Clinical, radiological, histological, and molecular characteristics of paediatric epithelioid glioblastoma

Abstract

Aims

A few case series in adults have described the characteristics of epithelioid glioblastoma (e-GB), one of the rarest variants of this cancer. We evaluated clinical, radiological, histological, and molecular characteristics in the largest series to date of paediatric e-GB.

Methods

Review of clinical characteristics and therapy, imaging studies, and histology was performed in patients younger than 22 years with e-GB seen at our institution over 15 years. Sequencing of hotspot mutations and FISH of relevant genes were undertaken.

Results

Median age at diagnosis of six patients was 7.6 years. Tumours originated in the cerebral cortex (n=2) or diencephalon (n=4). Three patients presented with acute, massive haemorrhage and three had leptomeningeal dissemination at diagnosis. Paediatric e-GB had the typical histological characteristics seen in adult tumours. Universal immunoreactivity for INI1 and lack of diverse protein expression were seen in all cases. One tumour had a chromosome 22q loss. Three tumours (50%) harboured a BRAF: p.V600E. One thalamic tumour had an H3F3A p.K27M. All patients received radiation therapy with (n=3) or without chemotherapy (n=3). All patients experienced tumour progression with a median survival of 169 days. One patient with non-metastatic disease had early leptomeningeal progression. Two patients had symptomatic tumour spread outside the central nervous system (CNS) through a ventriculo-peritoneal shunt. One additional patient had widespread metastases outside the CNS identified at autopsy.

Conclusions

Paediatric e-GBs are rare cancers with an aggressive behaviour that share histological and genetic characteristics with their adult counterparts. BRAF inhibition is a potential treatment for these tumours.

Introduction

Glioblastoma (World Health Organization [WHO] grade IV), one of the most malignant central nervous system (CNS) cancers in children and adults, is characterized by architectural diversity and cellular heterogeneity [1]. Although several uncommon variants of glioblastoma, each with an idiosyncratic morphology, have been described on the basis of these diverse histological characteristics [2–11], the 2007 WHO classification only recognizes two of them: giant-cell glioblastoma and gliosarcoma [1].

Two of the rarest variants of glioblastoma display cells with an epithelioid or rhabdoid phenotype and have very similar histological characteristics [12–16]. Both variants display round or oval cells with a distinct cell membrane, laterally positioned nucleus, and eosinophilic cytoplasm [12–16]. Although one study proposed that focal loss of INI1 immunoreactivity in rhabdoid areas and diverse protein expression should differentiate rhabdoid glioblastoma (r-GB) from epithelioid glioblastoma (e-GBs) [15], several other reports used the terms ‘epithelioid’ and ‘rhabdoid’ interchangeably [12, 13, 17–21]. e-GB is also distinct from the exceptionally rare true epithelial glioblastoma described in adults, which is a tumour with epithelial differentiation in the form of squamous nests and acinar structures [14]. Atypical teratoid/rhabdoid tumour (AT/RT) is an important differential diagnosis alongside e-GB, particularly in children. To complicate matters, several instances of AT/RT arising within previously diagnosed low-grade gliomas have been described [22–26].

We report the clinical, radiological, histological, and molecular characteristics in the largest series to date of paediatric e-GBs. We showed that children with e-GB experienced an aggressive course, despite the use of intensive therapy, with a high prevalence of haemorrhagic episodes and tumour dissemination in the leptomeninges and outside the CNS.

Patients and Methods

Once Institutional Review Board approval was obtained, we retrospectively identified in our institutional Neuro-Oncology and Pathology databases from January 1^st, 1997 until December 31^st, 2012 all patients younger than 22 years with newly diagnosed high-grade glioma that displayed cells with epithelioid or rhabdoid morphology. Upon central pathology review, we selected only those patients whose tumours met the diagnostic criteria for glioblastoma and harboured a significant epithelioid component. Furthermore, these tumours had to fulfill the immunohistochemical criteria for e-GB as previously described [15]. Information about clinical characteristics and therapy was abstracted from the patients’ medical records. Central radiological review of pertinent CT and MRI scans at diagnosis and during therapy was performed. All patients had at least a T2- and a T1-weighted brain MRI sequence before and after administration of contrast at diagnosis available for analysis.

Standard histological review, immunohistochemical studies, and interphase fluorescence in situ hybridization (iFISH) were performed in 5-µm sections of formalin-fixed, paraffin-embedded (FFPE) tissue. Samples from diagnosis and an episode of progressive disease 3 months later were obtained in one case. Immunohistochemical assessment employed antibodies to BRG1 (SMARCA4 gene product), INI1 (SMARCB1 gene product), GFAP, synaptophysin, NFP, EMA, cytokeratins (AE1/3 & CAM 5.2 antibodies), desmin, smooth muscle actin, melan A, and P53.

Dual-colour iFISH was done using standard methods as previously described [27]. Probes were derived from BAC clones (BACPAC Resources, Oakland, CA), labeled with either AlexaFluor-488 or Rhodamine fluorochromes, and validated on normal control metaphase spreads to confirm chromosomal location. The following target genes were probed relative to controls (BAC clones in parentheses): PDGFRA (RP11-231C18 + RP11-601I15) with 4p12 control (CTD-2057N12 + CTD-2588A19); EGFR (RP11-148P17 + RP11-1083E20) with 7q31.2 control (RP11-460J21 + CTB-133K23); MET (RP11-163C9) with 7p11.2 control (RP11-251I15); CDKN2A (RP11-149I2 on 9p21) with NOTCH1 (RP11-370H5 + RP11-1008C19 on 9q34.3); PTEN (CTD-2553L21) with 10p11.2 control (RP11-254A5 + RP11-322I2); SMARCB1 (CTD-2034E7 + CTD-2355C2) with 22q13.3 control (RP3-402G11).

Analyses of BRAF: p.V600E, H3F3A p.K27M and H3F3A p.G34, HIST1H3B p.K27M, IDH1 p.R132, and IDH2 p.R172 were performed in DNA extracted from FFPE scrolls using the Maxwell® 16 Plus LEV DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. DNA was then eluted in molecular grade DNase/RNase-free water (Ambion, Foster City, CA) and quantified using PicoGreen (Invitrogen, Carlsbad, CA). Polymerase chain reaction (PCR) experiments used previously published primers and GoTaq® Long PCR Master Mix (Promega, Madison, WI) [28–30]. Direct sequencing of PCR products was performed using BigDye version 3.1 and a 3730XL DNA analyzer (Applied Biosystems, Foster City, CA). Results were screened using CLC Main Workbench sequence analysis software version 6.0.2 (CLC bio, Cambridge, MA).

Results

Six patients that met our diagnostic criteria were identified. Four (2%) of 199 patients with newly diagnosed high-grade glioma treated at our institution during the study period had an e-GB. In addition, we consulted on two additional patients with the same diagnosis during the study period. The median age at diagnosis of all 6 patients was 7.6 years. (Table 1) The median interval from onset of symptoms to radiological diagnosis was 2.5 weeks (range, 1 day to 3 months). Histological diagnosis was delayed by 7 weeks in patient 2, who presented with an acute intra-tumoural and intra-ventricular haemorrhage. Patient 6 had Li-Fraumeni syndrome (heterozygous germline TP53 mutation at IVS3-2 A>C).

Table 1.

Clinical and radiological characteristics at diagnosis, pattern of tumor recurrence, and survival of patients with epithelioid glioblastoma

Patient #	Age at Diagnosis (years)	Gender / Race	Primary Tumor Location	Intra-Tumoral Hematoma at Diagnosis	Metastatic Disease at Diagnosis	Site of First Tumor Progression	Tumor Spread Outside the CNS	Survival (days)
1	10.7	F / Caucasian	Thalamus	YES	No	Outside the CNS	YES	144
2	11.3	M / AA	Temporal lobe	YES	YES¶	Leptomeningeal	YES‡	118
3§	10.2	F / AA	Bi-thalamic	No	YES¶	Outside the CNS	YES	42
4	3.5	M / Mixed race	Frontal Lobe	YES	YES¶	Leptomeningeal	No	194
5	3.5	F / Caucasian	Hypothalamic	No	No	Leptomeningeal	No	230
6§	5	M / Caucasian	Thalamus	No	No	Local	No	290

^¶CSF positive for malignant cells and tumor nodule in cauda equina (patient 2), CSF positive for malignant cells only (patient 3), and multiple foci of metastatic spread in the brain (patient 4);

^‡Tumor spread outside the CNS was documented at autopsy;

^§Patients 3 and 6 were only seen as consultation

Abbreviations: M, male; F, female; AA, African-American; CNS, central nervous system; CSF, cerebrospinal fluid

Radiological Characteristics at Diagnosis

Two tumours originated in the cerebral cortex and four in the diencephalon. (Table 1) Three patients had an acute, symptomatic intra-tumoural and intra-ventricular hemorrhage at diagnosis. (Figure 1) Three patients had disseminated leptomeningeal tumour at diagnosis. All tumours had varying degrees of enhancement with gadolinium. Five evaluable patients had tumours with restricted diffusion.

Figure 1.

Intra-tumoral hematoma and spread of hemorrhage inside the ventricular system at diagnosis in patient 4

Patient 3 had long-lasting radiological abnormalities preceding the diagnosis of e-GB. She was diagnosed with hydrocephalus and required a ventriculo-peritoneal (VP) shunt at the age of 1 month. A brain MRI obtained at 3 years of age disclosed an abnormal area in the posterior left thalamus, which was isointense on T-1 and hypointense on T-2 weighted MRI. A brain CT obtained at 7 years of age demonstrated that this same area was heavily calcified. A brain CT obtained at the time of diagnosis of e-GB showed these same calcifications within the tumour.

Treatment after Diagnosis

Patients 1 and 5 received local radiation therapy (RT; total dose of 59.4 Gy). Patient 1 received erlotinib (Tarceva, OSI Pharmaceuticals, Melville, NY; Roche, Basel, Switzerland; Genentech, South San Francisco, CA) concurrently with RT [31]. Patient 5 received vorinostat (Zolinza, Merck Sharp & Dohme, Whitehouse Station, NJ) concurrently with RT, followed by a combination of temozolomide (Temodar, Merck Sharp & Dohme, Whitehouse Station, NJ) and bevacizumab (Avastin, Genentech, South San Francisco, CA) after completion of RT. Patients 2 and 4, who had leptomeningeal dissemination at diagnosis, underwent a near-total resection of the primary tumour, followed by craniospinal RT (local tumour doses of 54 and 55.8 Gy, respectively; craniospinal dose between 30.6 and 39.6 Gy). Patient 4 received oral etoposide after completion of RT. Patient 3 received only local RT despite the presence of tumour cells in the cerebrospinal fluid because of her poor clinical condition. Patient 6 initially underwent surgical resection alone, while waiting for the confirmation of his diagnosis. Local tumour progression was diagnosed 97 days after surgery, at which time he underwent a second tumour resection followed by craniospinal RT.

Radiological Characteristics at Progression

Patients 1 and 3 experienced disease progression at sites outside the CNS, while receiving local RT; histological confirmation was obtained in both cases. (Table 1) Patient 1 developed tumour spread in the scalp near the site of VP shunt insertion. Patient 3, who also had a VP shunt, was diagnosed with neoplastic ascites, peritoneal tumour deposits, and multiple liver metastases. (Figure 2) Patients 2 and 4 experienced rapid progression of their leptomeningeal disease despite the use of craniospinal RT. Patient 5 developed tumour dissemination in the leptomeninges during chemotherapy and within 3 months of completing local RT. Patient 6 experienced massive local tumour progression following surgery alone. He developed leptomeningeal spread of the tumour shortly after completion of craniospinal RT. Patients 1 and 4 experienced further intra-tumoural haemorrhage after tumour progression.

Figure 2.

Neoplastic ascites (white arrow) and peritoneal tumor deposits (white arrowhead), and multiple liver metastases (black arrowheads) as the sites of first progression in patient 3

Histopathological Evaluation

While all e-GBs contained areas of pleomorphic cells with an astrocytic phenotype, they were dominated by groups of relatively monomorphic cells with distinct plasma membranes and eccentrically placed nuclei. Varying subtly from region to region, the cytology could be interpreted as blending epithelioid, rhabdoid, and gemistocytic phenotypes. (Figure 3) Mitotic figures were readily found among the tumour cells, and microvascular proliferation and necrosis were further histological features. The vasculature in these tumours appeared indistinguishable from that of a classic glioblastoma. All tumours contained at least a few small foci of haemosiderin-laden macrophages, suggesting pre-operative intra-tumoural haemorrhage. One e-GB contained two small areas with a gliosarcomatous phenotype, but no tumour contained foci of epithelial metaplasia, and lipidization was never a prominent cytological feature. Whereas the tumour initially resected from patient 1 harboured areas with astrocytic and epithelioid phenotypes, the tumour obtained at progression contained only the epithelioid component. Immunohistochemistry showed that all e-GBs contained a few foci of GFAP-positive cells. (Figure 3) Immunoreactivities for INI1 and BRG1 were universal in all e-GBs. The tumour from patient 1 contained rare cytokeratin-positive cells. Otherwise, all e-GBs failed to express synaptophysin, NFP, EMA, desmin, smooth muscle actin, and melan A. Only one tumour (patient 1) showed strong and widespread nuclear immunoreactive for P53.

Figure 3.

Histopathological characteristics of epithelioid glioblastoma (e-GB) in children. Several cytological phenotypes characterize these tumors, including epithelioid (a), rhabdoid (b), and astrocytic/gemistocytic (c). e-GBs resemble classic glioblastomas in some areas (d; all H&E, ×200). Immunoreactivity for GFAP was variable in tumor cells (e; anti-GFAP antibody, ×200). Homozygous deletion of CDKN2A was evident in two patients (f; iFISH: green and red fluorochromes targeted CDKN2A and the control locus on 9q, respectively)

Molecular Characteristics

Molecular analysis revealed that three of six e-GBs (50%) contained a BRAF: p.V600E mutation. (Table 2) Two of these tumours were thalamic and one originated in the cerebral cortex. One of the thalamic tumours (patient 1) also harbored an H3F3A p.K27M mutation, which was present at diagnosis and recurrence. No mutations were detected at H3F3A p.G34, HIST1H3B p.K27M, IDH1 p.R132, and IDH2 p.R172. iFISH analysis revealed EGFR amplification in one case. (Table 3) The tumour from patient 4 showed homozygous deletion of CDKN2A. None of the cases with BRAF: p.V600E showed concomitant homozygous deletion of CDKN2A. The tumour of patient 6 showed hemizygous deletion of PTEN. Monosomy 22q, as indicated by loss of one probe signal at both the SMARCB1 locus and at 22q13.3, was detected in the tumour from patient 2.

Table 2.

Analysis of hotspot mutations in epithelioid glioblastoma

Patient #	Surgery	Single Nucleotide Variations
		BRAF: p.V600E	H3F3A p.K27M	H3F3A p.G34	HIST1H3B p.K27M	IDH1 p.R132	IDH2 p.R172
1	1	+	+	−	−	−	−
	2	+	+	−	−	−	−
2		+	−	−	−	−	−
3		+	−	−	−	−	−
4		−	−	−	−	−	−
5		−	−	−	−	−	−
6		−	−	−	−	−	−

Abbreviations: +, mutation present; −, mutation absent

Surgery 1 and 2 indicate tumor resected at diagnosis and at the time of progression, respectively

Table 3.

FISH analysis of copy number gains and losses in patients with epithelioid glioblastoma

Patient #	Surgery	Copy Number Abnormalities
		PDGFRA (4q12)	EGFR (7p12)	MET (7q31)	CDKN2A (9p21)	PTEN (10q23)	SMARCB1 (22q11)
1	1	normal	polysomy 3–4	polysomy 3–4	normal	normal	normal
	2	polysomy 3–4	polysomy 3–7	polysomy 3–7	polysomy 3–4	polysomy 3–4	polysomy 3–4
2		normal	polysomy 3–4	polysomy 3–4	normal	normal	monosomy
3		normal	polysomy 3–4	polysomy 3–4	polysomy 3–4	polysomy 3–4	polysomy 3–4
4		polysomy 3–4	amplified	hemizygous deletion	homozygous deletion	normal	normal
5		normal	polysomy 3–4	polysomy 3–4	polysomy 3–4	monosomy	normal
6		polysomy 3–4	polysomy 3–4	polysomy 3–4	monosomy	hemizygous deletion	normal

Surgery 1 and 2 indicate tumor resected at diagnosis and at the time of progression, respectively

Outcome

All patients died of disease progression after a median survival of 169 days (range, 42 to 290 days). (Table 1) Whole-body autopsy was obtained after death in patient 2. Patient 4 underwent a brain-only autopsy. Patient 2 had residual tumour in the primary cerebral site and leptomeninges, and widespread involvement of all parenchymal organs outside the CNS and of mediastinal and abdominal lymph nodes. Patient 4 had extensive tumour involvement of the brain and subarachnoid space and a large area of recent intra-tumoural hemorrhage.

Discussion

Only a few case series have previously described e-GB, a very rare variant of glioblastoma [12, 14–16]. Although most patients have been adults, three children with e-GB were included in one of these studies [16]. Here, we describe a very aggressive disease course in the largest series to date of paediatric e-GB. Three of our patients (patients 1, 2, and 4) presented acutely because of massive intra-tumoural and intra-ventricular hemorrhage, and three patients (patients 2, 3, and 4) had leptomeningeal tumour dissemination at diagnosis. Two patients with non-metastatic tumours (patients 1 and 5) had either tumour progression outside the CNS or leptomeningeal dissemination during or shortly after completion of RT. Two of our patients developed further intra-tumoural hemorrhage at progression.

Although glioblastoma in children remains a deadly cancer, this pattern of aggressive tumour behaviour has rarely been reported in previous studies. Spontaneous, symptomatic haemorrhage was found in less than 10% of paediatric high-grade gliomas, including glioblastoma [32, 33]. Leptomeningeal dissemination at diagnosis was described in approximately 5% of high-grade gliomas in children [34], and only a few case reports described dissemination of glioblastoma through a VP shunt outside the CNS in children [35]. Except for patients with e-GBs, we have not observed any association between intra-tumoural haemorrhage and leptomeningeal spread in other patients with nonepithelioid glioblastoma at our institution.

It is possible that the frequent occurrence of intra-ventricular haemorrhage in our patients could partly account for their predisposition to leptomeningeal dissemination at diagnosis. The spread of tumour outside the CNS in patient 3 could also be due to a decision not to deliver craniospinal RT at the consulting institution.

Unlike e-GB in adults which arises predominantly in the cerebral cortex [14, 16], two-thirds of our patients had tumours originating in the diencephalon. One of our patients had imaging abnormalities many years before the diagnosis of e-GB to suggest that the tumour might have undergone malignant transformation.

We have shown that the histological characteristics of e-GB in children are akin to those previously described in adults [12, 14–16]. Histological evaluation at recurrence in one of our patients showed selection of the epithelioid phenotype. Similar to e-GB in adults, our patients’ tumours consistently expressed INI1 [14, 15]. GFAP was only focally expressed. Importantly, all tumours lacked a polyphenotypic expression pattern by immunohistochemistry [14, 15]. Replicating a recent study of mostly adult patients [16], our molecular analysis showed that BRAF: p.V600E is also frequent in paediatric e-GBs. Interestingly, we identified an H3F3A p.K27M mutation in the thalamic tumour of one patient at diagnosis and in the distant recurrence. This histone mutation was previously shown to occur predominantly in midline paediatric glioblastomas, particularly thalamic tumours [29, 37]. Of all chromosome copy number gains and losses analyzed, only a lower prevalence of EGFR amplification seems to distinguish paediatric and adult e-GBs [14, 16]. Unlike e-GB in adults [16], one of our patients had a tumour with a monoallelic chromosome 22q loss which included the SMARCB1 locus.

r-GB, another extremely rare variant of glioblastoma, is an important differential diagnosis of e-GB [15]. Despite the report of immunohistochemical differences between these two entities [15], specifically focal loss of INI1 expression in rhabdoid areas and the expression of multiple proteins from different histogenetic lineages in r-GB, several recent studies described patients with r-GBs whose tumours met the diagnostic criteria for e-GB [17–21].

Other tumours that enter the differential diagnoses of e-GB in adults are metastatic carcinomas and melanomas, but these are less relevant for children [14]. The distinction between e-GB and AT/RT is critical in the paediatric age group and should be more straightforward now that a universal lack of INI1 expression can be demonstrated in the tumour cells of an AT/RT. However, at least two patients in the current study (patients 4 and 6), who were diagnosed before the widespread availability of INI1 immunohistochemistry, were initially referred for treatment with a presumptive diagnosis of AT/RT.

Recent studies have unveiled key molecular abnormalities in paediatric glioblastomas [29, 36, 37]. BRAF: p.V600E mutation has been reported in a subset of other, more common, paediatric high-grade gliomas [38]. One pre-clinical study showed that cells lines and orthotopic xenografts of high-grade glioma harboring a BRAF: p.V600E were sensitive to an oral BRAF inhibitor [39]. Likewise, BRAF inhibition may also be a promising therapy for a subset of children with e-GB.

List of abbreviations

WHO

World Health Organization

CNS

central nervous system

r-GB

rhabdoid glioblastoma

e-GB

epithelioid glioblastoma

AT/RT

atypical teratoid/rhabdoid tumour

iFISH

interphase fluorescence in situ hybridization

FFPE

formalin-fixed, paraffin-embedded

PCR

polymerase chain reaction

VP

ventriculo-peritoneal

RT

radiation therapy