ip/d) or vehicle. Rats were exposed to the open field (OF) or to the Morris Water Maze (MWM) tests. Islet hormone expression in the brain and peripheral glucose were assessed.

Results: Islets transplantation in TBI rats increased brain insulin and glucagon levels without affecting blood glucose. In the OF, TBI rats did not differ in motility from the controls. However, MK801 treated TBI rats exhibited hyper‐responsiveness in motility, and this effect was normalized by grafted islets. TBI animals explored more the center of the field, this effect was augmented by MK801 and reversed in the rats with grafted islets. The performance of the TBI rats in the MWM test was similar to the control rats. However, TBI rats treated with MK‐801, showed significant aggravation of the impaired spatial memory, and grafted islets partially corrected it.

Conclusion: Brain grafted islets in rats stimulate brain neurotrophic homeostasis, and attenuate neuropsychiatric and cognitive impairment in rats with the TBI combined with hypoglutamatergic insult.

P25‐02

Myelination in coculture of rodent stem cell‐derived neurons and Schwann cell lines

Watabe K¹, Ishii T^1,4, Yanagisawa H¹, Sango K¹, Akiyama K¹, Kawakami E¹, Endo K², Tsukamoto M¹, Niimi N¹, Akutsu H³ and Misawa H⁴

¹ ALS/Neuropathy Project, Tokyo Metropolitan Institute of Medical Science; ² Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science; ³ Dept. of Reproductive Biology, National Center for Child Health and Development; ⁴ Dept. of Pharmacology, Keio University Faculty of Pharmacy, Japan

We have previously established in vitro myelinating coculture systems using rat dorsal root ganglia neurons or rat pheochromocytoma PC12 cells with a spontaneously immortalized rat Schwann cell line IFRS1 (Sango K et al., 2011, 2012). In the present study we performed cocultures of rat neural stem cell (NSC)‐derived neurons or mouse ES cell‐derived motoneurons with IFRS1 Schwann cells to establish myelinating cultures between these cell lines. For preparation of a rat stable NSC line, adult rat brain stem tissues were dissociated and cultured in neurobasal medium containing B27, 10 ng/ml of FGF2 and 10 ng/ml of EGF, and growing neurospheres were serially passaged. To differentiate the cells into neurons, dissociated NSCs were maintained in F12 medium containing 100 nM of retinoic acid (RA). For preparation of mouse ES cell‐derived motoneurons, embryoid bodies derived from a mouse ES cell line NCH4.3 were differentiated in DFK5 medium containing 100 nM of RA and 100 nM of SAG. These NSC‐derived neurons or ES‐derived motoneurons were mixed with IFRS1 Schwann cells at 1:2‐4 of the cell density ratio, plated, and maintained in F12 medium containing B27, 50 ug/ml of ascorbic acid and 10 ng/ml GDNF. So far, myelin formation was demonstrated by electron microscopy at 4 weeks of coculture of rat NSC‐derived neurons and IFRS Schwann cells. The present coculture system utilizing the stable cell lines can be useful in studies of peripheral nerve degeneration and regeneration.

P26. Systems and Pathology

P26‐01

Intracranial hypertension syndrome and multiple cranial neuropathy related to atypical radiologic presentation of an intradural chordoma

Oliveira NP^¹, Canedo NHS^², Esperança JCP^¹, Marcondes J^² and Vasconcellos LFR^²

¹Instituto de neurologia Deolindo Couto/ UFRJ; ²Hospital Universitário Clementino Fraga Filho/ UFRJ, Brazil

Introduction: We report the unusual case of an intradural chordoma with atypical clinical and radiologic presentation.

Case Report: 36 year‐old male, report in 2011 the beginning of bilateral hearing loss associated with vertigo and followed by bilateral hyposmia, decreased visual acuity in the left eye and trigeminal autonomic headache for 5 months. The physical examination showed bilateral hyposmia, Foster–Kennedy syndrome in the right eye, hypoestesia of the left hemiface and bilateral deafness. Laboratory exams showed cerebrospinal fluid with one cell, hyperproteinorachia (837 mg/dL), 210 erythrocytes/mL, glucose 60 mg/dL; VDRL, cryptococcal latex, culture for common germs, tuberculosis and cysticercosis all negative. Serology for histoplasmosis and paracoccidioidomycosis were also negative. MRI scan showed thickening of the meninges and multiple round lesions less than 1 cm each, hyperintense in T2 and FLAIR, well circumscribed, under the dura mater and around the cerebellum, intraparenchymal in some areas, without gadolinium enhancement. Biopsy of the lesion showed physaliphorous cells embedded in mucin matrix, suggesting an intradural chordoma. Patients’ follow‐up showed no improvement of the neurological symptoms but the patient decided not to go under any new surgeries. A few months after the surgery, the patient died of the disease.

Discussion/Conclusion: Intradural chordoma is a rare entity, with still controversial treatment regimen. This case report shows this rare entity with an even rarer radiologic presentation, posing a tricky differential diagnosis and serves as an alert for the issue.

P26‐02

UK Brain Archive Information Network (BRAIN UK)

Nicoll J

University of Southampton, Southampton, UK

Introduction: The neuropathology archives of the UK National Health Service contain a wealth of tissue from a great variety of neurological conditions. Built up over many years, they include common conditions such as stroke, head injury, psychiatric, developmental disorders and many rare disorders and represent an under‐utilized resource for research. BRAIN UK is a collaborative virtual brain bank, backed by the British Neuropathological Society, facilitating access to Neuropathology archives for research.

Methods: 19 regional neuropathology services and the Corsellis Collection currently participate in BRAIN UK. A centralized linked‐anonymized database of >60,000 cases includes diagnosis, lab number, simple demographic data (e.g. gender and age at death), location and custodian. There is a centralized application process www.brain‐uk.org. BRAIN UK acts as a “matchmaker” with ethical approval covering the majority of projects. Investigators’ grants fund the costs associated with the retrieval, processing and transportation of tissue.

Results: 27 applications have been supported so far (2011–2013) and tissue provided for 22 projects. Studies have encompassed a wide variety of conditions including neuromyelitis optica, neuroinflammation, stem cells, schizophrenia, Alper's disease, Huntington's disease, subarachnoid hemorrhage, affective disorders, sudden unexpected death in epilepsy, mitochondrial DNA mutations and tumors.

Conclusion: Archived tissue that would otherwise be unused has supported research projects. The scale and scope of the tissue resource is unprecedented. BRAIN UK is completely reliant on the goodwill of its participating centers. BRAIN UK represents excellent value for money, but finding someone's time to identify suitable cases can be a challenge. With encouragement from the brain tumor research community and funding from the brain tumor charity brains trust we are extending BRAIN UK to incorporate archived biopsies (400,000 approx.), which will provide an unprecedented resource for brain tumor research.

P27. Trauma

P27‐01

Chronic traumatic encephalopathy and amyotrophic lateral sclerosis

Bieniek KF^1,2, Stein TD^3,4,5,6, Alvarez VE⁵, Fry BT⁵, Dickson DW¹ and McKee AC^3,4,5,6

¹ Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA; ² Mayo Graduate School, Mayo Clinic, Rochester, MN, USA; ³ VA Boston Healthcare System, Boston, MA, USA; ⁴ Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston MA; ⁵ Department of Neurology, Boston University School of Medicine, Boston, MA; ⁶ Alzheimer Disease Center, Boston University School of Medicine, Boston, MA, USA

Chronic traumatic encephalopathy (CTE) is a progressive tauopathy resulting from repetitive traumatic brain injury (TBI). CTE pathology can be found concomitantly with other neurodegenerative pathologies, including amyotrophic lateral sclerosis (ALS). To assess the frequency of CTE pathology in ALS, we screened 88 pathologically‐confirmed ALS cases in the Mayo Clinic Florida brain bank with tau immunohistochemistry. Six male cases had pathologic features of ALS plus CTE (6.8%). A screen of 113 ALS cases from the Boston VA ALS brain bank found 5 cases of ALS+CTE (4.4%). Clinicopathologic comparison was performed with 12 additional cases with ALS+CTE from the Boston University (BU) CTE brain bank. The BU cohort included 8 American football players (3 NFL, 4 college, 1 high school), 3 professional boxers, and 1 soccer player. All 23 cases of ALS+CTE had marked neuronal cell loss of the anterior horn cells, and neuronal cytoplasmic inclusions immunoreactive for TDP‐43 in residual neurons as well as pathologic features of CTE, including prominent focal hyperphosphorylated tau perivascularly and at the depths of cerebral sulci. Of the 23 cases, 3 cases had very mild CTE pathology (Stage I), 11 cases had mild pathology (Stage II), 6 cases had moderate pathology (Stage III), and 3 cases had severe pathology (Stage IV). Cognitive and behavioral abnormalities, and history of TBI were not well‐documented in the cases screened from ALS brain banks. In summary, the comorbidity of these two neurodegenerative diseases raises the possibility of a clinicopathologic relationship and shared pathogenesis.

P27‐02

CTE in boxers: modern findings in classic cases

Gentleman S¹, Liu A¹, Cole P², Carvalho J² and Dhillon M¹

¹ Neuropathology Unit, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK; ² Corsellis Collection, West London Mental Health NHS Trust, Southall, UK

Chronic traumatic encephalopathy (CTE) is a tauopathy associated with a history of repetitive mild traumatic brain injury. Although initially described in boxers as ‘dementia pugilistica’ (DP), the term CTE is now used to cover other sporting and non‐sporting activities associated with repetitive brain injury. Although generally considered to be the same syndrome, some confusion exists over the use of the two terms, and it has recently been suggested that classic DP is a different disease entity to CTE, with the main distinguishing feature being the presence of astrocytic tangles in the latter. The aim of this study was to determine the extent and distribution of pathology in a seminal series of DP cases (Corsellis et al. Psychol Med 1973; 3: 270–303). Tissue sections from the brains of 22 boxers from the Corsellis archive were immunostained for tau, Aβ and alpha‐synuclein. Sixteen (73%) were found to exhibit tau pathology consistent with CTE. Tau pathology consisted of both neurofibrillary and astrocytic tangles, preferentially located focally at sulcal depths, and in perivascular and periventricular locations. The majority of cases also showed some diffuse Aβ plaques. Based on these findings there appears to be little or no difference in the tau pathology seen in classic DP and modern CTE cases.

P27‐03

Damage to histaminergic tuberomammillary neurons and other hypothalamic nuclei with traumatic brain injury

Reddy H¹, Yamamoto M¹, Finn K¹, Scammell T¹, Valko P², Gavrilov Y³, Haybaeck J⁴ and Weiss S⁵

¹ Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA; ² Department of Neurology, University Hospital Zurich, Switzerland; ³ Department of General Pathology and Pathological Physiology, Institute of Experimental Medicine, St. Petersburg, Russia; ⁴ Department of Neuropathology, Institute of Pathology, Medical University of Graz, Austria; ⁵ Department of Neuropathology, State Neuropsychiatric Hospital Wagner‐Jauregg, Linz, Austria

The need for increased sleep after traumatic brain injury is a common and disabling complaint, yet its etiology is unknown. Previous studies have demonstrated diffuse damage to various hypothalamic systems, but the integrity of the histaminergic tuberomammillary nucleus, a major arousal‐promoting system located in the posterior hypothalamus, has never been examined in head trauma patients. In post‐mortem examination of 16 patients with traumatic brain injury and 12 controls, we found a range of neuropathology in TBI patients from acute hemorrhage to infarction, rarefaction of neuropil, gliosis, and axonal swellings. We also demonstrate that severe head trauma is associated with a marked loss (41%) of histaminergic neurons. Reduced histamine signaling may contribute to increased sleep need, and therapies that enhance histaminergic tone may improve arousal after head trauma or other conditions.

P28. Tumors

P28‐01

Epithelioid (adenoid) glioblastoma: a rare histological variant

Salazar Morales MF, López García NL, Escalante Abril PA, Madrazo Moya M, Blancas García E, Velasco Vales M, Gómez Apo E and Chávez Macías LG

Hospital General de México “Dr. Eduardo Liceaga,” Mexico

Glioblastoma stands for the most common primary brain tumor in adult patients above forty years old. As its former designation «multiform» implies, it has a plethora of morphological variations including giant cell, small cell, rhabdoid, granular, myxoid, with primitive neuroectodermal component, etc. The epithelioid–adenoid–glioblastoma is an uncommon variant (1.6%) which can mimic metastatic carcinoma.

We present the case of a 55‐year‐old woman who started with severe cephalea irradiating to the whole head in association with nausea and ataxia. Imaging scans showed a right parieto‐occipital, well circumscribed tumor with perilesional edema. Craniotomy and tumor resection was performed.

Microscopically the tumor showed anaplastic astrocytes, necrosis, endothelial proliferation and cells arranged in nests with a central lumen structuring a glandular pattern. Immunohistochemical reactions revealed GFAP‐positive astrocytes as well as vimentin, CKAE1/AE3 and GFAP‐positive cells in the glandular formations.

Prone to adopt different morphologies, histopathological analysis of glioblastoma with unusual features must rule out a metastatic neoplasm when observing epithelioid qualities, such as those exemplified by this case.

P28‐02

Giant cell ependymoma of the lumbar spinal cord

Salazar Morales MF¹, Candanedo González F², Rosales S² and Ortiz CS²

¹ Hospital General de México “Dr. Eduardo Liceaga”; ² Centro Médico Nacional Siglo XXI, Mexico

The giant cell type is a rare and recently recognized histological variant of ependymoma which, despite featuring large pleomorphic cells, carries a good prognosis. Up to date, nearly 20 case reports have been written worldwide. We present the case of a 48‐year‐old woman with non‐relevant past medical history who began six months previously with sharp pain involving the lumbar region and consecutive gait impairment. Magnetic resonance imaging detailed an intra‐axial bulky lesion eroding from the second to the fifth lumbar vertebrae, so the tumor was considered not to be amenable to surgical resection and only excisional biopsy was performed with an ensuing diagnosis of poorly differentiated neoplasm. Reappraisal of the patient's slides disclosed non‐cohesive bizarre giant cells with broad eosinophilic cytoplasm and intranuclear cytoplasmic inclusions. Immunohistochemical analysis unveiled positivity for glial fibrillary acidic protein (GFAP), S100 protein, CD99 and CD56 in these cells without expression of synaptophysin, chromogranin or neuron‐specific enolase. The patient was subsequently treated with 54Gy radiotherapy ameliorating her symptoms. Uncommon and with a striking appearance, the differential diagnosis of giant cell ependymoma includes anaplastic oligodendroglioma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma and giant cell glioblastoma. Moreover, as the rest of the reported cases were treated using a surgical approach, our case seems to be the only one receiving radiotherapy without affecting the prognosis.

P28‐03

Gliofibroma: an uncommon bimorphic neoplasm

Salazar Morales MF, Escalante Abril PA, López García NL, Madrazo Moya M, Zamora Guerra YU, Gómez Apo E and Chávez Macías LG

Hospital General de México “Dr. Eduardo Liceaga,” Mexico

We inform about the case of a 60‐year‐old woman with non‐relevant past clinical data who consulted for cephalea and loss of memory. Magnetic resonance imaging showed a left parieto‐temporal mass displaying an annular enhancement after contrast media administration, therefore concluding the diagnosis of a high‐grade astrocytic neoplasm. Despite tumor resection was performed, the patient ultimately died.

Histologically the lesion was constituted by areas corresponding to glioblastoma – hypercellularity, endothelial proliferation, high mitotic activity and necrosis – mixed with a benign mesenchymal component featuring long spindle cell fascicles without cytological atypia surrounded by collagen and reticulin fibers.

Gliofibroma is a rare neoplasm with approximately 30 cases reported up to date. As shown in our case, its prognosis is apparently determined by the degree of anaplasia of the glial component.

P28‐04

A case of histiocytic sarcoma primary of the central nervous system

Cavalcante JM, De Lima FEMM and Nogueira CD

Laboratório Argos, Brazil

Introduction: Histiocytic sarcoma (HS) is a rare lymphohematopoietic malignant tumor which accounts for less than 1% of non‐Hodgkin's lymphoma. Primary central nervous system (CNS) HS is rare, and there have been difficulties in identifying the histiocytic derivation. We present a patient with primary presentation in the meninges, without any other organ involvement.

Case Report: A 58‐year‐old male was admitted to the hospital for headache. Brain MRI revealed a solid, extra‐axial and supratentorial mass measuring 5 cm in the right parieto‐occipital region. Histopathological examination showed a highly proliferative malignant tumor composed of atypical, large, multinucleated epithelioid cells with eccentrically situated irregular, lobulated nuclei, some with prominent nucleoli, and abundant pale cytoplasm. There were some aggregates of foam cells, a lot of them with cytoplasm filled with hemosiderin. Areas of necrosis and some spindling were also present. The tumor cells were infiltrative in adjacent cerebral parenchyma. Immunohistochemical staining showed that the neoplastic cells were positive for CD45, CD68, alpha‐1 antitrypsin, vimentin and CD163, but negative for CD1a, CD3, CD5, CD23, CD20, CD79a, CD138, CD30, ALK‐1, CD34, Myeloperoxidase, AE1‐AE3, EMA, HMB 45, Melan‐A, S‐100 and GFAP.

Discussion: CNS presentations of HS are exceptionally rare. Light microscopy alone is insufficient and unreliable in diagnosis. The histologic features of the neoplasia raises the possibility of a wide range of malignant tumors and wide panel of immunostains is recommended. CD163 is the most specific marker for HS and have a significant diagnostic utility in separating specific tumors with macrophage/histiocytic deviation from other entities.

P28‐05

A study of proliferation and apoptosis related markers ATRX, Bcl‐2 and p53 in ependymomas

Sharma MC, Nambirajan A, Singh M and Sarkar C

India

Background: Ependymomas are relatively rare gliomas, whose pathogenesis is not well elucidated. They show site‐specific differences in biological behavior; prognostic value of WHO grading criteria may vary with tumor location. ATRX (Alpha thalassemia mental retardation X‐linked protein) is an epigenetic modifier, whose mutation is a hallmark of alternate lengthening of telomeres (ALT). ATRX mutation, seen in diffuse astrocytomas and oligodendrogliomas, is associated with p53 and IDH mutation and better prognosis; however, its role in pathogenesis of ependymomas has not been studied. p53 and Bcl‐2 immunopositivity are associated with poor prognosis.

Methods: Immunohistochemistry for ATRX, Bcl‐2 and p53 was performed on 126 cases of ependymomas of different grades and location. Results were correlated with clinicopathological parameters and patient outcome.

Results: ATRX loss was found in 29% of ependymomas, more frequently in Grade II and infratentorial cases and did not show any prognostic value. p53 positivity was more frequent in intracranial, recurrent, Grade III tumors and was associated with a poor outcome. Bcl‐2 expression was significantly frequent in Grade II spinal tumors and associated with a poorer prognosis. ATRX loss was distinctly uncommon in the supratentorial tumors and spinal tumors that showed p53 and Bcl‐2 positivity.

Conclusions: ATRX loss is observed in ependymomas, mainly in infratentorial tumors and was mutually exclusive from p53 and Bcl‐2 expression, indicating that ALT may play a role in infratentorial ependymoma pathogenesis, unlike in those at other sites. Supratentorial tumors showed high frequency of p53 positivity, poor outcome, and rarity of ATRX loss or Bcl‐2 expression.

P28‐06

ALK protein expression by standard immunohistochemistry in glioblastoma

Zunarelli E, Mengoli MC, Mataca E and Nosseir S

Division of Surgical Pathology, University Hospital Policlinico di Modena, Modena, Italy

Introduction: The EML4‐anaplastic lymphoma kinase (ALK) translocation is a recognized oncogenic driver in anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, non–small cell lung cancer, and neuroblastoma. Multiple preclinical studies demonstrated that specific small molecule ALK tyrosine kinase inhibitors can delay tumor growth and/or induce tumor regression in xenograft and transgenic models. Based on such promising preclinical studies, ALK inhibitors have recently entered into clinical trials.

The present study aimed at detecting ALK immunohistochemical expression in glioblastomas, in order to verify whether it could be routinely applicable in clinical practice to detect glioblastoma patients for clinical studies of ALK inhibitors.

Materials and Methods: Screening for ALK immunohistochemical detection was performed on 87 cases of formalin‐fixed, paraffin embedded surgically removed conventional glioblastomas using a standard antibody recognizing the ALK protein (Clone ALK01, Ventana, Tucson, AZ).

Immunostained tissue sections were assessed for the level of tumor‐specific ALK expression in the nucleus, cytoplasm and cell membrane of glioblastoma cells.

Results: No immunostain for ALK was detected in this series of cases, even when the titers of ALK antibody in our IHC reactions were increased to maximize the sensitivity without sacrificing the specificity of the assay. Furthermore, we did not find that alternative antigen retrieval methods improved the sensitivity of the assay.

Conclusions: Our findings suggest that the routine immunohistochemical screening for ALK expression by standard methods has no value for ALK detection and screening of glioblastoma patients who could eventually benefit of ALK‐specific therapies.

P28‐07

An algorithmic approach to sellar region masses

Kleinschmidt‐DeMasters BK^1,2,3, Lopes MBS^4,5 and Prayson RA⁶

Departments of ¹ Pathology, ² Neurology, and ³ Neurosurgery at the Anschutz Medical Campus, University of Colorado School of Medicine, Aurora, CO, USA; Department of ⁴Pathology and ⁵Neurosurgery, University of Virginia School of Medicine, Charlottesville, VA, USA; Department of ⁶Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, OH, USA

Context: Most sellar region masses (85–90%) are pituitary adenomas; however, other neoplasms or even inflammatory or cystic non‐neoplastic lesions may occasionally be encountered in this location. A practical, non‐electron microscopically‐based approach is essential for the daily practice of diagnosing and subclassifying adenomatous and non‐adenomatous sellar region lesions.

Objective: To provide an algorithmic approach to sellar region masses for the pathologist and to formulate a cost‐effective, limited panel of stains and immunostains that can be used in daily practice at most small‐ to medium‐sized centers.

Design: Pool collective experience of three neuropathologists practicing at academic medical centers with expertise in diagnosis and treatment of sellar region masses to craft a single page algorithmic diagram and to liberally illustrate the range of lesions present in the sellar region.

Results: After formulating a differential diagnosis, the general pathologist can generate a confident final diagnosis of adenoma using 1 histochemical (reticulin) and 1 immunohistochemical (synaptophysin) stain, supplemented by 5 immunohistochemical stains [CAM5.2, follicle stimulating hormone (FSH), growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)] which provide subtyping of the adenoma in the overwhelming majority of examples. CAM5.2 and clinical information further help identify clinically‐aggressive variants such as sparsely granulated GH adenomas and silent ACTH adenomas, respectively. MIB‐1, thyroid transcription factor‐1 (TTF‐1) and S‐100 protein can be of further assistance in select cases where increased mitotic activity or possible non‐adenomatous, spindle cell lesions are suspected.

Conclusions: Adenomas, normal anterior or posterior gland, and non‐adenomatous masses can be easily diagnosed in a non‐tertiary pathology laboratory setting.

P28‐08

Analysis of the frequency of mutations and polymorphisms in BRIP1 gene in meningiomas

Zugaib R

UNESP, Brazil

Constituting 20% of all intracranial tumors, the meningiomas are derived from arachnoid cells, being most frequent in women. We observed in previous studies, related to the methylation profile in BRCA1 gene in these tumors, that all male cases were hypermethylated, showing the importance of this gene in oncogenesis. Based in these results, was aroused the interesting for other genes that interacting with BRCA1. BRIP1 is a tumoral suppressor gene that encodes a helicase, essential for the chromosomal maintenance by interacting with BRCA1. Mutations in this gene were evaluated in other types of cancer with several conclusions. In meningiomas, mutations and polymorphisms in BRIP1 are still unknown. Thus, the aim of this study was to sequence the exon 19 of BRIP1 gene and to determine the frequency of rs4986764 polymorphism in patients with meningioma. Samples of 61 patients with meningiomas were collected, operated in the HC/FMB – UNESP, and 19 control samples, from necropsies, performed by the Pathology of FMB – UNESP, from patients whose deaths were not related to any type of cancer. The samples were analyzed by the automatic sequencing of BRIP1 exon 19. The results showed that 59% of the patients presented the variant allele (T), being 8.2% homozygotes (TT) and 50.8% heterozygotes (CT). In control samples, 47.3% presented the polymorphism, being 10.5% homozygotes (TT) and 36.8% heterozygotes (CT). The present study seems to suggest that the heterozygosity (CT), somehow, contributes for the meningothelial oncogenesis. However, these data should be interpreted with caution, for be a small casuistry.

Support: CNPq.

P28‐09

Atypical teratoid rhabdoid tumor, an immunohistochemical study of potential prognostic markers

Al‐Hussaini M¹, Dissi N¹, Souki C¹ and Amayiri N²

¹ Department of Pathology and Laboratory Medicine, King Hussein Cancer Center, Amman, Jordan; ² Department of Paediatrics, King Hussein Cancer Center, Amman, Jordan

Atypical teratoid/rhabdoid tumor is a rare highly malignant tumor of the central nervous system mostly seen in infants associated with dismal outcome in most instances. The diagnosis nowadays relies mainly on the negative INI‐1/BAF47 immunostain. We aim at investigating a number of immunohistochemical antibodies looking for potential prognostic markers. Nineteen cases were identified a tour center over a 10‐year period including 10 males, with a median age of 24 months. Ten (52.6%) cases were supratentorial. Six (42.9%) showed metastasis at time of presentation. Chemotherapy was administered for 10 (62.5%), while radiotherapy for 7 (43.8%). The median overall survival was 9.5 months. A single long term survival of 104 months is identified.

Pathologically, most cases showed admixture of rhabdoid cells and/or small cells and/or pale cells. Of all tested immunostains, FLi‐1 positivity correlated with long survival (p = 0.0012), while cyclin‐D1 showed a tendency towards prolonged survival (p = 0.056). SALL4, OCT3/4, CD99, beta‐catenin andP16 didn't correlate with outcome. CDX2 was positive only in the single long term survival. Fli‐1 and cyclin‐D1 are potential prognostic markers associated with better outcome.

P28‐10

Clinical relevance of molecular stratification of anaplastic grade III gliomas – Indian study

Santosh V, Rajmohan KS, Arivazhagan A and Pandey P

National Institute of Mental Health and Neurosciences, India

Background: The histopathological diagnosis of diffuse anaplastic gliomas; WHO grade III [AG] that includes anaplastic astrocytoma, AA; Anaplastic oligidendroglioma, AO; anaplastic oligoastrocytoma, AOA, often lacks the precision that is needed for tailoring treatment. Assessment of molecular markers will probably allow more robust and prognostically relevant classification. Such data is not available from India.

Aim: To assess the prognostic significance of 1p/19q co‐deletion, MGMT promoter methylation, IDH1 status and expression of Alpha‐Internexin (INA) in AG patients and correlate with clinical profile, overall and progression free survival (OS,PFS).

Material and Methods: Molecular aberrations was investigated by fluorescent in situ hybridization, methylation specific PCR, and immunohistochemistry in a series of 91 AGs (AA‐18,AO‐40,AOA‐33), and correlated with clinical, treatment and follow up data.

Results: Whilst histopathological subgroups of AGs (AA, AO, AOA) did not show significant association with survival (p = 0.841), molecular stratification of the whole group of AGs yielded significant results. 1p/19q co‐deletion (p < 0.001), MGMT methylation (p < 0.001), and INA expression (p < 0.001), were strong independent prognostic factors for both OS/RFS in AG patients, preferably in those treated with radiochemotherapy. However, IDH‐1 expression did not have influence on OS (p = 0.700) and RFS (p = 0.796). Multivariate analyses confirmed the prognostic value of these markers. Interestingly, cases with 1p and/or 19q polysomy had the worst prognosis. INA, though an independent prognostic marker, did not serve as a surrogate marker for AOs with 1p19q co‐deletion.

Conclusion: Molecular stratification of AGs scores over traditional histology and adds value in providing a more robust prognostic classification, helpful in guiding adjuvant therapy. This could form the basis of ‘biomarker based neuropathology report’ for AGs in future.

P28‐11

Clinicopathologic features of pineal parenchymal tumors in a single institutional study

Sasaki A¹, Ishizawa K¹, Shimizu M¹, Nishikawa R¹ and Honma T²

¹ Saitama Medical University; ² Nihon University, Japan

Introduction: Pineal parenchymal tumors (PPTs) are very rare tumors that are divided into pineocytomas (PCs), pineal parenchymal tumors of intermediate differentiation (PPTIDs), and pineoblastomas (PBs). Pathologists still encounter diagnostic difficulties with these, particularly for PPTIDs, at biopsy, since only a tiny piece of tissue is often available, and many PPTs are not readily classified. The aim of our study was to provide further information regarding the clinical features, pathologic findings, and response to therapy of these tumors.

Patients and Methods: This study included data on 12 patients (6 males and 6 females) who were pathologically diagnosed with PPTs and treated at Saitama Medical University International Medical Center between 2007 and 2013. Follow‐up information was available for all 12 patients, ranging from 7 to 69 months.

Results: There were 2 PCs (47 and 48 years), 4 PPTIDs (range 35–45 years), and 6 PBs (range 0–11 years). The mean MIB‐1 labeling index (LI) in PBs was significantly higher than those in the other types of PPT. Among the 4 PPTID patients, who received subtotal tumor resection or biopsy plus postoperative radiation, two progressed with recurrence and two are disease‐free. There was no clear correlation between MIB‐LI score and the presence of recurrence in PPTIDs. The 5‐year survival rates of patients with PCs, PPTIDs, and PBs were 100% (2/2), 33% (1/3), and 60% (3/5), respectively.

Conclusion: Our single institutional study indicates that MIB‐1 LI may be useful for the diagnosis of PPTs, and that MIB‐LI scoring might be difficult in assessment of the prognosis of PPTID patients.

P28‐12

Comorbidity with CNS‐PNET and diabetes in Arabian gulf population

Al Homssi MF and Mohamad A

College of Medicine, University of Sharjah, Sharjah, United Arab Emirates

More than two hundred (209) cases of central nervous system primitive neuroectodermal tumors (CNS‐PNET) are identified in our pathology archives over a period of sixteen years. Files of these patients are reviewed to check co‐morbidity with diabetes mellitus (DM).

Three subtypes of CNS‐PNET with specific cytogenetic findings are usually identified. Both karyotypic analysis and the fluorescence in situ hybridization (FISH) procedure on paraffin‐embedded tissues are used looking for the presence of i(17q), and deletions of 22. Medullocytoma is characterized by areas of “lipomatous differentiation,” low proliferative potential, and manifestation in adults. Atypical teratoid/rhabdoid tumor (ATT/RT) is characterized by the presence of rhabdoid cell differentiation and triad immunohistochemical analysis of epithelial membrane antigen (EMA), vimentin, and smooth muscle actin (SMA). Cytogenetic studies show that the most frequent cytogenetic abnormality in medulloblastomas is isochromosome 17q, and in ATT/RT is deletion in chromosome 22. At the onset of the symptoms of CNS‐PNET fourteen patients had type 1 DM, two patients had maturity onset diabetes of the young (MODY), and one patient had type 2 DM. The overall incidence of DM in this group of patients is (17/209) 8.1%.

Although this incidence may appear higher than expected, the prevalence throughout the later years may become much higher as the incidence and prevalence rates of DM in the Arabian Gulf population is in excess of 23–27%. This behooves us to conduct a collaborative study with other Arabian Gulf Countries (GCC) and other Middle Eastern Countries at large.

P28‐13

Comparative analysis of frequency of rs12979860 polymorphism in IL28B gene in meningiomas and gliomas

Galvani AF¹, Zugaib R¹, Del Vescovo AA¹, Ferrasi AC¹, de Moura Campos Pardini MI¹, Rabenhorst SHB² and Faria MHG³

¹ Transfusion Blood Center, UNESP – Sao Paulo State University, Botucatu‐SP, Molecular Biology Laboratory; ² Department of Pathology and Forensic Medicine, Molecular Genetics Laboratory (LABGEM), School of Medicine, Federal University of Ceara, Fortaleza, Brazil; ³ Dr. Mário Gatti Hospital, Department of Neurosurgery, Campinas‐SP, Brazil

The Interleukin 28B (IL28B), member of IFN type III family, seems to be involved in anti‐viral and anti‐tumor immune response. Recently was discovered that rs12979860 polymorphism in IL28B gene predicts the efficiency of treatment with pegylated IFN‐A in association with ribavirin, against hepatitis C virus. Individuals with CC genotype presented higher chances of viral clearance when compared with those who have CT and TT genotype, however, literature lacks of new studies about the importance of these polymorphism in carcinogenesis. The aim of this study was compared the allelic and genotypic frequency of rs12979860 polymorphism in IL28B gene in patients affected by meningiomas and gliomas. Casuistry was composed by 95 and 56 patients affected by meningiomas and gliomas, respectively, which were done germinative analysis of these polymorphism. Polymerase chain reactions (PCR) were performed to amplify polymorphism region and after, a restriction fragment length polymorphism (RFLP) was done with BSTU 1 enzyme, and revealed on polyacrylamide gel. In analyzed patients, C allele was more frequent in both types of tumors, being 62,1% in meningiomas and 62,5% in gliomas. Also, a high frequency of CT genotype was observed in both types of tumors, 46,3% and 50% in meningiomas and gliomas respectively. Another association between genotype and degrees of both types of tumors were analyzed and no statistics significance was observed. Considering the importance of IL28B gene in immune response to virus and tumors, the data support the need of analysis in other regions of this gene.

Support: FAPESP.

P28‐14

Diagnostic value of plasma and urinary 2‐hydroxyglutarate to identify patients with IDH‐mutated glioma

Gardiman MP¹, Pizzi M¹, Lombardi G², Zagonel V², Corona G³, Bertorelle R⁴ and Della Puppa A⁵

¹ General Pathology and Cytopathology Unit, Department of Medicine‐DIMED Padua University Hospital, Padua, Italy; ² Medical Oncology 1, Veneto Institute of Oncology‐IRCCS, Padua, Italy; ³ Experimental and Clinical Pharmacology, National Cancer Institute, Aviano, Italy; ⁴ Molecular Immunology and Oncology, Veneto Institute of Oncology, Padua, Italy; ⁵ Department of Neurosurgery, Padua University Hospital, Padua, Italy

Background: We analyzed 2Hydroxyglutarate (2HG) concentration in plasma and urine in patients (PTS) affected by glioma to identify a biomarker of IDH1 gene mutation.

Methods: Plasma and urine samples were taken from all PTS and 2HG concentrations determined by liquid chromatography tandem mass spectrometry; Mann–Whitney test was used to test the differences in metabolite concentrations. ROC curve was used to evaluate the cut off value of 2HG biomarker.

Results: 84 PTS were enrolled: 38 with mutated IDH1 and 46 with wild‐type IDH1. Among PTS with mutant IDH1 we had 21 high‐grade gliomas (HGG) and 17 low‐grade gliomas (LGG); among PTS with IDH1 wild‐type we had 35 HGG and 11 LGG. In all PTS we analyzed the mean 2HG concentration in plasma (P_2HG), in urine (U_2HG) and the ratio between P_2HG and U_2HG (R_2HG). We found a significant difference in R_2HG between PTS with and without IDH1 mutation. The optimal cut‐off value of R_2HG to identify glioma PTS with and without IDH1 mutation was 19 with sensitivity (S) 63%, specificity (SP) 76% and accuracy (A) 70%; only in PTS with HGG the optimal cut‐off value was 20 (S 76%, SP 89%, A 84%, PPV 80%, NPV 86%). No association between the grade or size of tumor and R_2HG were found. In 7 out of 7 HGG PTS we found a correlation between R_2HG value and response to treatment.

Conclusions: By analyzing the R_2HG derived from individual plasma and urine 2HG levels it is possible to discriminate glioma PTS with/without IDH1 mutation.

P28‐15

Differential gene expression in meningiomas by microarray and real time PCR with and without 22q deletion

Renzi Jr I, Morato F, Panepucci RA, Neder L, da Cunha Tirapelli DP and Carlotti Jr CG

School of Medicine of Ribeirão Preto, University of São Paulo, Brazil

Introduction: Meningiomas are intracranial tumors originated from the arachnoid cap cells, with an annual incidence of 6 per 100,000 people. They are most commonly seen in female adults and after the fifth decade of life. The most common genetic event found in 60% of the cases is the chromosome 22q deletion, but little is known about the other genetic events that are important on the molecular pathology and drive the malignant progression in meningiomas. To better understanding the landscape of molecular alterations in pathogenesis of meningiomas we perform a systematic study of expression of miRNAs and mRNAs using microarray‐based expression profiling platforms in a series of 90 grade meningiomas.

Patients and Methods: For this study we used 90 samples of bonafide grade I meningiomas, which are categorized according the 22q del. Arachnoid samples obtained from patients submitted to surgery for aneurysm were used as controls. The status of chromosome 22 was addressed by FISH assays, followed by global analysis of gene expression using microarrays and subsequent validation by real‐time PCR.

Results: The genes ACVRIC, CCL18 and VGLL3 were downregulated in meningiomas regardless the 22q status. The gene OGDHL was upregulated only in meningiomas without 22q del and the BTC gene was differentially expressed only in meningiomas with 22q deletion. We have also identified two microRNAs hypo expressed in meningiomas irrespective of 22q status, miR‐1 and miR‐133b that are related to cell cycle arrest, migration and invasion.

Support: FAPESP.

P28‐16

Discrepancies in intraoperative diagnosis of central nervous system lesions: analysis of 1018 cases

da Fonseca TC¹, do Nascimento MF¹, Eisenberg ALA¹, Maia CB¹ and de Oliveira JA²

¹ Department of Pathology, National Cancer Institute, Rio de Janeiro, Brazil; ² Department of Neurosurgery, National Cancer Institute, Rio de Janeiro, Brazil

Introduction: Intraoperative consultation (IC) for neurosurgical specimens provides an accurate preliminary diagnosis, assessment of tissue adequacy and facilitates screening for supplementary studies. IC can be difficult and several factors influence the accuracy of this procedure. Certain lesions are recognized to cause diagnostic challenges at IC.

Objective: This study aims to determine the IC diagnostic discrepancies in neurosurgical specimens and to detect the diagnostic difficulties in neuropathology at our Institution.

Material and Methods: We retrospectively reviewed the cases of IC on neurosurgical specimens from January 2000 until December 2005. The IC diagnoses were compared with the final diagnoses, and the discrepancies were identified as: confusing non‐neoplastic with neoplastic processes, misclassifying two different neoplastic conditions, and overgrading tumors.

Results: Of the 1018 cases identified, 105 discrepant diagnoses were found. Between these 105 cases 17 (13%) involved overgrading neoplasias and 88 (84%) involved histopathological diagnosis discrepancies. Among the histopathological diagnosis, the most frequent discrepancies were confusing absence of neoplasia with primary neoplastic lesions and metastases (37%) and, metastases with primary neoplasia (16%). In the group of discrepancies involving overgrading, four fibrillary astrocytoma, one oligoastrocytoma and one pilocytic astrocytoma, had a IC diagnosis of high‐grade glioma and, five pilocytic astrocytomas had a IC diagnosis of fibrillary astrocytoma.

Conclusions: IC in neurosurgical specimens can be highly accurate. The review of discrepant diagnosis in IC with the detection of diagnostic errors can improve the accuracy of this diagnostic and the knowledge of possible errors could minimize misinterpretation and improve our diagnostic accuracy.

Keywords: intraoperative consultation, neurosurgery, discrepancy, diagnosis.

P28‐17

Dysplastic cerebellar gangliocytoma (Lhermitte–Duclos disease)

Prados SR¹, Gómez A¹, Fajre L² and Campero A³

¹ Dept. of Pathology; ² Dept. of Radiology; ³ Dept. of Neurology, University of Tucuman, School of Medicine, Argentina

Dysplastic cerebellar gangliocytoma (DCG) (Lhermitte–Duclos disease) is a rare disorder characterized by a slowly progressive unilateral tumor. It remains unclear whether this lesion is neoplastic or hamartomatous. The association of dysplastic cerebelar gangliocytoma with multiple hamartoma–neoplasia complex (Cowden syndrome) (CD) has been recognized more than two decades ago. Approximately 250 cases of DGC have been reported, 20% of which were associated with CD. We report another case of this rare entity in a 40 year‐old man who presented with hand trembling of two months duration. MRI showed a cerebelar mass with a striped appearance. Suboccipital craniotomy and complete removal of the infratentorial tumor was performed. Histopathological examination revealed enlarged and distorted cerebellar folia with the molecular and internal granular layer showing proliferated dysplastic ganglion cells of varying sizes. The dysplastic ganglion cells were positive for synaptophysin. The patient underwent complete examination without evidence of associated lesions suggestive of CD. We recommended that the diagnosis of DCG triggers cancer surveillance and preventive care.

P28‐18

Expression of CXCR7 and CXCR4 in diffuse astrocytomas and its interaction with HIF1α expression and IDH1 mutation

Oba‐Shinjo SM¹, Marie SKN², Bianco A², Clara C², Galatro T², Rosemberg S², Teixeira MJ² and Uno M³

¹ Hospital Pio XII de Câncer; ² USP; ³ ICESP, Brazil

Background: There is abundant evidence showing that chemokine CXCL12 and its receptor CXCR4 are involved in progression of tumors, metastasis and angiogenesis. CXCR7, a recent additional receptor for CXCL12 with higher affinity than CXCR4, has raised key issues on glioma cell migration. Furthermore, CXCR4 and CXCR7 are implicated in glioma migration in hypoxic condition. The aim of this study is to investigate CXCR7 mRNA expression in diffusely infiltrating astrocytomas and to correlate to expression levels of CXCR4, HIF1A and IDH1 and mutation status of IDH1.

Methods: CXCR7, CXCR4, HIF1A and IDH1 expression levels were evaluated by quantitative real‐time PCR in 154 frozen astrocytoma samples (50 diffuse astrocytoma‐AGII, 18 anaplastic astrocytoma‐AGIII and 86 glioblastoma‐GBM) and 22 samples from non‐neoplastic cerebral tissue (NN). IDH1 mutation status of 105 samples of astrocytomas, previously determined, were analyzed with IDH1 and HIF1A expressions, matched with clinicopathological parameters and overall survival. Furthermore CXCR7 protein expression was analyzed by immunohistochemistry in astrocytoma of different grades and in glioma cell line by confocal microscopy.

Results: There was significant difference in the expression levels of the genes studied between each astrocytoma group and NN (p < 0.001). The analysis of associated gene expressions in AGII showed significant correlation between CXCR7/IDH1 (p < 0.001) and CXCR7/HIF1A (p = 0.007). AGII patients presenting downregulated CXCR7 expression had observation time of 43 months while those with CXCR7 overexpression presented 24 months (p = 0.043). HIF1A overexpression was associated to higher CXCR7 and CXCR4 expression levels (p = 0.001), while presence of IDH1 mutation was associated to lower CXCR7 and CXCR4 mRNA expressions (p = 0.01). CXCR7 protein expression was identified in all samples studied, and it increased with malignancy. CXCR7 protein was predominantly localized in the cellular membrane of U87 cells.

Conclusion: CXCR7 was overexpressed in astrocytoma of different malignant grades. HIF1A overexpression was correlated to higher expression levels of CXCR7 and CXCR4, otherwise presence of IDH1 mutation was correlated to lower expression of both genes. CXCR7 overexpression had a negative impact on the observation time of AGII and protein expression level was associated with the degree of malignancy.

P28‐19

Expression of EVGF, EVGR2 and eNOS in chordomas. Clinicopathological and immunohistochemistry approaches

Suck MLT, Salinas LC, Garibay CS and Carares K

Instituto Nacional de Neurología y Neurocirugía, Ciudad de México, Mexico

Background: Chordoma is the most common tumor in the fifth and sixth decade of life, corresponds to remnants of the notochord. Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)‐induced angiogenesis and vascular hyper permeability.

Objective: This study evaluated immunoexpression of EVGF, EVGFR2 and eNOS, ki67‐Li and MVD‐Li (CD34) in chordomas.

Material and Methods: 70 cases of chordoma were included in this study and were classified into conventional, chordoid and atypical type. Clinicopathological and immunohistochemistry correlation.

Results: 42 (60%) cases were females and 28 (40%) male. Tumor localization was; 5 infratentorial and 22 in skull base (p = 0.611). The ages ranged from 17 to 77 year (mean of 41.93 ± 15.28 years). The average range of follow‐up was 42.6 ± 16.8 years. The mean tumor size was 37.87 ± 7.12 mm. Brain invasion was in 47 (67%) cases. Recurrences occurred in 38 (54.3%) cases (p = 0.002). The mean Ki67‐Li was 16 ± 1.670% for classic type and 5.810 ± 1.861% for chordoid type (p = 0.000), while, the MDV‐Li mean value was 39.34 ± 5.759% for classic type and 41.15 ± 4.378% for chordoid type (p = 0.323).

The expression of EVGF was weak in classic type and strong in those chordoid type. The EVGFR2 and eNOS was strongly positive in classic type and negative in chordoid type and the classical. Tumors that showed necrosis and inflammation were more intensely positive than those who did not and there was statistically significant correlation to increased Ki67 proliferation indices with expression of eNOS and EVGFR2.

Conclusion: EVGFR2 and eNOS and Ki‐67‐li are helpful to predict tumor recurrence and prognosis and serve as potential prognostic biomarker and therapeutic target for chordoma patients.

Keyword: nitric oxide (NO), EVGF, EVGFR, chordomas, immunohistochemistry.

P28‐20

Expression of SSTR2a, but not of SSTRs 1, 3 or 5 in somatotroph adenomas assessed by monoclonal antibodies was reduced by octreotide and correlated with the acute and long‐term effects of octreotide

Casar‐Borota O^1,3,4, Heck A^2,3, Schulz S⁵, Nesland JM^1,3 and Bollerslev J^2,3

¹ Division of Pathology, Oslo University Hospital, Norway; ² Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital; ³ Faculty of Medicine, University of Oslo; ⁴ Department of Immunology, Genetics and Pathology, Uppsala University, Sweden; ⁵ Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich‐Schiller‐University, Jena, Germany

Introduction: Reduced expression of somatostatin receptors (SSTRs) in somatotroph adenomas and their potential down‐regulation after medical treatment may explain the unsatisfactory response to octreotide in particular acromegalic patients. We aimed to determine the expression of SSTRs1, 2a, 3 and 5 in somatotroph adenomas, to correlate expression with clinical characteristics and the response to octreotide, and to ascertain whether preoperative octreotide treatment affected SSTR expression.

Patients and Methods: The study included 65 adenomas from acromegalic patients. Twenty‐eight patients received preoperative octreotide, and 37 patients were operated on without pretreatment. Twenty‐six patients were randomized to direct surgery (n = 13) or to octreotide pretreatment (n = 13). Immunohistochemistry was performed with rabbit monoclonal antibodies towards SSTRs 1, 2a, 3 and 5 (clones UMB‐7, 1, 5, 4). SSTRs expression was evaluated using a 12‐grade scoring system. The responses to the octreotide test dose (GH reduction) and to 6 months of octreotide (IGF‐1 reduction) were measured.

Results: The majority of adenomas showed membranous expression of SSTRs 2a and 5. SSTR2a expression was reduced in the pretreated group and correlated with the acute octreotide test results and the effect of octreotide treatment. The correlation with the acute test response improved after adjusting for medical pretreatment.

Conclusion: Rabbit monoclonal antibodies are reliable markers of SSTRs in somatotroph adenomas. SSTR2a expression correlated with the response to octreotide and was reduced after octreotide treatment, indicating the need for adjustment when correlating SSTR2a expression with baseline characteristics. Evaluating SSTR subtypes may be an important tool for improving the medical treatment for acromegaly.

P28‐21

Expression of the inhibitor of apoptosis protein survivin, but not of tumor suppressor protein p53, has a negative prognostic impact in glioblastoma

Faccion RS^1,3*, de Faria GP^2,3*, Bernardo PS^1,3, Teixeira CL⁴, de Oliveira JA⁵, Chimelli L⁶ and Maia RC¹

¹ Laboratório de Hemato‐oncologia Celular e Molecular, Programa de Hemato‐oncologia Molecular; ³ Programa de Pós‐graduação Strictu Sensu em Oncologia; ⁴ Laboratório Geral; ⁵ Serviço de Neurocirurgia; ⁶ Divisão de Patologia, Instituto Nacional de Câncer (INCA), Brazil; ² Laboratório de Neuroquímica e Biologia Celular, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Brazil

*Both authors contributed equally to this work.

Introduction: Glioblastoma (GBM) is a lethal human malignancy with 12 months of median survival. The relationship between p53 and survivin in GBM is still controversial. This study aims to investigate the role of these proteins in GBM survival.

Patients and Methods: Seventy formalin‐fixed paraffin‐embedded GBM samples were obtained at Brazilian National Cancer Institute (INCA) from 2000 to 2008. Cases were selected according to tumor sample quality, availability of follow up information and absence of previous grade II/III astrocytoma.

Results: From the 55 cases studied so far, 44 were classic GBM, 8 were GBM with oligo component (GBM‐O), 2 were GBM with giant cells and 1 was gliosarcoma. Half of the patients underwent complete tumor resection. Protein expression status was classified as negative (0–5% positive tumor cells) or positive (more than 5%). So far, 20/50 tumors were p53 positive and 9/45 were survivin positive (nucleus and cytoplasm). 10/39 GBM cases and 6/8 GBM‐O cases co‐expressed survivin and p53. Survivin expression conferred a poorer prognosis (p = 0.009) while p53 expression did not influence survival (p = 0.745).

Conclusion: These results suggest that overexpression of survivin may be considered a prognostic factor for GBM. Patients whose tumors express survivin may benefit from novel targeted anti‐survivin treatment.

Financial Support: Capes, INCT para Controle do Câncer, Ministério da Sáude, CNPq.

P28‐22

FISH as a fundamental tool in the differential diagnosis of melanomas with complete neural differentiation

Weidenfeld J, Nekula D, Barshack I and Nass D

Chaim Sheba Medical Center, Tel Hashomer, Department of Pathology, Ramat Gan, Israel

Melanomas are tumors with occasional difficult diagnosis, especially when the histological and immunophenotypical features are not straightforward. Seldom, melanomas may expose neural differentiation, and typical features of melanomas should be present elsewhere in order to get a precise diagnosis. It should be stressed that typical areas of melanoma and foci of neural differentiation show an overlapping immunoprofile and this underlines the importance of the histological features for the accurate diagnosis. We report a case of melanoma which presents histologically as a benign‐looking spindle cell and epithelioid tumor diffusely showing features of neural/Schwannian differentiation, with neither mitotic activity nor necrosis. In view of the clinical history of melanoma, several immunostains were performed but did not aid in the differential diagnosis between a benign Schwannian/neural tumor and malignant melanoma with complete neural differentiation. Fluorescence in situ hybridization (FISH) was performed in order to seek common mutations in melanomas. We found alterations in chromosomes 6, 8, 9 and 11 that were previously described in the medical literature. The results of the FISH analysis favor the diagnosis of melanoma with complete neural differentiation. To our knowledge, this is the first case of melanoma with complete neural/Schwannian differentiation described in the literature in which the in situ hybridization assay was fundamental in arriving to the precise diagnosis, in view of the lack of typical melanoma histology and the overlapping immunohistochemical results. When a clinical history of melanoma is present, FISH assay may help in the diagnosis of tumors with complete neural differentiation and ambiguous immunoprofile.

P28‐23

Ganglion cell tumors in sella turcica

Naganska E¹, Matyja E², Maksymowicz M¹, Bonicki W³, Kunicki J³, Zieliński G⁴

¹ Department of Experimental and Clinical Neuropathology, M. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; ² Department of Pathology and ⁴ Department of Neurosurgery, M. Sklodowska‐Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; ³ Department of Neurosurgery, M. Sklodowska‐Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; ⁴ Department of Neurosurgery, Military Institute of Medicine, Warsaw, Poland

Gangliocytic neoplasms in the sellar region are uncommon. They are usually associated with pituitary adenomas whereas isolated ganglion cell tumors without evidence of adenomatous component are extremely rare. We report two cases of mixed/collision tumors composed of intrasellar gangliocytoma and pituitary adenoma and one case of ganglioglioma not related to adenoma. One patient presented with acromegaly, whereas the other was initially diagnosed with non‐functioning adenoma. In these two cases, the histopathological examination of surgical specimens revealed mixed sellar lesions composed of distinct parts of pituitary adenoma and ganglion cell component. The adenomatous part appeared to be a growth hormone (GH)‐secreting adenomas on immunostains and sparsely granulated somatotroph adenomas in the electron microscopy. The rest of the lesion reveled morphology typical for gangliocytoma. It was composed exclusively of groups of neoplastic, mature ganglion cells. This gangliocytic component was characterized by dysplastic changes and presence of bi‐ or multinucleated cells with aggregates of Nissl granules at the periphery of the body. The third case had no endocrine symptoms and there was no adenoma or normal pituitary gland in the surgically excised material. The sellar lesion appeared to be a mixture of large, ganglioid, often multipolar neurons and neoplastic astroglial elements. Immunohistochemical studies supported biphasic pattern of gangliogliomas with expression of both neuronal and glial markers. The Ki‐67 labeling index was low.

Concluding the intrasellar ganglion cells tumors may appear in association with pituitary adenomas or without evidence of the adenomatous components. The histogenesis of gangliocytic neoplasm in sellar region remains unclear.

P28‐24

Genetic heterogeneity within oligoastrocytomas

Wilcox P¹, Kim L^2,3, Morey A², Li C^1,3 and Buckland ME^1,3

¹ Brain & Mind Research Institute, The University of Sydney, Camperdown, NSW Australia; ² Anatomical Pathology, St. Vincents Hospital, Darlinghurst, NSW, Australia; ³ Department of Neuropathology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia

Oligoastrocytomas are diffuse glial tumors defined by their mixture of two distinct morphological components; neoplastic cells resembling those found in oligodendrogliomas and in astrocytomas. Despite the intratumoral morphological heterogeneity, many believe that both types of cells within oligoastrocytomas are molecularly homogeneous. With the increasing utility of molecular classification in glioma pathology, the concept of a mixed oligoastrocytoma is becoming marginalized in favor of a molecular description. We present here two cases of biphasic WHO grade III oligoastrocytomas with genetically distinct oligodendroglial and astrocytic components, each expressing the typical markers for ‘astrocytic’ and ‘oligodendroglial’ phenotypes, but united by a common IDH1 (R132H) mutation. In one case, the tumor recurred after 3 years, with a predominantly astrocytic phenotype. These cases demonstrate that, at least in a subset of oligoastrocytomas, lineage‐associated molecular changes align with morphology, with important implications for future glioma classification.

P28‐25

Genomic alterations in atypical teratoid/rhabdoid tumors: the Medical University of Vienna experience

Haberler C, Ströbel T, Czech T, Chocholous M, Dorfer C and Slavc I

Institute of Neurology, Medical University of Vienna, Vienna, Austria

Introduction: The only recurrent genomic aberrations detected in atypical teratoid/rhabdoid tumors (AT/RTs) are alterations within the SMARCB1/INI1 gene. They comprise deletions including copy neutral LOH, mutations, and duplications. Patient outcome is poor with disease recurrence despite treatment, and death due to progression. Improved patient outcome could be achieved with an intensive multimodal therapy developed at the Medical University of Vienna.

Methods: Tumor tissues and constitutional DNA from 16 patients treated at the MUV for primary or recurrent tumors were analyzed using immunohistochemistry, MLPA and Sanger sequencing, as well as SNP arrays in 3 patients. Patient age at diagnosis ranged between 3 months and 22 years (median 32.5 months). 5 were females, 11 males. Tumor location was in 10 patients supra‐ and in 6 infratentorial.

Results: All tumor tissues showed characteristic loss of SMARCB1/INI1 expression. In 8 patients (50%) homozygous deletions of the SMARCB1/INI1 gene were observed. In 3 patients (18.8%) the combination of a coding sequence mutation and a deletion was found. In 5 (31.2%) tumors only one hit was detectable within the tumor tissue (4 deletions, 1 mutation). In 3 patients a germline alteration was found, including 2 deletions and 1 coding sequence mutation. Patients with germline mutations were 3, 6 and 35 months at diagnosis. 13 patients, including 1 patient with germline mutation are alive, 3 patients died of disease.

Conclusion: To date no genotype/phenotype correlations exist, therefore documentation of genomic alterations and outcome of AT/RT patients is important to detect patients who might have an improved survival.

P28‐26

Glioma with signet‐ring cell morphology: case report

Figurelli S, Alvarez C, Ericsson PG, Bruni A, Guevara M and Gardella J

Hospital Fernandez, Argentina

Introduction: Primary central nervous system tumors with signet‐ring cell morphology are extremely rare. This morphology is described in glioblastoma, oligodendroglioma, oligoastrocytoma and ependymoma. The presence of signet‐ring cells in the CNS should raise the suspicion of metastatic carcinoma, particularly, from the upper gastrointestinal tract. Other differential diagnoses include atypical teratoid rhabdoid tumor in children and rhabdoid meningioma in adults.

Patient and Methods: Female 29 year old patient who presents with persistent oppressive headaches and left facio‐brachio‐crural hemiparesis. Neuroimaging shows a 4.2 × 4 cm parasagittal right fronto‐parietal mass in the corpus callosum, hyper‐intense on T2, with heterogenous Gadolinium enhancement and slight peri‐lesion edema.

Results: The intraoperative study and the histologic sections show a proliferation of epithelioid cells with eccentric oval nuclei containing granular chromatin and PAS and Alcian blue negative. Arranged in sheets showing tumoral necrosis and mitotic figures. There are no morphological glial features (fibrillar cell component). Inmunohistochemistry: tumor cells resulted Vimentin positive; S‐100, GFAP and CK AE1‐AE3 focally positive; EMA positive in isolated cells; ACL, CEA, CD138, HMB‐45, CK20, CK7, CA‐125, smActin, PR negative and Ki‐67 35% Diagnosis: Glioblastoma with signet‐ring cell morphology. The patient was studied further on images and lab results did not show evidence of another tumor. Treated with chemotherapy and radiotherapy, the patient recurres due to bilateral tumor and dies.

Conclusion: Ultrastructural and inmunohistochemical studies have been the basis for considering the glial origin of the signet‐ring cells, which show intermediate filaments (GFAP, Vimentina, S‐100) and degenerative changes in mitochondria and endoplasmic reticulum.

P28‐27

Holomedular pilomyxoid astrocytoma: case report

Souza FS, Junior AM, La Roque‐Ferreira A and Moura HB

Sarah Network of Rehabilitation Hospitals, Brazil

Introduction: Pilomyxoid astrocytoma (PMA) belongs to the group of low‐grade astrocytomas described recently as a grade II WHO variant of pilocytic astrocytoma with distinct histopathological characteristics and more aggressive behavior. PMA occurs predominantly in the hypothalamic region but can be found elsewhere along the neuraxis. Seven cases of intramedullary PMA were reported in the literature until now. The purpose of this paper is to describe a patient with a rare holomedular PMA.

Case Report: A 3‐year‐old boy presented with congenital cervical deformity associated to cephalic and dorsal pain, cervical rotational limitation and difficulties to walk. MRI imaging revealed a solid‐cystic holomedular tumor occupying the entire spinal cord. The patient was submitted to a cervico‐thoracic (C3‐T11) laminoplasty for a partial tumor resection and presented with flaccid tetraplegy and became ventilatory mechanical support dependent after surgical resection. He also presented with hydrocephalia and was submitted to a ventriculoperitoneal shunt. Histopathological findings were consistent with a PMA with high Ki67 proliferation index and molecular analysis didn't show IDH1, IDH2 or BRAF mutations. Given the extension of the neoplasm and the clinical patient status no other complementary therapy was considered.

Conclusion: Our report shows an uncommon presentation of PMA and emphasizes: 1. the importance of correct diagnosis of this tumor in order to distinguish it from others gliomas; 2. the increasing relevance of molecular markers complementing classic histological diagnosis; 3. the importance of an interdisciplinary team work for individualized decision‐making on treatment strategies, as there are not definitive treatment guidelines yet.

P28‐28

Hybrid peripheral nerve sheath tumor – case report

de Souza ACS, Chimelli L and Guedes JF

Institute of Neurology, Federal University of Rio de Janeiro, Brazil

Introduction: Hybrid peripheral nerve sheath tumors are uncommon, affect mainly patients with hereditary syndromes like neurofibromatoses or schwannomatosis, but may be sporadic. The most common subtype aggregates schwannoma and perineurioma, but neurofibroma with schwannoma, or neurofibroma with perineurioma may also occur.

Case Report: A male swarthy 51 year‐old patient, without stigmas of neurofibromatoses or schwannomatosis, complained of progressive pain on the distal third right leg for seven months. On physical examination, the pain was tingling, starting in the middle third of the distal limb, worsening at night, with positive Tinel sign along the tibial nerve. On palpation an extremely painful lump, fixed to the surrounding tissues, was noted. There was no motor or sensory deficit. Imaging examination showed a large heterogenic tumor, without evidence of muscular or osseous invasion. It was completely removed with microsurgical technique and intraoperative stimulation. Histologically it consisted of an intraneural neurofibroma, with small isolated foci of schwannoma. There were intermingled elongated neural and collagen bundles, embedded in a mucoid matrix, in which small foci of Antoni A areas of a schwannoma were seen. The patient recovered completely, without any sign of sensory/motor deficit or pain.

Conclusions: The schwannomatous component may be scarce and overlooked contributing to the rarity of this type of tumor. Its clinical behavior is probably not different from most pure neurofibromas and schwannomas and resection should be tailored in the same way as in most common tumors. Imaging exams may not contribute to differentiate the two histological patterns.

P28‐29

Immunohistochemistry evaluation of signaling pathways PI3K/Akt/mTORC1, Raf‐1‐MEK‐1‐MAP(Erk1/2) and JAK‐STAT3 and the relationship whit HER‐2, EGFR, KI‐67 in meningiomas of different histological subtypes and grades

Cavalcante JM, Machado A, Torres AFC, De Lima FEMM, Távora FRF and Nogueira CD

Laboratório Argos, Brazil

Introduction: Meningiomas are tumors of the central nervous system that originate from arachnoidal cap cells. Recent studies showed that activation of the PI3K/Akt/mTORC1, Raf‐1‐MEK‐1‐MAP(Erk1/2) and JAK‐STAT3 pathways seems to be found in meningiomas. The purpose of this study is to assess regulatory pathways MEK‐1‐MAPK, Akt‐mTOR and JAK‐STAT3, by protein expression of MTOR, pAKT, ERK1/2, STAT3 by immunohistochemistry along with the expression of HER‐2 and EGFR proteins and grade and histological subtype of the tumors.

Material and Methods: Meningiomas from a single institution were sampled in a tissue microarray (TMA) and stained for mTOR, STAT3, ERK1/2, phospho‐AKt, EGFR, HER‐2 and Ki‐67. Clinical information was available in all cases. Immunoreactivity in the cytoplasm and nucleus for the sections was assessed and graded by two pathologists independently.

Results: There were 46 cases (38 female, mean age 52, range 18–87). 40 were grade 1, 5 grade 2 and 1 grade 3. Tumor grade correlated with mitotic rate and size, and size correlated with mitosis and ki‐67 proliferation index (p < 0.05). Expression of STAT3 was more prevalent in higher grade tumors as well as in and tumors with higher mitotic index and invasion of others structures. There was no significant difference in expression of mTOR, ERK1/2, phospho‐AKt, EGFR, HER‐2 among variables analyzed.

Conclusion: STAT3 expression correlated with tumor grade, ki‐67 and invasion of others structures, may be used as adjunct in predicting tumor behavior. The lack of significant difference among other proteins may indicate other pathways of tumor activation that may be target for future therapies.

P28‐30

Impaired mitochondrial ADP phosphorylation despite preserved oxidative capacity in the highly glycolytic T98G and U87 glioma cells

Silva ER, Santos ES, Ruas JS, Rogério F and Castilho RF

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil

In contrast to most differentiated cells that rely on mitochondrial oxidative phosphorylation to generate metabolic energy, glioblastoma cells exhibit increased conversion of glucose to lactate in the presence of normal levels of oxygen (Warburg effect). The aim of the present study was to characterize the mitochondrial function of the T98G and U87 glioma cell lines. The addition of the protonophore FCCP to intact cells suspended in DMEM medium resulted in a sustained 3‐4 fold increase in oxygen consumption, eliciting oxygen consumption rates that matched those of FCCP‐treated digitonin‐permeabilized cells incubated in the presence of exogenous mitochondrial substrates. Oxygen consumption by T98G and U87 cells was well coupled to ADP phosphorylation, as evidence by ADP/O values similar to those obtained with isolated rat brain organelles. Respiration of glioma cell lines also displayed a high affinity for oxygen. In permeabilized T98G and U87 cells, ADP‐stimulated oxygen consumption was only about 50% of that obtained in the presence of FCCP, indicating that glioma cells may have an important limitation of the oxidative phosphorylation (OXPHOS) system relatively to the activity of the electron transport system (ETS). Using flux control coefficient analysis, we found that this impaired OXPHOS was mainly related to the lower activities of both ATP synthase and adenine nucleotide translocator, but not to the phosphate transporter. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in the highly glycolitic T98G and U87 glioma cells, nonetheless these cells possess a relative restrained OXPHOS capacity.

P28‐31

Intracranial cellular schwannomas: a clinicopathologic study of 9 cases

Costa FD¹, Burger PC^2,3 and Rodriguez FJ^2,3

¹ Department of Anatomic Pathology, A.C. Camargo Cancer Center, Sao Paulo, Brazil; ² Department of Pathology and ³ Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA

Correspondence: Fausto J. Rodriguez, MD, Department of Pathology, Division of Neuropathology, Johns Hopkins Hospital, Sheikh Zayed Tower, Room M2101, 1800 Orleans Street, Baltimore, MD 21231

Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

Introduction: Cellular schwannomas are neoplasms of nerve sheath derivation that can be misinterpreted as malignant neoplasms. Intracranial examples are rare, comprising approximately 10% or less of cellular schwannomas as a group.

Patients and Methods: We describe the clinicopathological features of 9 cases of intracranial cellular schwannomas and provide a concise literature review.

Results: Nine cases were found to represent cellular schwannomas of the intracranial space. The mean age of the patients was 29.8 years, with males more often affected than females (2:1). The most common parent nerves were the 8th (n = 3) and 5th (n = 3) cranial nerves. The remainder occurred in the cerebellopontine angle, left occipital lobe (intraaxial) and left frontal lobe (extraaxial in the anterior cranial fossa). Histologically, the tumors were hypercellular and composed of interlacing fascicles of spindle cells, with storiform areas, focal whorl formation, and scattered enlarged and hyperchromatic nuclei. A curious feature occurring in the case arising in the anterior cranial fossa was the presence of granular cell change. Immunohistochemical findings included immunoreactivity for S100 (9/9 cases), pericellular collagen IV or reticulin special stain (7 out of 7 tested cases), SOX10 (4 out of 4 cases) and CD34 (1 out of 3 cases).

Conclusion: In contrast to cellular schwannomas at other sites, intracranial tumors in our experience present at a younger age and with a male predominance. Awareness of this relatively uncommon anatomic location for cellular schwannoma is crucial since the differential diagnosis is relatively wide, and encompasses a variety of benign and malignant spindle cell neoplasms.

P28‐32

JAM‐A – a novel prognostic cancer stem cell‐related marker in glioblastoma

Rosager AM¹, Dahlrot RH¹, Kristensen BW¹, Hansen S², Kristensen BW² and Lathia JD³

¹ Department of Pathology, Odense University Hospital, Odense, Denmark; ² Institute of Clinical Research, University of Southern Denmark, Odense, Denmark; ³ Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

Introduction: Glioblastomas (GBMs) are among the most lethal cancers, being resistant to radiation and chemotherapy. This has been explained by the existence of resistant cancer stem cells. Using a high throughput flow cytometry screen we identified the expression of junctional adhesion molecule‐A (JAM‐A) on the surface of GBM cells. Our studies demonstrated that JAM‐A is a unique GBM stem cell niche adhesion factor. The aim of the present study was to investigate the prognostic value of JAM‐A protein expression in gliomas.

Materials and Methods: A well characterized population‐based cohort of 433 patients with gliomas of increasing grade was identified and tissue samples from 235 patients with WHO grade II, III and IV gliomas were included. JAM‐A protein expression was evaluated by immunohistochemical chromogenic staining and advanced quantitative image analysis with continuous estimates of staining intensity.

Results: The JAM‐A antibody stained tumor cell membranes or cytoplasm in different extent in most gliomas. JAM‐A expression increased with glioma malignancy grade and was higher in GBM tissue as compared to non‐neoplastic cortical tissue. High JAM‐A protein level significantly correlated to poor GBM patient prognosis as compared with low JAM‐A expression (cut‐point 45/55%) with Hazard Ratio 1.48 and a p‐value < 0.05 using an invariable cox regression. JAM‐A expression was not prognostic in WHO grade II or III tumors.

Conclusion: The results show that JAM‐A protein intensity levels increase with WHO grade and represent an independent prognostic estimate for GBM patients. This suggests that JAM‐A is closely associated with glioma aggressiveness.

P28‐33

Loss of INI‐1 is a novel marker for chordoid meningiomas of the CNS

Sharma MC, Ghosh R, Kakkar A, Sarkar C, Suri V, Singh M and Sharma BS

Departments of Pathology and Neurosurgery, All India Institute of Medical Sciences, New Delhi, India

Introduction: Loss of expression of the INI1 gene is associated with several rhabdoid and non‐rhabdoid tumors. INI1 loss in different meningioma subtypes has not been reported adequately. Chordoid meningioma is one of the subtypes of meningioma with characteristic histomorphology. We present a study of immunoexpression of INI1 in different meningioma subtypes with special reference to chordoid meningiomas.

Materials and Methods: Twenty‐five cases of meningioma were retrieved from the archives of All India Institute of Medical Sciences, New Delhi, comprising of all grades of meningioma, including ten cases of chordoid meningioma and five cases of clear cell meningioma (WHO grade II), seven grade I tumors, and three grade III tumors (two rhabdoid, one papillary). INI1 immunohistochemistry was performed in all these cases.

Results: Eight out of ten cases (80%) of chordoid meningioma demonstrated loss of expression of INI1. None of the other meningioma subtypes were immunonegative for INI1, including three cases of grade III meningiomas.

Conclusion: Chordoid meningioma is new entrant in the category of INI 1 loss group of tumors. INI1 may be used as an immunohistochemical marker for chordoid meningiomas in addition to histomorphology in making a diagnosis, and to differentiate it from other subtypes of meningiomas. The role of this loss of INI1 expression in the pathogenesis of these tumors, however, requires further evaluation.

P28‐34

Malignant transformation of a benign epidermoid cyst: a case report

Baweja R, Reddy K, Whitton A, Provias J and Lach B

McMaster University HHS, Canada

Malignant transformation of an epidermoid cyst is rare with only a handful of cases described in the literature. We report a case of a 65‐year‐old woman who initially presented in 1999 with right sided facial pain. An MRI was done at the time, which revealed a right CP angle tumor. This lesion was followed until 2006 when she developed worsening right sided facial pain, numbness in all three divisions of the trigeminal nerve and right‐sided facial paresis. The lesion was resected at the time by right retrolabyrinthine approach with pre and post sigmoid craniectomy. The histopathologic diagnosis was epidermoid cyst. The patient was followed for several years and appeared to do well with improvement in her deficits at presentation. She then presented in 2012 with left sided weakness and gait instability. She was re‐operated on that year with minimal improvement in her deficits. The histopathologic diagnosis was squamous cell carcinoma. She received a total of 2500 cGy of SRS in five treatments but despite this intervention, her tumor progressed. Gross total resection followed by radiotherapy is the treatment of choice for primary intracranial SCC. Even with this combined modality, prognosis is poor. A review of the available literature along with possible mechanisms of malignant transformation are discussed.

P28‐35

Men over 55 years with meningioma show hypermethylated BRCA1

Ferrasi AC^1,5, Lombardi IAS^1,2, Faria MHG³, Galvani AF¹, Zugaib R¹, Del'Vescovo AA¹, Rabenhorst SHB⁴, Zanini MA² and Pardini MIMC^1,5

¹ Transfusion Blood Center, UNESP – Sao Paulo State University, Botucatu‐SP, Molecular Biology Laboratory; ² Botucatu Medical School, UNESP – Sao Paulo State University, Botucatu‐SP, Division of Neurosurgery; ³ Dr. MárioGatti Hospital, Department of Neurosurgery, Campinas‐SP, Brazil; ⁴ Department of Pathology and Forensic Medicine, Molecular Genetics Laboratory (LABGEM), School of Medicine, Federal University of Ceara, Fortaleza, Brazil; ⁵ Botucatu Medical School, UNESP – Sao Paulo State University, Botucatu‐SP, Department of Internal Medicine, Brazil

BRCA1 gene is expressed in the cells of the breast and other tissues, where it plays a role in cell cycle regulation, transcription, DNA repair of double‐stranded breaks, ubiquitination, and transcriptional regulation as well as other functions, such as regulation of cell response to mitogenic signals triggered by estrogens. Considering that meningiomas show greater tumor growth during pregnancy, express estrogen receptors and proliferate in response to estrogenic stimulation, the hypothesis that this type of tumor can share molecular mechanisms that involve exposure to estrogen should be investigated. Therefore, the aim of the present study was to investigate the methylation status of the BRCA1 gene in meningiomas. The most important finding of this study was that 100% of the male patients over 55 years with meningioma showed a methylated BRCA1 gene in their tumor cells.

P28‐36

Methylation analysis of BRCA1 in gliomas

Del'Vescovo AA¹, Galvani AF¹, Zugaib R, Zanini MA², Faria MHG³, Rabenhorst SHB⁴, Pardini MIMC^1,5 and Ferrasi AC^1,5

¹ Transfusion Blood Center, UNESP – Sao Paulo State University, Botucatu‐SP, Molecular Biology Laboratory; ² Botucatu Medical School, UNESP – Sao Paulo State University, Botucatu‐SP, Division of Neurosurgery; ³ Dr. Mário Gatti Hospital, Department of Neurosurgery, Campinas‐SP, Brazil; ⁴ Department of Pathology and Forensic Medicine, Molecular Genetics Laboratory (LABGEM), School of Medicine, Federal University of Ceara, Fortaleza, Brazil; ⁵ Botucatu Medical School, UNESP – Sao Paulo State University, Botucatu‐SP, Department of Internal Medicine, Brazil

Gliomas are the most frequent primary brain tumors of the CNS, originated from glial cells that surround and support nerve cells. They are classified into four grades of malignancy (I, II, III, IV). Genetic factors have been investigated to elucidate the mechanisms involved in this tumor. In addition, epigenetic changes may play a role in carcinogenesis, like the silencing caused by hypermethylation of the promoter regions of tumor suppressor genes transcription or hypomethylation of proto‐oncogenes. The BRCA1 gene is hypermethylated in 80% of cases of familial breast cancer, however, the literature lacks information on the methylation profile in gliomas. The present study aimed to analyze the methylation profile of BRCA1 in 42 cases of gliomas (18 women, 24 men) underwent surgery (operated) in two Brazilian hospitals (Department of Neurosurgery of the Clinical Hospital, Botucatu Medical School, UNESP and the Dr. Mário Gatti Hospital – Campinas). The DNA was extracted using QIAamp DNA Mini Kit (Qiagen) and methylation analysis was performed by MSP‐PCR after treatment of DNA with sodium bisulfite. When the samples were distributed in accordance with the degree of malignancy, hypermethylation was found in 30% and 70% of low and high level tumors, respectively. These indicate the significance of methylation in BRCA1 in gliomas, particularly in advanced cases of disease. However, these data should be analyzed with caution, due to the small number of cases. Increasing the casuistry and the expansion of analysis for the validation of the results is considered.

P28‐37

Mib‐1, MVD labeling index and P53 immunoexpresion in pituitary adenomas: clinico‐pathological, immunohistochemistry and correlation between Harydy–Vesinia

Tena‐Suck ML¹, Palacios‐Ortiz IJ², Castillejos‐López M³, Casares‐Cruz K⁴ and Sánchez AV⁵

Departamento de ¹ Neuropatología; ² Servicio de Neurocirugía; ³ Servicio de Epidemiología; ⁴ Service of Neuroimagen, National Institute of Neurology and Neurosurgery, Mexico City; ⁵ Service of Neurology, National Institute of Neurology and Neurosurgery, Mexico City, Brazil

Background: Pituitary adenomas are benign tumors of slow growth.

Objective: The aim of this work was to study the immunohistochemical expression of p53, the MIB‐1 index and the DMV in pituitary adenomas and correlate with the of Hardy Vezina radiological classification.

Results: 84 cases of pituitary adenomas operated on INNN were included. 43 (51.2%) cases were female and 41 (49%) men. The age of the patients ranged from 18 to 72 years (mean 46.48 ± 1.49). 27 (32%) cases were functional adenomas and 57 (68%) non‐nonfunctional adenomas. The tumor size ranged from 5 to 75 mm. According Harydy–Vesinia classifications; grade I were 45 (54%), grade II were 18 (21%), grade III were 13 (16%) and grade IV were 8 (10%). Ki67‐Li for microadenomas was <2% while for macroadenomas was 15%. MVD‐Li for microadenomas was 5% and for macroadenomas 14%. According Harydy–Vesinia classifications, grade I showed Ki67 (0%), MVD‐Li (10%), and p53 negative, Grade II were Ki67‐Li (2%), MVD‐Li (10%) and p53 was negative, grade III were Ki67‐Li (5%), MVD‐Li (20%) and p53 immunoexpression in 50% and grade IV was Ki67‐Li (6%), MVD‐Li (25%) and all case were strong p35 immunoexpression. The Mib‐1 index was statistical significative between grade IV and P53 immunoexpression as well as invasion, necrosis and tumor size.

Conclusions: There was association between the expression of p53, higher Ki67 and MVD‐Li those were increased with grade IV of Hardy–Vezinia classification. Therefore, classify ADH from the radiological point of view is most useful for predicting unfavorable prognosis and provide advantage over Ki67 and MVD index.

P28‐38

MicroRNAs and gene expression profile differentially expressed in grade III and grade II oligodendrogliomas

Neder L, Carvalho B, Nawaz M, Fatima F, de Oliveira VC, Lima H, Haddad R, Panepucci RA and Gilberto Carlotti Jr BOCC

School of Medicine of Ribeirão Preto, University of São Paulo, Brazil

MicroRNAs (miRNAs, miRs) are short non‐coding regulatory RNA molecules found ubiquitously. More lately, miRNAs have also been implicated in oncogenesis, acting as tumor suppressors or oncogenes. Hitherto, the role of miRNAs in CNS tumors has been intensively investigated in glioblastomas and medulloblastomas, but there are few data regarding miRNAs in oligodendrogliomas. We performed a systematic evaluation of miRNAs and mRNAs expressions in a series of grade II and grade III oligodendrogliomas (OGs). Our goal was to determine miRNAs and putative target genes that are differentially expressed in grade III oligodendrogliomas, but not in grade II OGs and in non‐neoplastic white matter (nnWM). Total RNA was extracted from 14 cases of grade II and III OGs naïve of treatment (7 cases per grade) and 15 WM after samples’ microdissection. For each case, the expression of miRNAs and mRNAs was evaluated using microarray‐based expression profiling platforms (723 transcripts and 41,000 genes, respectively). Using this strategy we have found 50 miRs and 70 miRs that were significantly overexpressed exclusively in anaplastic oligodendrogliomas and in both grades of OGs, respectively. Conversely, we disclosed 33 miRs and 21 miRs were exclusively downregulated in grade III OGs and grade II OGs, respectively. After matching with the expressions of putative target‐mRNAs, we were able to validate 8 out of 10 key‐miRNAs by RT‐qPCR (assays in duplicate). Among the hypo‐expressed miRNAs, we found some miRs that were previously described in cell differentiation of embryonic stem cells and related to cell adhesion and protein‐protein interaction surfaces.

Support: FAPESP.

P28‐39

Molecular analysis of gliomas: from diagnosis to prognosis

La Rocque‐Ferreira A¹, Souza FS², Moura HB² and Takata RI¹

¹ Molecular Genetics Laboratory; ² Cirurgic Pathology Laboratory, Sarah Network of Rehabilitation Hospitals, Brasília‐DF, Brazil

Gliomas are the most frequent primary tumors from the central nervous system. Historically, the morphology and immunohistochemical analysis were the first tools used for diagnosis (astrocytic, oligodendroglial, mixed oligo‐astrocytic or ependymal tumors) according World Health Organization (WHO). Recently, the use of genetics biomarkers that carry both diagnostic and prognostic information is providing a progress in the classification of those tumors, permitting a “subtyping” of histologic similar tumors and aiding in differential diagnosis. These “molecular status” of the tumor in a recently future will become an integral part of the modern neuro‐oncology practice, helping to guide clinical decision making, estimating the outcome and predicting benefits from specific types of therapies. The analysis of some molecular biomarkers of gliomas becomes to be used at Sarah Network of Rehabilitation Hospitals about 7 years ago. Until now brain tumors of 68 patients were submitted to analysis of 1p, 19q and 10q loss of heterozygosity (LOH), hypermethylation of MGMT gene, mutations in IDH and IDH2, amplification of EGRF gene and BRAF fusions. This work provides a retrospective analysis of those markers to illustrate how the assessment of these data can assist the clinicians.

P28‐40

Molecular classification of gliomas

Martinetto H¹, Fernández Gamba A¹, Núñez L¹, Arakaki N², Riudavets M², Calvar J³, Diez B⁴, Cervio A⁵, Arias E¹, Surace E¹, Taratuto A² and Sevlever G²

¹ Molecular Biology Laboratory, Department of Neuropathology and Molecular Biology, FLENI; ² Neuropathology Laboratory, Department of Neuropathology and Molecular Biology, FLENI; ³ Department of Neuroimaging, FLENI; ⁴ Department of Neurooncology, FLENI; ⁵ Department of Neurosurgery, FLENI

FLENI: Montañeses 2325 (C1428AQK) Buenos Aires, Argentina

Introduction: Gliomas are currently classified according to their hypothetical line of differentiation as oligodendrogliomas, astrocytomas and oligoastrocytomas; this classification also includes grades, depending on malignancy degree. However, it is recognized that lack of reliable immunohistochemical markers results in variations in the diagnosis. This highlights the need for more accurate tools to perform such a classification. Towards fulfilling this goal, we propose a classification model based on genomic alterations.

Material and Methods: This study included 230 gliomas, which had at least a two‐ year follow‐up. We detected point mutations in IDH1, IDH2 and TP53 genes along with copy number alterations in chromosomes 1p, 19q and 10q. Amplification of EGFR gene and homozygous deletion of CDKN2A gene were also assessed. Two subsets of tumors were employed for expression profiling and metabolomic profiling.

Results: Presence of IDH mutations allowed us to divide gliomas in two main groups which displayed strong differences in over‐all survival (OS) and progression free survival (PFS). This correlated with marked differences in expression and metabolomic profiles. Further sub‐grouping could be performed considering other genomic alterations such as 1p‐19q loss, EGFR amplification or TP53 mutation.

Conclusions: The resulting sub‐groups showed better correlation with OS and PFS than histological classification. Considering the current hypothesis about gliomas originating from stem cell or glial precursors, it is possible to speculate about different cells of origin for each subgroup; these cells of origin may be only susceptible to certain alterations and they may predominate in particular brain regions depending on age.

P28‐41

Molecular inversion profiling – a helpful tool in diagnostics and prognostification of tumors of the CNS

Sundar P¹, Last A¹, Mancini M¹, Dörner E², zur Mühlen A², Gessi M² and Pietsch T²

¹Department of Neuropathology, University of Bonn, Germany; ²Affymetrix, Santa Clara, USA

Brain tumor entities are characterized by specific copy number alterations (CNA). We aimed to analyze molecular inversion profiling (MIP) as a tool to identify CNA in brain tumor diagnostics and to compare this method to FISH and MLPA.

Genomic DNA extracted from up to 20 years old FFPE materials from >1100 brain tumors covering most WHO entities were analyzed by MIP profiling (Oncoscan V2, Affymetrix). MIP revealed genome‐wide copy number information from as little as 20 ng of degraded DNA; drop‐out rate was <5%. In contrast to FISH and MLPA, MIP allowed a genome‐wide analysis, adding significant information to the differential diagnosis. Characteristic CNA were detected, including BRAF duplications in pilocytic astrocytomas, chromosome 22 loss in ATRT, chromosome 10 loss and EGFR amplification in glioblastoma, 1p19q codeletion in oligodendroglial tumors, chromosome 2 gain and C19MC amplification in ependymoblastoma. Novel prognostic markers including MYCN amplification in CNS‐PNET were identified. Established prognostic markers including MYC and MYCN amplifications in medulloblastomas and chromosome 1q gain in ependymomas were easily detected and validated by orthogonal methods. MIP also detected copy‐neutral LOH and tumor‐associated point mutations. Our data indicate that MIP is a sensitive and robust method to assess CNA in FFPE tumor material.

P28‐42

Multicentric intracranial juvenile xanthogranulomas

Abdulkader MM¹, Gener MAH¹, Kovanda TJ², Boaz JC² and Bonnin JM¹

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine and ² Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

Juvenile xanthogranulomas (JXG) usually present as cutaneous lesions affecting children. They are the most common form of non‐Langerhans cell histocytoses in children and adolescents. Extracutaneous lesions may involve multiple organ systems including the nervous system, eyes, and orbital soft tissues. We report a12‐year old boy who initially presented with multiple cutaneous nodules. They appeared as yellow papules measuring up to three mm in diameter. They were distributed around eyes, neck, trunk, inguinal region and scrotum. A skin biopsy displayed the classical morphological features of JXG with a dense lymphohistiocytic infiltrate, Touton multinucleated giant cells and occasional eosinophils. Despite appropriate therapy, new skin lesions continued to appear. Later, the child complained of fatigue and developed panhypopituitarism and diabetes insipidus. Magnetic resonance imaging of the head revealed numerous round, enhancing, intraparenchymal nodules scattered throughout the cerebrum, cerebellum and brainstem. The ventricles were not dilated. A subtle enhancement was noted in the infundibular region, the optic chiasm and the optic nerves. A skeletal survey showed no abnormalities. Excisional biopsy of one of the lesions revealed a rubbery well‐demarcated nodule. Histologically, it had the features of JXG and a rich myxoid stroma. Classical Touton giant cells were not observed. Surgical resection of single lesions have been reported to be curative in some cases but chemotherapy is the treatment of choice for multicentric lesions. Radiotherapy is usually not indicated in infants but it has been used in older children and adolescents, particularly in those who are refractory to chemotherapy.

P28‐43

NF1 copy number alterations in diffuse gliomas: a comparative study

Vizcaíno A, Shah S and Rodriguez FJ

Faculty of Medicine, UNAM, Mexico City, Mexico; Johns Hopkins University, Baltimore, MD, USA

Background: Recent studies have identified somatic alterations in the gene encoding for neurofibromin (NF1) (mutations/deletions) in a subset of glioblastoma (GBM), usually associated with the mesenchymal molecular subtype. In the current study, we tested for NF1 copy number alterations in a cohort of diffuse gliomas, evaluated public databases and searched for associations with pathologic and molecular features.

Methods: A cohort of 130 diffuse gliomas (11 diffuse astrocytomas (DA), 16 anaplastic astrocytomas (AA), 17 oligodendroglial tumors (OT), and 86 GBM (57 adult, 29 pediatric) were successfully evaluated for NF1 copy number alteration using tissue microarrays. A custom made probe targeting the NF1 gene (17q11.2) and a control probe targeting the centromere of Ch17 were used.

Results:  NF1/17q or Ch17 gains/polysomies were identified in 40/130 (31%) cases across tumors tested, including 2/11 DA, 6/16 AA, 7/17 OT and 25/86 (29%) GBM (8/29 peds‐GBM, 17/57 adult GBM). However, NF1/17q (n = 2) or whole Ch17 (n = 3) losses were only identified in the GBM group (5/86 (6%)). When looking at this subset with NF1/Ch17 loss, the tumors were predominantly adult GBM (4/5), lacked EGFR amplification (0/4), strong p53 immunolabeling (1/5) or mutant IDH1 (R132H) protein expression (0/5). Conversely, the tumors expressed the mesenchymal marker podoplanin 4/5 more frequently than other diffuse gliomas 55/122 (45%). When looking at genomic data of diffuse gliomas from the TCGA using the cBIOPORTAL for cancer genomics website from MSKCC (http://www.cbioportal.org/public‐portal), there was a trend for homozygous NF1 deletions to be more frequent in glioblastomas (6/281 (2%)) compared to lower grade gliomas (1/262(0.4%)), although this did not achieve statistical significance (p = 0.12, Fisher exact test).

Conclusion:  NF1/Ch17 gains occur in a subset of diffuse gliomas, irrespective of grade and pathologic subtype. Conversely, NF1/Ch17 loss identifiable by FISH is restricted to a small GBM subset, with possible unique pathologic and molecular features.

P28‐44

Non‐adenomatous tumors of the pituitary: an important diagnosis not to overlook

Weidenfeld J, Barshack I and Nass D

Chaim Sheba Medical Center, Tel Hashomer, Department of Pathology, Ramat Gan, Israel

Pituitary tumors are relatively frequent and the vast majority consists of adenomas of the anterior hypophysis. However, less frequent tumors are occasionally found and must be sought whenever the morphological and phenotypical features are not typical of adenomas. We report a series of cases of pituitary tumors that were clinically and radiologically suspected as being adenomas, but histologically were shown to be rarer and distinct tumors other than adenomas. We reviewed the computerized pathological records for sellar tumors and selected the non‐adenoma cases, during the years 2005 until 2013. The histological types found included: two cases of pituicytoma, one case of pilocytic astrocytoma, one ganglion tumor (associated with GH‐expressing adenoma), one granular cell tumor, one case of meningioma and one case of melanocytic lesion (favoring melanocytoma). We described the histological findings and for every case the specific immunostains which were performed to support the morphological diagnosis. Hormonal adenohypophyseal markers were done and shown absence of functional anterior pituitary tissue in every case except for the mixed ganglion cell tumor‐adenoma. In conclusion, although the adenomas are by far the most common tumor in the pituitary, its differential diagnosis includes many other benign and malignant neoplasms. A careful neuropathologist should be aware of the presence of these rare tumors in order to get a precise diagnosis.

P28‐45

Oil cyst content of craniopharyngioma (oil machinery fluid) used as experimental model. And intermediate filament and extracellular matrix analysis

Tena‐Suck ML, Hernández‐Campos ME, Lara CS, Sánchez A and Santamaría A

Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina, Instituto politécnico Nacional, Departamento de Neuropatología, Instituto Nacional de Neurología y Neurocirugía, México

Background: Craniopharyngioma is a cystic tumor that containing an oily material and little is known about the characteristics of this fluid. The aim of this study was to conduct an experimental model using the oil machinery fluid content craniopharyngioma cyst and know the side effects on normal brain tissue.

Material and Methods: The craniopharyngioma cyst fluid was collected during the surgery and administered by stereotaxic surgery in left frontotemporal lobe in rats. Rats were randomly divided into three groups; control; sham, saline solution and craniopharyngioma cyst fluid called as oil fluid. Rats were sacrificed at 15, 30 and 45 days after surgery. Histological and immunohistochemistry (nestin, PGAF, PGAF delta, metalloproteinase 2, and 9, laminin and myelin) analysis were performed. Damage area and subventricular zone were studied for each case at differences times. Labeling index of astrocytes was counted for each primary antibody.

Results: We observed that the oil content of the fluid craniopharyngioma is rich in protein and fat separating and producing extracellular matrix destroys vascular damage foamy macrophages, and inflammation. We noted that group that was injected with the oil content showed higher expression of astrocytes nestin+ and extracellular matrix destruction associated with inflammation than in the control groups; alteration of the blood brain barrier and astrocyte proliferation of the sub ependymal zone those immature astrocytes nestin+, GFAP delta+, vimentin+ and GFAP– were involved in repair in the injured areas and in cerebral plasticity process.

Conclusion: Oil machinery fluid of craniopharyngioma may acts as toxic in normal brain and promotes a new astrocyte population.

P28‐46

Orbital and intracranial perineural arachnoid gliomatosis

Gener MAH¹, Abdulkader MM¹, Kralik SF², Burgett RA³ and Bonnin JM¹

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine, Department of Radiology, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, IN, USA

Optic pathways gliomas (OPG) represent 2 to 5% of all brain tumors in children and may involve the optic nerve, chiasm and the optic tracts. They are among the most common nervous system tumors in patients with neurofibromatosis type I (NF1). Classically, they have an intraneural growth pattern but sometimes, the bulk of the neoplastic proliferation involves the subarachnoid space, a pattern known as perineural arachnoid gliomatosis (PAG). We report a case of a 10‐month old female evaluated because of right sided proptosis. Imaging studies revealed a tumor involving the intraorbital portion of the optic nerve with extension into the intracranial compartment. The tumor did not reach the optic chiasm but it involved the cavernous sinus, compressing the internal carotid artery. Gross total resection of the tumor was achieved. Histological examination revealed that the main component involved the subarachnoid space. It consisted of elongated GFAP‐positive astrocytes intermingled with fibroblasts and abundant reticulin fibers. Six month later, regrowth of the tumor was documented and the patient was treated with chemotherapy. On serial MRIs over the next 9 years, there was no progression of the mass. At age 10 years, worsening proptosis led to resection of the tumor and eye enucleation. Histologically, no anaplasia was observed. This growth pattern of OPG has been reported more commonly in NF1 but it has been observed in patient without any other manifestations of the disease. Morphologically, they resemble desmoplastic infantile astrocytomas but these are usually supratentorial and are not known to involve the optic nerve.

P28‐47

Perineuronal satellitosis in diffuse gliomas versus non‐diffuse glioma lesions: a quantitative analysis

Mezmezian MB¹, Vega PD¹, Petrozzino F¹, Carassai MB¹, Vidal N², Deforel ML³

¹ Department of Pathology, Hospital Ignacio Pirovano, Buenos Aires, Argentina; ² Institute of Neuropathology, Department of Pathology, IDIBELL, Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain; ³ Committee of Teaching and Research, Hospital Ignacio Pirovano, Buenos Aires, Argentina

Introduction: Perineuronal satellitosis (PS) is a secondary Scherer structure characterized by a collection of glioma cells around neurons. It is useful for pathological diagnosis of diffuse gliomas, especially when the solid tumor tissue has not been resected. However, a hypercellularity of oligodendrocyte‐like cells with an increase in normal satellite oligodendroglia was described in temporal lobes resected to control seizures and in the environ of vascular malformations. These situations can mimic a well‐differentiated oligodendroglioma or the edges of other diffuse gliomas.

Objective: To quantify the PS in diffuse gliomas versus non‐diffuse glioma lesions to evaluate the morphological relevance of the PS in the diagnosis of the former.

Material and Methods: 104 cases were evaluated. Group 1: diffuse gliomas, n = 49; group 2: non‐glial tumors (meningiomas, metastasis, primitive neuroectodermal tumors), n = 40; group 3: non‐tumoral lesions (mesial temporal sclerosis, brain abscesses, vascular malformations), n = 15. Cells per neuron were counted using hematoxylin‐eosin slides. Immunohistochemistry for Ki67 was done. Statistical analyses were performed.

Results: There were more oligodendrocyte‐like cells per neuron in group 1 (1.24 ± 0.49) than in group 2 (0.82 ± 0.34) (p < 0.00001 Kruskal–Wallis). Group 3 (0.98 ± 0.24) showed no significant differences with either. Some of these cells expressed Ki67 in group 1 but they did not in group 3.

Conclusion: This analysis reveals that PS is not a pathological feature exclusive of diffuse gliomas. Neither does it help differentiating gliomas from lesions associated with hypercellularity of oligodendrocyte‐like cells, such as some epileptogenic non‐tumoral lesions. In incomplete resections, Ki67 is useful for doing this differential diagnosis.

P28‐48

Pineal parenchymal tumor of intermediate differentiation: a case report

Zunarelli E, Nosseir S, Mengoli MC and Mataca E

Division of Surgical Pathology, University Hospital Policlinico di Modena, Modena, Italy

Introduction: Neoplasms of the pineal region comprise less than 1% of all intracranial tumors, 14–27% of which are of pineal parenchymal origin (pineal parenchymal tumors, PPTs).

PPTs are further classified as pineocytomas (WHO grade I), PPTs of intermediate differentiation (WHO grade II or III, with definite criteria yet to be established) and pineoblastomas (WHO grade IV).

We report on a case of PPT of intermediate differentiation, which was designated WHO grade II.

Materials and Methods: A 64‐year‐old gentleman presented with mnesic deficits. Neuro‐imaging disclosed a well circumscribed, contrast‐enhancing mass in the pineal region, less than 2 cm in maximum dimension, with peripheral calcifications and microcystic change.

A completed surgical resection was achieved.

On gross examination, the specimen consisted of a brownish nodule measuring 1,5 × 1 cm, which was embedded in toto.

Results: Hematoxylin and eosin stained sections showed a moderately cellular tumor composed of sheets and lobules of uniform cells with mild nuclear atypia and few mitotic figures. No necrosis was identified. Positive immunoreactions for synaptophysin and neuron‐specific enolase were observed. Variable labeling was seen with antibodies to neurofilament protein and chromogranin. Immunoreactions for GFAP, PLAP, CD117/c‐kit, EMA and MNF‐116 yielded negative results. Ki67/MIB‐1 labeling index was 3%.

The patient is alive 4 years after surgery and followed up by neuroradiology, with no evidence of recurrence.

Conclusions: The low mitotic and proliferative activity indices, as well as the absence of necrosis and the positive immunostain for neurofilament protein, accurately define grade II PPT of intermediate differentiation.

P28‐49

Primary central nervous system lymphoma (PCNSL): a retrospective analyze of Cancer National Institute 2009–2014 cases

Madruga M, de Mesquita GG and da Silva Camacho AH

National Cancer Institute (INCA), Brazil

Introduction: Primary central nervous system lymphoma (PCNSL) is an extranodal non‐Hodgkin lymphoma in the brain, leptomeninges, spinal cord or eyes. The incidence of PCNSL increased approximately three‐fold in the last decades. Although individuals infected with HIV and other immunocompromised individuals have a 3,600‐fold increased risk of developing PCNSL compared with the general population, the incidence has not increased only in this kind of group. The clinical presentation depends on the location of the tumor with neurological rather than systemic symptoms. Computed tomography (CT) and magnetic resonance imaging (RMI) are essential in diagnosis, however the gold standard is tumor biopsy.

Material and Methods: We retrospectively analyzed cases of primary central nervous system lymphoma (PCNSL) diagnosed during 2009–2014 in National Cancer Institute. Immunocompromised and HIV positive patients were excluded from the study. The study analyzed age; sex; ECOG (Eastern Cooperative Oncology Group) performance status in the moment of the diagnosis; site of the lesion in the central nervous system; the subtype of lymphoma, with a histopathology review; treatment and survival of the patients.

Conclusion: We identified 35 cases of primary central nervous system lymphoma and in a previous analysis the range age was 4–81 and most of them (29 cases) got the B cell large diffuse lymphoma. The study is in curse and our data are similar to those previously described in literature.

P28‐50

Primary extraosseous Ewing's sarcoma of the central nervous system. Eighteen cases in a single institution

Sevlever G¹, Cervio A¹, Martinetto H¹, Cerrato S¹, Diez B¹, Riudavets M¹, Arakaki N and Marrón A²

¹ FLENI; ² Laboratorio BIO Lab, Argentina

Primary CNS Ewing's sarcoma not related to bone or metastatic disease is a rare condition generally affecting children and young adults. They are mostly located supratentorially and less frequently in the posterior fossa, spinal cord or the meninges. The characteristic chromosomal translocation t(11,22)(q24;q12) or other rare variants such as t(21,22)(q22;q12) that result in the fusion of EWS gene to FLI1 gene or to ERG gene are essential in the differential diagnosis of ES from central PNET and medulloblastoma.

A retrospective study from November 2007 to May 2014 revealed eighteen patients with CNS ES diagnosis, including thirteen males and five females with a median age of 22 years (2 to 65 y). In nine cases exeresis were performed in our institution and the others were received as second opinion with diagnosis of medulloblastoma, glioma, meningitis, neurofibromatosis and cPNET.

Six cases were infratentorial and seven had leptomeningeal involvement.

The tumor consisted of undifferentiated small round cells arranged in sheets and nodules. Nuclei were monomorfous and hypercromatic with scant cytoplasm, but some cases showed vesicular nuclei and pale cytoplasm. Immunostaining for synaptophysin was moderate, vimentin was focally positive, CD99 was membranous and cytoplasmatic in areas.

Diagnosis of pCNSPNET was confirmed by RT‐PCR; sixteen cases presented the EWS/FLI1 fusion while EWS/ERG was detected in one case. Four cases were reconfirmed by the presence of Ewing's sarcoma family tumors (ETF) specific non coding transcript(ncRNA) AK057037 in frozen tissue.

P28‐51

Radiographically localized biopsies reveal subtype‐specific differences in molecular and cellular composition at the margins of glioblastoma

Gil B^1#, Pisapia D^2,5#, Malone H¹, Goldstein H¹, Lei L², Sonabend A¹, Yun J¹, Samanamud J¹, Sims J¹, Teich AF², Sheth S¹, McKhann G¹, Sisti M¹, Bruce JN¹*, Sims PA^3,4,* and Canoll P^2,*

¹ Department of Neurological Surgery, Columbia University Medical Center, New York, NY, USA; ² Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA; ³ Department of Systems Biology, Columbia University Medical Center, New York, NY, USA; ⁴ Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY, USA; ⁵ Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA (Present address)

^#These authors contributed equally to this work.

Glioblastomas (GBM) remain notoriously difficult‐to‐treat tumors in part because of their diffusely infiltrative nature, which prevents complete resection. Following surgical resection of a tumor's contrast‐enhancing component, oncologists are faced with treating the remaining tumor within reactive, non‐neoplastic brain parenchyma that provides the environment for continued tumor growth and progression. Indeed, this portion of the tumor is seldom studied. Here, histological, immunohistochemical, and/or RNAseq analysis was performed on 69 patients with high grade glioma. Biopsies were designated as coming from either contrast enhancing (CE) areas or non‐enhancing (NE) areas. We found that samples from CE areas were molecularly distinct from those of NE areas with the former showing predominantly mesenchymal, classical, or proneural phenotypes, and the latter showing predominantly neural phenotypes. To assess the contribution of individual cell types to these molecular profiles, we used a computational approach to assign relative levels of gene expression for each gene to several cell types including oligodendrocytes, astrocytes, neurons, and microglia. Differentially expressed genes in tumor samples relative to normal brain were assigned to a diversity of cell types. Moreover, the contribution of a given cell type to a sample's molecular profile within NE samples varied with the molecular class designation assigned to the tumor's corresponding CE component. Thus, it becomes possible to derive information about residual tumor from surgical resection. Furthermore, we demonstrate a method for parsing out cell‐type specific contributions to a tumor sample's molecular profile.

P28‐52

Role of IGFBP family members and their downstream molecules in influencing glioblastoma pathogenesis and patient prognosis

Santosh V¹, Shastry AH¹, Thota B¹, Arivazhagan A¹, Thennarasu K¹, Praveen Kumar VR² and Kondaiah P²

¹ National Institute of Mental Health and Neurosciences; ² Indian Institute of Science, India

Background: Insulin‐like growth factor binding protein family (IGFBPs) has been implicated in the pathogenesis of glioma.

Aims: To evaluate the expression pattern, functional role and downstream molecules of IGFBPs in glioma and their prognostic relevance in GBM.

Methods: The mRNA and protein levels of the selected IGFBPs were evaluated using RT‐PCR and immunohistochemistry in astrocytoma. Functional studies (proliferation, invasion, migration and colony formation assays) were performed on glioma cell lines using RNA interference and exogenous protein expression techniques. Downstream protein targets were identified on western blot. The IGFBPs and selected downstream targets were evaluated for prognostic significance in a uniformly treated cohort of 154 GBM patients with long term follow‐up. Survival analyses were done using Kaplan–Meier and Cox regression models.

Results: IGFBP‐2, ‐3, ‐4 and ‐5 emerged as GBM specific markers. IGFBP‐3 was identified as an early predictor of death in GBM patients (Santosh et al. CEBP 2010; 19: 1399–408). IGFBP‐2 emerged as a predictor of early progression and poor overall survival on long term follow‐up. Functional studies on glioblastoma cell lines identified IGFBPs to promote cellular proliferation, migration, invasion and colony formation and EMT markers in vitro (Praveen Kumar et al. J Neurooncol. 2014; 116: 455–64). IGFBP‐3 regulated the expression of STAT1 (signal transducer and activator of transcription 1) in glioma cell lines. STAT‐1 was also overexpressed in human GBM tissues conferring aggressiveness to the tumor by enhancing its invasive potential and predicted poor prognosis.

Summary: Our study substantiates the critical role of IGFBP isoforms in glioblastoma pathogenesis and demonstrates STAT1 as a novel prognostic marker in glioblastoma.

Keywords: glioblastoma, prognosis, IGFBP, STAT1.

P28‐53

The biomarker potential of MGMT protein in glioblastoma is improved by exclusion of non‐tumor cells

Klitkou J^1,2, Dahlrot RH^2,3, Hansen S^2,3 and Kristensen BW^1,2

¹ Department of Pathology, Odense University Hospital, Odense, Denmark; ² Institute of Clinical Research, University of Southern Denmark, Odense, Denmark; ³ Department of Oncology, Odense University Hospital, Odense, Denmark

Introduction: Methylation of the DNA repair protein O6‐methylguanine‐DNA methyltransferase (MGMT) gene promoter is predictive of benefit from temozolomide (TMZ) in glioblastoma (GBM). Non‐tumor cells are numerous in GBMs and a potential source of bias in attempts to determine MGMT status by immunohistochemistry. The aim of the present study was to explore the prognostic potential of MGMT protein expression excluding these cells.

Methods: Immunofluorescence multiplexing and automated analysis were combined to quantify MGMT (green fluorescence) in tumor cell nuclei using image analysis algorithms. Microglia/macrophages, lymphocytes and vascular structures (red fluorescence) known to express MGMT were excluded from the analysis. The study included paraffin sections from 173 primary GBMs.

Results: High area fraction of tumor cell nuclei expressing MGMT was associated with poor prognosis (HR = 1.59, p = 0.005). Prognostic value of MGMT expression was particularly strong in patients treated with TMZ as first line therapy (HR = 2.31, p = 0.0003). The 2‐year survival in this group was 44% versus 7% for patients with low and high expression respectively. Non‐tumor cell nuclei often showed higher MGMT expression than adjacent tumor cells. Exclusion of these cells was shown to improve the prognostic value of MGMT expression.

Conclusion: MGMT protein expression in tumor cell nuclei holds prognostic potential in GBM. Exclusion of non‐tumor cells from the analysis is crucial to avoid false positives. The path to possible clinical implementation will require further testing in independent patient cohorts as well as development of a highly robust multiplexing assay with not yet defined cut‐off levels.

P28‐54

The influence of p53 expression status on the association of STAT1 and MMP‐9 with survival in glioblastoma: from molecular biology towards personalized medicine

Shastry AH¹, Thota B², Durgad S², Thennarasu K³, Arivazhagan A⁴, Somanna S⁴, Kondaiah P⁵ and Santosh V^1,2

Departments of ¹ Clinical Neuroscience, ² Neuropathology, ³ Biostatistics and ⁴ Neurosurgery, National Institute of Mental Health and Neurosciences, Bangalore, India; ⁵ Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India

Background: We have previously demonstrated IGFBP‐3, a protein over‐expressed in glioblastoma, to regulate the expression of STAT1 (signal transducer and activator of transcription 1). Here we evaluate the functional significance of STAT1 in malignant glioma cells, its prognostic role in glioblastoma and its relation with the well established markers of invasion. In view of inter‐tumoral heterogeneity in GBM we explored their prognostic significance with respect to p53 expression.

Methods: Immunohistochemistry (IHC) for STAT1 expression was performed on tumor tissues from a cohort of uniformly treated glioblastoma patients (n = 154). Survival analyses were done using Kaplan–Meier and Cox regression models. The functional significance of STAT1 was investigated by gene knockdown approach on U251 and LN229 cell lines. Expression of MMP‐2 and MMP‐9 was studied by IHC. P53 IHC expression, stratification and subgroup specific evaluation of STAT1, MMP‐2 and MMP‐9 with survival was performed.

Results: STAT1 protein expression was a significant predictor of shorter survival in glioblastoma patients, further STAT1 knockdown impaired invasion in glioma cell lines. No correlation was noted between STAT1 and MMP‐2, MMP‐9 expression patterns. Survival analysis following stratification based on p53 expression revealed strong correlation of MMP‐9 and STAT1 with both poor overall and progression free survival.

Conclusions: We have demonstrated STAT1 as a novel invasive marker in glioblastoma, and subsequently shown STAT1 and MMP‐9 to associate with prognosis only in p53 expressing GBMs. This study highlights that the stratification of GBM into subgroups based on p53 status provides a unique opportunity to identify newer therapeutic targets.

P28‐55

Thirteen years of histopathological and cytogenetic studies of meningiomas at Sarah Network of Rehabilitation Hospitals – Brasilia/DF, Brazil

Abe KT, de Moura HB, de Oliveira MDV, Sakai Jr N, Formigli LM, Roese LL, Schneider M, Pereira MF, de Souza FS and Brandão ICS

Citogenetic – Molecular Patology and Surgical Patology of SARAH Network of Rehabilitation Hospitals, Brasília – DF, Brazil

Meningiomas are common central nervous systems tumors that account for up 30% of all primary intracranial neoplasms. The World Health Organization (WHO) classifies meningiomas into three histological grades: grade I (benign), grade II (atypical), and grade III (anaplastic). Most meningiomas (80%) are benign tumor grade I, and meningiomas grade II (15–20%) and grade III (1–2%) are more aggressive. We reviewed data from 88 patients diagnosed as grade I (86.3%), grade II (11.3%), grade III (2.7%) meningiomas from January 2001 to March 2014. The sex ratio was 63 female:13 male, 6 female:4 male and 2 female:0 male for grade I, II and III, respectively. The average age of all patients for grade I meningioma was 51.4 years and 59.8 years for grade II/III. Females were affected more frequently than males for grade I (4.4F:1M) and for grade II/III (1.5F:1M) meningiomas. The more usual cytogenetic aberration was monosomy 22/deletion 22q (54 samples). Only grade I meningiomas presented monosomy 22 as a single abnormality. In grade, II or III this aberration was always associated to other abnormalities as gains and losses, and/or telomeric association supporting their higher aggressiveness. The amount and cumulative chromosome abnormalities in higher grades meningiomas is indicative of genomic instability that allows progression to more aggressive tumors. Moreover, the differences observed in the karyotype of grade I, II and III meningiomas also support the idea that a combination of both histopathological and cytogenetic descriptions of meningiomas could result in improved prognostic accuracy.

P28‐56

Two cases of ependymosarcoma, a proposed entity

Aizpurua M¹, Bodi I¹, Laxton R¹, Cheserem JB³, Ashkan K^2,3, Bhangoo R^2,3 and Al‐Sarraj S¹

¹ Clinical Neuropathology, ² Neuro‐oncology and ³ Neurosurgery, King's College Hospital NHS Foundation Trust, London, UK

Introduction: Gliosarcomas are infrequent primary CNS tumors characterized by its biphasic pattern of glial (mainly astrocytic) and sarcoma and considered as a subtype of glioblastoma WHO grade IV. However tumor with both ependymal and sarcoma components (ependymosarcoma) are very rare and not included in the current WHO classification.

Materials and Methods: Case 1. A 50 year‐old male presented with a short history of headache, blurred vision and altered sense of smell. MRI showed an enhancing temporal mass. Despite radio‐ and chemotherapy, the patient had tumor progression and died 1 year after diagnosis. Case 2. A 59 year‐old male presented with weight loss, dizziness, nausea, double vision and vomiting for the last 2 years. MRI showed a heterogeneously enhancing lesion arising from the floor of the fourth ventricle.

Results: Case 1 exhibited mainly an anaplastic ependymoma, mixed with sarcomatous and small epithelial areas. Case 2 showed low grade areas with subependymal and ependymal features, mixed with high grade ependymal and sarcomatous areas of osteocartilaginous differentiation. Immunohistochemistry showed focal GFAP and EMA expression in the ependymal component in both cases. Focal immunopositivity was noted for desmin and SMA in the mesenchymal component of Case 2. The MGMT was methylated in Case1 in both components. However in Case 2, MGMT was unmethylated in the high grade area but methylated in the low grade component.

Conclusion: The two cases showed histological features of ependymosarcoma. It is possible that both the components are derived from the neoplastic ependymal cells similar to the view of histogenesis of gliosarcoma. This is supported by the mixed high grade ependymal component with the sarcoma in case 1. However in case 2 (located in the fourth ventricle) there is a clear separation of areas of ependymoma with low grade features (some with features of subependymoma) from those of malignant ependymoma and sarcoma raising the possibility of a collision tumor which is supported by differences in MGMT methylated status in the two components. The grading is not clear depending on the current WHO criteria but it remains to be seen if ependymosarcomas have a different prognosis from gliosarcomas.

P28‐57

Ultrastructure of Rosenthal fibers in the boundary of craniopharyngioma and experimental model using the oil machinery fluid of the cyst contents of craniopharyngioma

Tena‐Suck ML, Hernández‐Campos ME, Hernández AD, Fernández‐Valverde F and Santamaria A

Escuela Superior de Medicina, sección estudios de postgrado, Instituto Politécnico Nacional y Departamento de Neuropatología, Instituto Nacional de Neurología y Neurocirugía, México DF, México; Departamento de Patología, Instituto Nacional de Rehabilitación, México DF, México; Laboratorio de Neuropatología experimental, Instituto Nacional de Neurología y Neurocirugía, México DF, México

Background: Craniopharyngioma is a slow‐growing epithelial tumor, which is often cystic, encapsulated and slow‐growing. Certain of these tumors can behave in an aggressive manner and either invade surrounding structures or recur. The cystic structures contain a dense oily fluid that that comes out through separation and necrosis of epithelial cells that damaged to normal brain tissue.

Objective: To investigate pathological changes of oil fluid content in brain of patients with craniopharyngioma and Rosenthal fibers formation.

Methods: Ten cases of adamantinomatous craniopharyngiomas were examined by light and electron microscopy and special attention was paid to the formation of Rosenthal fibers and boundary tissue. Oil machinery fluid cyst content of craniopharyngioma was administered to Wistar rat with propose to obtain Rosenthal fibers.

Results: In both, experimental model and biopsies, the presence of cellular debris abundant lipid‐laden macrophages and also inflammatory cells we observed. Macrophages were filled with lipids and reach break and in the absence of lysosomes and become unable to engulf. This response promotes the presence of immature astrocytes, which are able to repair the damage. FRs are formed by the accumulation of intermediate filaments of the extracellular matrix are filled with lipids. We did not find Rosenthal fibers in rat brain.

Conclusions: These results demonstrate focal pathological disturbances and tumor‐specific cellular environment at the tumor–brain junction, which produce different degrees of cellular disorganization and Rosenthal fibers formation, aberrant filament–filament interactions that then lead to protein aggregation an effect which induces mutant proteins accumulation. So we believe that the formation of FR is the result of greater and continuously exposure of oil content in human brain tissue that is not formed by a single dose in the rat brain.

P28‐58

Unusual variant of an unusual orbital tumor: biphasic solitary fibrous tumor with malignant epithelioid component

Gardiman MP¹, Pizzi M¹ and Montesco MC²

¹ General Pathology and Cytopathology Unit, Department of Medicine‐DIMED, Padua University Hospital, Padua, Italy; ² Melanoma and Sarcoma Pathology Unit, Veneto Institute of Oncology, IOV‐IRCCS, Padua, Italy

A 42 years‐old Caucasian man presented at Padua University Hospital for swelling of the right eye. His past medical history was unremarkable but for a previous diagnosis of benign solitary fibrous tumor (SFT) of the right orbit, removed 19 years before. The patient underwent MRI that disclosed a right retro‐orbital mass (diameter: 5 cm) infiltrating the surrounding bone tissue. Following surgical excision, histological examination revealed a biphasic mesenchymal proliferation composed by both a spindle and an epithelioid cell component. The spindle cell component was characterized by a fascicular growth pattern with hemangiopericytoma‐like vessels. Spindle cells were cytologically bland and sharply positive for CD34, vimentin and CD99. The epithelioid cell component was characterized by nests of highly atypical cells (>10 mitoses/10HPF), positive for cytokeratin AE1/AE3, CAM5.2, CK7 and MNF116. Cytogenetic analysis did not show recurrent chromosomal abnormalities. Based on these histological and immunohistochemical features a diagnosis of biphasic SFT with malignant epithelioid component was posed. Histological revision of the previously excised lesion confirmed the diagnosis of benign SFT with no epithelioid features. Three years after surgical removal, follow‐up CT documented local recurrence and systemic metastatic disease. Conventional chemotherapy failed to attain complete/partial remission. At present (6 years after presentation) the patient is alive with progressive disease.

To our knowledge, this is the first case of benign SFT of the orbit with documented progression to malignant disease and epithelioid features. The peculiar histological features and the aggressive clinical behavior prompts further studies to better characterize this extremely rare entity.

P28‐59

Growth factor signaling pathway activation in meningiomas

Hilton D, Shivane A, Kirk L, Ammoun S and Hanemann CO

Plymouth Hospitals NHS Trust, UK

A significant minority of meningiomas are difficult to treat with surgery or radiotherapy, and there are limited chemotherapeutic alternatives. This study aims to better understand the pathways that are active in these tumors, in order to direct future treatment strategies. We have investigated the expression of several growth factors and their signaling pathways in 30 meningiomas, using immunohistochemistry. Expression was correlated with presumed NF2 gene status, using fluorescent in situ hybridization to look for chromosome 22q loss. Membrane expression of VEGFR and PDGFRβ was seen in 83% of tumors, Axl in 70%, EGFR in 50% and IGFR in 47%. Expression was similar in low and high grade tumors, but membrane EGFR expression was not seen in tumors showing chromosome 22q loss. Expression of IGF, NRG, VEGF, Gas 6, and downstream signaling proteins, Mek, Erk, Jnk and Akt, and pS6RP, was widespread. Our findings suggest that the majority of meningiomas express and show activation of multiple growth factor receptors and their downstream signaling pathways irrespective of tumor grade. In addition to previously reported receptors, Axl may also offer an attractive therapeutic target. Our findings also suggest that anti‐EGFR based therapies may be less effective in meningiomas with 22q loss.

P29. Varia

P29‐01

Rathke cleft cyst with ectopic salivary gland rests

Bonnin JM¹ and Fulkerson DH²

¹ Division of Neuropathology, Department of Pathology and Laboratory Medicine and ² Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

Ectopic islands of salivary gland were found in approximately 3.4 per cent of pituitary glands examined in a large autopsy series, and both benign and malignant salivary‐like tumors involving the sellar region have been reported. A 16‐year‐old female developed visual loss in the left eye over a 3‐day period. Her visual acuity was 20/200 on the left eye and normal on the right. An MRI scan revealed a 1.7 × 1.5 × 1.3 cm bilobed peripherally enhancing cystic mass in the sella turcica. The lesion extended to the suprasellar cistern with mass effect on the optic chiasm. Because of the rapid progression of her visual loss, a trans sphenoidal resection of the lesion was performed. The cyst contained a greenish mucinous fluid. Histopathological examination revealed fragments of a cyst wall lined by cuboidal, focally ciliated, epithelium. The cyst wall contained multiple islands of serous glandular acini compatible with salivary gland rests. Two years later she was found to have bilateral pheochromocytomas, and these were resected. An identical twin also had bilateral pheochromocytomas, resected at 6 years of age and later was found to have an asymptomatic sellar cyst. Although Rathke cleft cysts involving the sellar region are not uncommon and despite the relatively frequent occurrence of salivary gland rests in the pituitary gland, such rests are rarely observed in the cyst walls. Unlike some cases of craniopharyngioma, Rathke cleft cysts have not been linked to any genetic abnormalities and are not known to be part of one of the familial tumor syndromes.

P30. Vascular

P30‐01

Role of skin‐muscle biopsy in diagnosis of cerebral microangiopathies

Dziewulska D^1,2, Bojakowski J¹, Gogol P¹ and Lewandowska E³

¹ Department of Neurology, Medical University of Warsaw; ² Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, PAS, Warsaw; ³ Department of Neuropathology, Institute of Psychiatry and Neurology, Warsaw, Poland

Introduction: Introduction of skin‐muscle biopsy into diagnostics of cerebral small vessel diseases (CSVD) has allowed in vivo detection of several diseases/syndromes like CADASIL, CARASIL, PADMAL, and others. But spectrum of cerebral microangiopathies seems be much wider than hitherto known disorders.

Material and Methods: In 133 patients at the age of 8–57 yrs with clinical suspicion of CADASIL, skin‐muscle biopsy and ultrastructural examination of the biopsy material were performed. None of the patients has typical risk factors for vascular diseases but in brain MRI, all of them demonstrated diffused or focal hyperintense changes in the cerebral white matter.

Results: Ultrastructural examination confirmed CADASIL in 16 patients (12%). In 56 cases (42%) result of the examination was normal. In 61 cases (46%) various but repeating types of morphological abnormalities in microvessels were observed. In some patients, affected endothelial cells and/or pericytes showed changes resembling oncosis. In others, numerous intracytoplasmic vesicular structures, conglomerates of dilated mitochondria, thickened basal lamina or polymorphic deposits of an unknown material different than GOMs in CADASIL were seen.

Conclusion: Skin–muscle biopsy is a useful diagnostic method which not only allows to recognize cerebral microangiopathies of generalized character but also may help to identify new vascular disorders within a heterogenous group of CSVD.

P30‐02

Pitavastatin downregulates MCP‐1 and CCR2 in atherosclerotic lesions of human carotid arteries

Toi S¹, Inose Y², Kawamata T³, Okada Y³, Kitagawa K¹, Uchiyama S¹, Sawada T² and Shibata N²

Departments of ¹ Neurology, ² Pathology and ³ Neurosurgery, Tokyo Women's Medical University, Japan

Atherosclerosis; carotid artery; immunohistochemistry; statin; immunoblotting.

Carotid atherosclerosis occurs based on dyslipidemia, and plaque instability driven by lipid‐triggered inflammation in intima may induce large‐sized cerebral infarction. It is well known that statins exert not only lipid‐lowering but also anti‐inflammatory effects. To elucidate the efficacy of pitavastatin, a member of the statin family, on carotid atherosclerosis, we performed a retrospective study, focusing on the situations of protein expression of the proinflammatory CC chemokine monocyte chemoattractant protein‐1 (MCP‐1) and its receptor CCR2 in atherosclerotic plaques, and compared them between the pitavastatin‐administrated and non‐statin‐administrated control groups (n = 12, each), using morphological and quantitative techniques. Histopathologically, the lesions of the pitavastatin group tended to show improvement of ulceration, thrombosis and plaque hemorrhages the morphological hallmarks of plaque instability. Immunohistochemical analysis revealed that the MCP‐1 and CCR2 determinants were colocalized in CD31‐identified vascular endothelial cells (VECs), CD68‐identified macrophages and α‐smooth muscle actin‐identified intimal vascular smooth muscle cells (iVSMCs) of the lesions. Immunoblot analysis disclosed significant decreases in the β‐actin‐normalized MCP‐1 and CCR2 signals (P < 0.05 and P < 0.005, respectively) in the pitavastatin group as compared to the control group. Our results suggest that pitavastatin downregulates protein expression levels of MCP‐1 and CCR2 in VECs, macrophages and iVSMCs in atherosclerotic lesions of human carotid arteries and ameliorates plaque instability.

P30‐03

Quasi‐moyamoya disease secondary to atherosclerosis in a Hispanic woman

Mezmezian MB¹, Jaroslavsky MJ¹, Dopazo V¹, Gomez S¹ and Miyata H²

¹ Department of Pathology, Hospital Ignacio Pirovano, Buenos Aires, Argentina; ² Department of Neuropathology, Research Institute for Brain and Blood Vessels, Akita, Japan

Introduction: Quasi‐Moyamoya Disease (quasi‐MMD) is an occlusive cerebrovascular disease involving the terminal portion of the internal carotid artery, or proximal portion of the anterior and/or middle cerebral arteries, accompanied by an abnormal vascular network in association with an underlying disease. MMD and quasi‐MMD are mainly found in East Asia. Their incidence in USA ranges from 0.052 to 0.086 per 100,000 population being quasi‐MMD 14.2% of all MM cases.

Patient and Methods: A 37‐year‐old Hispanic woman, suffering from insulin‐dependent diabetes, obesity, hypertension and dyslipidemia, was hospitalized for left brachial hemiparesis, left lingual paresthesia and dysarthria. Cerebral angiography demonstrated luminal stenosis and occlusion of the anterior and middle cerebral arteries, more marked on the left side. The patient was diagnosed with cerebral vasculitis. The corresponding treatment was applied. Despite clinical improvement, some months later she died of another stroke.

Results: The brain autopsy showed recent to old multiple anemic infarcts, mainly in the bilateral internal carotid artery territories, showing severe cortical atrophy in the frontal and parietal lobes. The anterior and middle cerebral arteries presented white plaques and luminal narrowing. Histologically, they showed fibrous lesion‐predominant atherosclerosis manifested by marked fibromuscular intimal thickening and degeneration of the media and internal elastic lamina, showing duplication and splitting as well as focal loss.

Conclusion: Despite having both MMD and quasi‐MMD low incidence in Western countries, if multiple cerebral infarcts associated with severe intracranial atherosclerosis are present in a young patient, quasi‐MMD should be considered as a differential diagnosis.

P31. Veterinary

P31‐01

Distribution of brain lesions in rabbits naturally infected with Encephalitozoon cuniculi

Dell'Armelina Rocha PR¹, Zanet S², Ilaria G², Andrea A², Machado GF¹, Bianco P³, Ferroglio E² and Capucchio MT²

¹ Department of Clinics, Surgery and Animal Reproduction, Universidade Estadual Paulista (UNESP), Faculdade de Medicina Veterinária de Araçatuba, Brazil; ² Department of Veterinary Sciences, University of Torino, Torino, Italy; ³ ASLTo4, Torino, Italy

Introduction: Encephalitozoon cuniculi is an obligate, intracellular spore‐forming, parasitic organism of mammals that causes encephalitozoonosis, an opportunistic zoonosis, that mostly affects immunocompromised individuals. This pathogen has a particular tropism for the brain and kidneys. This report describes the distribution of brain lesions in rabbits naturally infected with E. cuniculi.

Materials and Methods: 104 regularly slaughtered rabbits seropositive for E. cuniculi by means of carbon immunoassay test and 8 rabbits with neurological signs were included. Brains were fixed in 10% formalin and routinely processed for histology. Sections were stained with hematoxilin and eosin. Immunohistochemistry for GFAP was also performed. Fresh brain samples of clinically affected rabbits were frozen at –18°C for PCR and genotyping for E. cuniculi.

Results: Focal/multifocal granulomatous/linfoplasmocytic meningoencephalitis with granulomas and perivascular cuffing were the most significant lesions. Some granulomas presented a necrotic center surrounded by epithelioid‐like and glial cells, sometimes associated with parasitic forms. Hippocampus and temporo‐parietal cortex were the most affected areas, involving both gray and adjacent white matter. Lesions were present less frequently in the thalamus, mesencephalon, pons/obex and cervical spinal cord. The cerebellum was rarely involved. Positive reaction for GFAP was observed especially adjacent to the granulomas. A1110 bp fragment was amplified from tissues of all cases; all belonged to genotype I (rabbit strain).

Conclusions: The distribution of brain lesions of this report may be applicable for the diagnosis of encephalitozoonosis. The identification of genotype I in the clinically affected rabbits underlines that this species may represent an important source of human infection.

P31‐02

Increased Fcγ‐RI staining in the brain of dogs with visceral leishmaniasis correlates with inflammatory changes

Machado GF, Melo GD, Kremer B, Março KS, Grano FG and Silva JES

Laboratory of Applied Pathology (LAPAP), College of Veterinary Medicine (FMVA), UNESP – Univ Estadual Paulista, Araçatuba, São Paulo, Brazil

Visceral leishmaniasis (VL) is an immune‐mediated anthropozoonosis caused by the parasite Leishmania infantum. Infected dogs usually mount an ineffective humoral immune response, with high levels of anti‐Leishmania antibodies in serum and in the cerebrospinal fluid (CSF). The Fcγ‐receptors bind IgG and act as a connection between humoral and cell‐mediated immune responses. Since we have previously detected high amounts of perivascular IgG in the brain and CSF of infected dogs, we aimed to investigate the presence of Fcγ‐RI in the brain of 15 naturally infected dogs and 4 uninfected dogs, and to correlate it with the brain histopathological findings. The brain lesions were assessed by HE‐staining and Fcγ‐RI was detected by immunohistochemistry (Abcam ab104273) and quantified by computerized image analysis. The most common lesions were mild‐to‐marked meningitis (77.5%), choroiditis (74.2%), subventricular gliosis (72.5%) and lymphohistioplasmacytic perivascular cuffs (55.0%). Further, the infected dogs exhibited increased Fcγ‐RI staining (P = 0.0480) at the ependymal surface and in the choroid plexi, as well as in the parenchymal perivascular areas, especially in mononuclear cells and astrocytes. The frequent occurrence of morphological changes in the brain of infected dogs supports previous evidence that the nervous system is also affected during VL. The observation of subventricular gliosis correlates with the location of cells expressing the receptor Fcγ‐RI. Within the brain, the activation of Fcγ‐RI may be a trigger to increase the production of pro‐inflammatory cytokines related to blood–brain barrier disruption, possibly explaining the inflammatory changes in the nervous milieu during the occurrence of VL.