Increased L-[1-¹¹C]leucine uptake in the leptomeningeal angioma of Sturge-Weber syndrome: a PET study

Abstract

Background and Purpose

We used L-[1-¹¹C]leucine (LEU) positron emission tomography (PET) to measure amino acid uptake in children with Sturge-Weber syndrome (SWS), and to relate amino acid uptake measures with glucose metabolism.

Methods

LEU and 2-deoxy-2[¹⁸F]fluoro-D-glucose (FDG) PET were performed in 7 children (age: 5 months-13 years) with unilateral SWS. Asymmetries of LEU uptake in the posterior brain region, underlying the angioma, and in frontal cortex, were measured and correlated with glucose hypometabolism. Kinetic analysis of LEU uptake was performed in 4 patients.

Results

Increased LEU standard uptake value (SUV, mean: 15.1 %) was found in the angioma region in 6 patients, and smaller increases in LEU SUV (11.5 %) were seen in frontal cortex in four of the 6 patients, despite normal glucose metabolism in frontal regions. High LEU SUV was due to both increased tracer transport (3/4 patients) and high protein synthesis rates (2/4). FDG SUV asymmetries in the angioma region were inversely related to LEU SUV asymmetries (r=−0.83, p=0.042).

Conclusions

Increased amino acid uptake in the angioma region and also in less affected frontal regions may provide a marker of pathological mechanisms contributing to chronic brain damage in children with SWS.

INTRODUCTION

Sturge-Weber syndrome (SWS) is a sporadic neurocutaneous disorder characterized by a facial port wine stain and leptomeningeal angiomatosis. The abnormal, tortuous veins on the cortical surface lead to impaired cerebral blood flow, hypoxia and chronic ischemia of the underlying brain tissue. ^{1, 2} The often progressive neurological complications include hemiparesis, visual field defects, seizures and mental retardation. ³ Leptomeningeal angiomas are generally considered to be developmental malformations, the results of a failed regression of the primitive embryonal vascular plexus during the first trimester of gestation. ⁴ Recent studies, however, suggested that these angiomas may not be static lesions but, may undergo dynamic proliferative changes. ^{5, 6} These studies have provided evidence for increased expression of vascular cell proliferation markers in the resected angioma and brain tissue. Histological studies have shown neuronal degeneration, gliosis and calcification in the brain tissue adjacent to the angioma in SWS. ^{5, 7} These degenerative changes are consistent with decreases in glucose metabolism demonstrated by positron emission tomography (PET). ⁸

Increased cell proliferation has been studied in vivo using PET to measure glucose metabolic rates, amino acid uptake (to measure protein synthesis), and uptake of thymidine analogs (to measure DNA synthesis); these approaches are commonly used in the clinical setting to estimate proliferative activity of malignancies. ^{9, 10} In brain tumors, amino acid uptake generally provides a more accurate estimate of proliferative activity than measures of glucose metabolism. ^{11, 12}

Among several available amino acid PET tracers, ¹¹C-labeled leucine is very suitable to estimate tissue protein synthesis rates because of its kinetic modeling. ^{13, 14} In this study, we measured L-[1-¹¹C]leucine (LEU) uptake in angioma regions and in brain regions not directly affected by the angioma in children with SWS who had unilateral, predominantly posterior, brain involvement associated with seizures. We hypothesized that the posterior brain regions, affected directly by the angioma, would show increased uptake of LEU on PET. To evaluate potential pathological correlates of abnormal amino acid uptake in the angioma region, histopathological features including vascular proliferative activity and glial markers were assessed directly in a resected angioma of an infant who underwent hemispherectomy following LEU PET. In addition, we hypothesized that higher increases of LEU uptake would be associated with more severe glucose metabolic abnormalities, measured by 2-deoxy-2[¹⁸F]fluoro-D-glucose (FDG) PET, due to more severe tissue damage underlying the angioma.

MATERIALS AND METHODS

Subjects

Seven children (5 boys, age range: 5 months to 13 years, median age: 6 years at the time of LEU PET; Table 1) with the clinical and radiological diagnosis of SWS who had unilateral hemispheric involvement and history of seizures underwent MR imaging as well as PET scanning using the tracers FDG and LEU in the Children’s Hospital of Michigan, Detroit. These children were selected from a series of 48 consecutive children with SWS who were recruited between October 2004 and August 2009 for a prospective neuroimaging research study, including yearly clinical and imaging follow-ups, approved by the Institutional Review Board at Wayne State University. All selected patients (15 % of the recruited patients) met the following inclusion criteria: (1) age below 14; (2) radiologic (MRI and FDG PET) evidence of unilateral brain involvement; (3) history of seizures; (4) no evidence of seizure(s) during PET; (5) parents’ approval for a second PET scan (LEU) in addition to FDG PET and MRI. Five patients had right hemispheric, whereas two patients had left hemispheric involvement. In one patient (patient #5) leptomeningeal angioma could not be visualized on MRI; however, the presence of a right facial port wine stain as well as right central transmedullary veins indicated right hemispheric involvement. Some imaging studies were collected 1 year apart from each other (see Table 1) as the patients participated in a prospective, longitudinal study with yearly follow-ups (except the infant who underwent hemispherectomy after initial imaging). The MRI and FDG PET image data, which were the closest to the LEU PET scan in time, were used for analysis (Table 1). FDG and LEU PET scans were done at the same time (1 day apart) in 5 patients and 1 year apart in the remaining two (patients #4 and #7). These latter patients were 5 and 11.8 years old at the time of the first scan; thus, both of them were older than 3 years when major metabolic progression had been observed. ¹⁵ Written informed consent was obtained from the parents or legal guardians.

Table 1.

Clinical and imaging data of the patients

Patient No.	Age (years)	Sex	PWS side	Initial imaging	Imaging 1 year later	Angioma location	FDG AI (posterior; %)	FDG AI (frontal; %)	LEU SUV AI (Kcompl AI/VD AI) (posterior; %)	LEU SUV AI (Kcompl AI/VD AI) (frontal; %)
1	0.4	F	right	MRI, FDG, Leu	n.a.†	rFTPO	−28.5	0.5	35.2* (n.a.)	16.3* (n.a.)
2	2.4	F	left	MRI (FDG)	FDG, Leu	lTPO	−45.2	−1.3	12.1* (8.7*/32.8*)	6.9* (−1.5/47.2*)
3	2.9	M	right	MRI (FDG)	FDG, Leu	rTPO	−2.9	1.7	8.2* (6.5*/37.1*)	1.4 (−2.5/26.4)
4	6.0	F	left	FDG (MRI)	MRI, Leu	lTPO	−61.2	−9.2	12.0* (n.a.)	8.8* (n.a.)
5	7.3	M	right	MRI (FDG)	FDG, Leu	[right trans- med. veins]	3.2	1.7	−1.5 (−3.7/19.3)	−1.6 (0.2/−8.7)
6	10.7	M	right	MRI	FDG, Leu	rTPO	−90	2.5	13.9* (1.6/57.8*)	0.4 (−1.2/−8.2)
7	12.8	M	right	MRI, FDG	Leu	rFTPO	−53.7	−6.9	8.9* (n.a.)	14.1* (n.a.)

Note: Age is given at the time of L-[1-¹¹C]leucine PET; F: female; M: male; PWS: port wine stain; r: right; l: left; F: frontal; T: temporal; P: parietal; O: occipital; FDG: 2-deoxy-2[¹⁸F]fluoro-D-glucose, LEU: L-[1-¹¹C]leucine; transmed.: transmedullary; SUV: standardized uptake value; Kcompl: K-complex macroparameter; VD: volume of distribution; AI: asymmetry index; n.a.: not available. Imaging studies in parenthesis were not used for this study. Values exceeding the corresponding normal mean AI+3SD are indicated with an asterisk.

^†patient had hemispherectomy after initial imaging studies.

FDG PET data acquisition

All PET scans were performed using a CTI/Siemens EXACT/HR scanner. This scanner has a 15 cm field of view and generates 47 image planes with a slice thickness of 3.125 mm. The FDG PET scanning protocol has been described in detail previously. ¹⁶ The reconstructed in-plane resolution is 5.5 mm (±0.35) full width at half maximum and 6.0 mm (±0.49) in the axial direction. Initially a venous line was established to inject FDG (0.143 mCi/kg). After 40 minutes of FDG uptake, a static 20-minute emission scan of the brain was acquired in 3D mode. Calculated attenuation correction was applied to the brain images using automated threshold fits to the sinogram data. All FDG PET scans were performed in the interictal state as indicated by EEG monitoring performed during the FDG uptake period.

LEU PET data acquisition

The tracer L-[¹¹C]-leucine was produced at Children’s Hospital of Michigan using a synthesis module designed and built in house. ¹⁷ Details of the imaging procedure have been described previously. ¹⁴ Four out of the 7 patients had blood input data obtained during the PET study for more detailed quantification. The PET sequence began with a transmission scan of the chest. Subsequently, the tracer L-[1-¹¹C]-leucine (0.1 mCi/kg) was injected, and a 20 min dynamic PET scan of the heart was performed (sequence: 12×10s, 3×60s, 3×300s) in order to obtain the left ventricular (LV) input function. Continuation of the input function was then accomplished by combining the time-activity curve derived from a small region-of-interest (ROI) in the center of the LV with venous blood samples obtained at later times. Subsequently, beginning at 25 min following tracer injection, seven 5 min scans of the brain were performed in 3D mode followed by a 15 min transmission scan of the brain. Measured attenuation, scatter and decay correction were applied to all PET images. Scalp EEG was not monitored during the LEU uptake period.

MRI data acquisition

All MRI studies were carried out on a Sonata 1.5 T MRI scanner (Siemens, Erlangen, Germany) using a standard head coil. For the purpose of the present study, only the post-gadolinium T1-weighted images (T1-Gad) were used (as a reference for co-registration of PET images). The acquisition parameters of this sequence were as follows: TR/TE:20/5.6 ms; flip angle: 25°; voxel size: 1×0.5×2 mm³.

Sedation during imaging studies

Children aged<2 years were sedated with chloral hydrate (50–100 mg/kg by mouth), and children aged 2–8 years were sedated with nembutal (3 mg/kg), followed by fentanyl (1 μg/kg), as necessary. All sedated subjects were continuously monitored by pediatric nurses, and physiological parameters (heart rate, pulse oximetry, respiration) were measured during the studies.

ROI definition

FDG PET images as well as summed and individual frames of the LEU PET images were initially rigid-body co-registered to each patient’s T1-Gad MR images using a 3D registration technique (VINCI 2.50). ¹⁸ Subsequently, ROIs encompassing the angioma region (including the angioma and the underlying cortex; these structures were not delimited separately, since they cannot be distinguished on PET images due to their close proximity) as well as mirror regions in the contralateral hemisphere were defined on the gadolinium enhanced MRI images using ROI Editor 1.4.1 (www.mristudio.org). All these regions were located mainly in the posterior (temporal, parietal, occipital) cortex. In patient #5, with no leptomeningeal angioma visualized on MRI, similar posterior cortex was included in this ROI. In addition, ROIs were drawn on at least 3 image planes in apparently spared (no atrophy and angioma) or less affected (mild hypometabolism in some cases; see Table 1) frontal cortical regions ipsilateral to the angioma as well as in contralateral homotopic regions. All ROIs were then superimposed on the co-registered FDG and LEU PET images including the dynamic LEU image sequences, in cases where blood input data were available. In subjects, who had MRI and PET scans acquired 1 year apart, small manual adjustments of the ROIs had to be carried out to adjust for interim changes in brain size.

Semi-quantitative analysis of FDG and LEU uptake

Standardized uptake values (SUVs) for FDG and LEU uptake were calculated for all regions by dividing the average radioactivity concentration obtained from each region by the injected FDG and LEU dose per total body weight, respectively. Subsequently, asymmetry indices (AIs) for SUVs were calculated as follows: AI (%) = 200 × [(I-C)/(I+C)], where I represents ipsilateral, C represents contralateral values as compared to the side of the lesion. For FDG, AI values above 10% were considered to be abnormal. ^{15, 16} For LEU, normal control asymmetries were calculated using LEU PET images of 5 healthy adults (all males, mean age: 39 years), where absolute values of right-left SUV AIs were calculated for both posterior and frontal regions, separately, and AI values above the normal mean + 3 standard deviations were considered to be abnormal in the patients. We used the conservative 3 standard deviations because of the unknown age-related differences in normal AIs of cortical LEU uptake.

Quantitative analysis of protein synthesis

To study mechanisms of LEU uptake abnormalities, unidirectional uptake rate constants (K-complex) and volumes of distribution (VDs) were derived from the Patlak graphical analysis ¹⁹ in 4 cases where blood input data were available. Details of this approach have been described previously. ¹⁴ Asymmetries (AIs) for K-complex and VD were calculated similar to those for SUVs. Again, AI values above the normal mean + 3 standard deviations, derived from the normal controls, were considered to be abnormal. Absolute SUV, K-complex and VD values of the patients were not compared to those of the healthy adults, due to potential developmental differences in cerebral protein synthesis rates. ²⁰

Statistical analysis

Hemispheric asymmetries in the angioma and frontal regions were compared using Wilcoxon’s signed rank test. Association between glucose metabolic and LEU uptake asymmetries was tested using Spearman’s rank correlation. P<0.05 was considered statistically significant.

Histological assessment of resected tissue samples: Resected specimens were available from one patient, an infant who underwent hemispherectomy at 6 months of age. In addition to standard histological processing of the resected brain and angioma tissues (including hematoxyilin-eosin, cresyl violet, Masson trichrome, glial fibrillary acidic protein [GFAP] and synaptophysin immunostaining, Luxol fast-blue/periodic-acid Schiff), angioma specimens were also immunostained using Ki-67 antibody (clone K-2, Ventana), co-labeled with the endothelial marker CD31 (JC70A, Ventana) to demonstrate proliferative activity in the endothelial cells of the angioma. ^{6, 21} Furthermore, the expression of vascular endothelial growth factor-A (VEGF-A) was also assessed (using mouse anti-human monoclonal antibodies; sc-7269, Santa Cruz Inc.) in the resected angioma specimen. Immuno-histochemistry results of resected brain tissues of 7 (non-SWS) patients (age range: 4 months to 7 years), who underwent epilepsy surgery, are also presented for comparison.

RESULTS

FDG and LEU uptake asymmetries

LEU SUV asymmetries in the angioma and frontal regions exceeded the calculated normal cutoff values (3.8% for the angioma region and 3.3% for frontal cortex) in 6 and 4 cases, respectively (mean AIs for these abnormal cases: angioma – 15.1%, frontal – 11.5%; see Figure 1). FDG SUV asymmetries were abnormal (>10%) in 5 patients in the angioma region (mean AI: 55.7%) and in none of the patients in the frontal region. Glucose metabolic asymmetries of the angioma region were significantly higher than those of the frontal regions (−39.7% vs. −2.3 %; p=0.028). However, LEU uptake asymmetries of the angioma region were only slightly higher than those of the frontal regions (12.7 vs. 6.6%; p=0.09).

Figure 1.

Representative co-registered T1 post-gadolinium MRI (A), FDG PET (B) and L-[1-¹¹C]leucine (LEU) PET (C) images of a 6 years old girl (patient #4) with left hemispheric angioma in the temporo-parieto-occipital region. Solid arrows indicate the angioma region which was hypometabolic (AI=−61%) but showed abnormally increased LEU uptake (AI=12%). Although the MRI showed deep transmedullary veins in the frontal lobe, the angioma did not extend to the frontal cortex. However, this region was also mildly hypometabolic (dashed arrow; AI=9%) and showed moderately increased LEU uptake (SUV AI=9%) compared to the contralateral homotopic area.

Quantification of LEU uptake

Four patients had blood input data to estimate regional protein synthesis rates. K-complex asymmetries exceeded the normal AI limit (angioma region: 4.8%; frontal: 4.4%) in the 2 youngest children (ages: 2.4 and 2.9 years) where quantification was available in the angioma region (increases; AI: 8.7% and 6.5%, respectively) and in none of the cases in the frontal cortex (Table 1). VD asymmetry was above the normal AI limit (angioma region: 28%, frontal: 30%) in the angioma region in 3 patients and in the frontal region in 1 child, always with higher values ipsilateral to the angioma.

Association between glucose hypometabolism and LEU uptake

We found a significant negative correlation between FDG SUV asymmetries and LEU SUV asymmetries for the 6 patients older than 2 years of age in the angioma regions (r=−0.83, p=0.042, see Figure 2) and a trend for the same association in the frontal regions (r=−0.77; p=0.07) indicating that higher LEU uptake was associated with more severe hypometabolism. For this analysis, we excluded patient #1, a 5-month old infant. In this age group, patients with SWS often show increased glucose metabolism and blood flow, followed by a transition from increased to decreased metabolism and blood flow. ²² This patient’s data point may be an outlier (see point denoted by X in Figure 2) in this comparison for this reason.

Figure 2.

Relationship between glucose metabolic (FDG) vs. L-[1-¹¹C]leucine (LEU) uptake asymmetries (AIs) in the angioma region. Black circles represent datapoints from the 6 older (above the age of 2 years) patients with chronic disease. A correlation for these patients’ data indicates that higher LEU uptake ipsilateral to the angioma is associated with more severe glucose hypometabolism (r=−0.83, p=0.042). In addition, the cross indicates the data of an infant with recent onset seizures. This case was not included in the correlation since glucose uptake is not an appropriate measure of cortical damage in the initial stage of the disease when potential transition of cortical hypermetabolism to hypometabolism occurs.

Histopathological assessment

The surgical specimen from the infant who underwent epilepsy surgery showed marked leptomeningeal vascularity, cortical malformation (polymicrogyria and heterotopias), neuronal loss, gliosis (positive GFAP immunostaining) and prominent calcification. Positive Ki-67 labeling was seen in the nuclei of endothelial cells (Figure 3A) but not in cortical cells (Figure 3B). The calculated proliferative index (proportion of labeled endothelial cells) was between 5–10%. In comparison, no endothelial Ki-67 staining was seen in control tissues (Figure 3E). In addition, we also found strong VEGF-A expression in endothelial cells of the angioma as well as in cortical neurons (Figure 3C, D). Pediatric epilepsy control tissues showed no, or weak, staining for VEGF (Figure 3F).

Figure 3.

Immunostaining for the proliferation marker Ki-67 (co-stained with endothelial marker CD31; A) and VEGF-A (C) in resected angioma tissue as well as cortex (B and D) of an infant (patient #1) who underwent hemispherectomy at 6 months of age. These representative images show positive Ki-67 labeling (brown stain) in numerous endothelial nuclei of the leptomeningeal angioma (one of the Ki-67 positive nuclei are shown by a red arrow) as well as intense VEGF-A staining (brown) in leptomeningeal vessels. No Ki-67 staining was seen in the cortex but numerous cells showed VEGF-A expression. The representative images at the bottom show no endothelial Ki-67 expression (E) and no neuronal staining for VEGF (F) in tissue samples of one of the control subjects (a 4 year old patient with intractable temporal lobe epilepsy).

DISCUSSION

The main finding of this study is the increased LEUSUV of the angioma region and in the frontal lobe in some cases in children with SWS. Based on the available data with quantification, these increases are driven by elevated volume of distribution (VD) of labeled leucine indicating elevated LEU transport in 3/4 cases and also by increased protein synthesis rates reflected by the K-complex in two of the four cases. These results increase our understanding of pathological changes occurring in brain underlying the angioma and beyond in frontal cortical regions showing relatively normal glucose metabolism. Elevations in amino acid transport and protein synthesis may be related to endothelial and glial proliferation, which were observed in the one case in which brain tissue was available for pathological studies. Altered blood-brain barrier (BBB) is also a probable mechanism for increased amino acid uptake near the angioma. Further studies are needed to understand these mechanisms more fully.

This is a preliminary study with a limited number of subjects. Additional major limitations include that FDG and LEU PET images were acquired 1 year apart from each other in some cases. Furthermore, detailed kinetic analysis could be carried out in only a subset of the study group. Importantly, the angioma and the underlying cortex could not be distinguished on the PET images since partial volume effects preclude the reliable separate analysis of these structures. Therefore, our results most likely represent a summed effect of abnormal amino acid transport and protein synthesis in these structures, at least in the angioma-affected regions. In addition, changes in glucose metabolism during development in the region of the angioma complicate our analysis. In SWS, FDG PET often detects profound cortical hypometabolism of the affected hemisphere, predominantly under the angioma but frequently extending well beyond the apparent structural abnormalities seen on MRI. ^{16, 22} We found a significant inverse association between the severity of hypometabolism and the increase in LEU uptake in children above 2 years of age. In the only infant (a 5-month old boy), LEU uptake showed the highest AI value, while FDG AI was relatively moderate. However, this latter may not reflect the true degree of tissue damage in this young age, as infants with SWS are known to show transient hypermetabolism, ²² followed by a transition from hyper- to hypometabolism in the affected brain regions. Therefore, it is possible that in this infant, FDG PET underestimated the severity of cortical involvement. In the 6 older children, higher LEU uptake associated with lower glucose metabolism suggests that the degree of amino acid transport and protein synthesis (related to blood brain barrier damage, glial or vascular proliferation) is associated with tissue damage as reflected by low glucose metabolism.

Recent studies have demonstrated that intracranial vascular malformations in SWS are not static lesions but may undergo dynamic remodeling. ⁵ Enhanced endothelial cell turnover as well as increased expression of key angiogenetic factors, such as VEGF and its receptor provided evidence for ongoing angiogenesis and suggested the progressive nature of angiomas. ^{5, 6} In addition to the leptomeningeal angioma, the structure and neural regulation of cortical vessels are also affected in this disorder. ^{23, 24} Whether these vascular changes contribute to clinical disease progression remains to be clarified.

In our study, the increased LEU uptake in the angioma region was mainly driven by the increase in the VD of the tracer, suggesting increased amino acid transport. The marked increase of LEU VD may be due to the retention of the amino acid by increased vascular beds, disruption of the blood-brain barrier, increased capillary permeability, and/or increased expression of the large amino acid transporter (LAT1). ^{23, 25} Contrast enhancement of the leptomeningeal angioma on MRI suggests BBB disruption, which itself can lead to increased amino-acid uptake. ²⁶ Increased uptake of two other amino-acid tracers (i.e., ¹¹C-methionine and α-[¹¹C]methyl-L-tryptophan, where increased VD, but not high K-complex, was associated with contrast enhancement) has been demonstrated in brain tumors and other pathological conditions associated with BBB breakdown. ^{27, 28} In contrast, FDG uptake is not sensitive to BBB disruption, ^{29, 30} thus, glucose metabolic rates on PET may remain low even in the case of impaired BBB. Accumulation of ¹¹C-methionine (which, similar to leucine, is a substrate of LAT1) on PET was reported in the angioma region of an adult with SWS, ²⁵ but quantification of methionine uptake has not been performed. The mechanism of the increase in the K complex macroparameter also remains to be determined. Based on a study from Comati et al. and our previous work demonstrating proliferative activity and angiogenesis in the vascular malformations, ^{5, 6} increased protein synthesis may be expected in the angioma due to active remodeling. This is supported by immuno-histochemistry findings in our youngest patient, showing endothelial Ki-67 proliferative index of 5–10%, similar to what could be expected in low-grade tumors. ³¹ Importantly, other factors, such as cortical glial proliferation or increased expression and deposition of extracellular matrix proteins like fibronectin ⁴ could also contribute to high protein synthesis in the angioma region. In addition, cortical malformations (also seen in our infant) have been reported in patients with SWS ³² and these epilepsy-associated developmental abnormalities can show increased amino acid uptake. ³³ Detection of such malformations by imaging could be of high clinical importance in SWS as these lesions are highly epileptogenic and should be considered when partial resection is planned in children with SWS and intractable epilepsy. Although mechanisms of increased LEU uptake are currently speculative and warrant further studies, our imaging findings suggest that increased LEU uptake on PET may be a useful marker of underlying pathology not only in angioma-affected regions but also in frontal cortex even when it shows no apparent abnormalities on conventional imaging in children with SWS.