WNT-Activated Medulloblastomas With Hybrid Molecular Subtypes

INTRODUCTION

Molecular characterization of CNS tumors is becoming increasingly important for both diagnostic clarity and prognostication in the practice of neuropathology. Medulloblastomas, in particular, can be divided into 4 core consensus molecular subgroups (ie, WNT-activated, sonic hedgehog [SHH]-activated, group 3 [G3], and group 4 [G4]) based on genetic alterations, methylation profiles, and transcriptional patterns, which predict prognostic outcomes better than histology alone and are increasingly used for medulloblastoma classification.^1,2 WNT-activated medulloblastomas are defined by activating mutations in the WNT/β-catenin signaling pathway (eg, CTNNB1 or germline APC), are often associated with loss of chromosome 6, and have the most favorable prognosis. SHH-activated medulloblastomas are denoted by mutations in the SHH pathway (eg, PTCH1, SMO, SUFU), with intermediate or poor prognosis guided by TP53 status. G3 and G4 medulloblastomas are associated with MYC amplification and MYCN or CDK6 amplification, respectively, frequently harbor isochromosome 17q, and have poor prognoses. Notably, individual tumors with highly characteristic driver events (eg, WNT methylation subclass with CTNNB1 mutation) were thought to be readily assigned to a specific methylation or genomic subclass with little genetic overlap between the subgroups.³

Recently, however, several patients with medulloblastoma have been reported to harbor combined molecular alterations characteristic of both WNT- and SHH-activated tumors.^4,5 We report 2 additional patients with medulloblastoma with hybrid subclass features, one with WNT/SHH dual-activation tumors and another, strikingly, with features of WNT, SHH, and isochromosome 17q—a tertiary feature conventionally thought to be characteristic of G3 and G4 medulloblastomas.

CASE 1

A 37-year-old woman presented with headaches and vision changes, and was found to have a 4-cm posterior fossa mass, prompting craniotomy for tumor resection. Pathology demonstrated an embryonal tumor, which was diagnosed morphologically as medulloblastoma, WHO grade IV, with classic histology (Fig 1A). Molecular analysis (Table 1) using a 1,213-gene, targeted hybrid-capture, next-generation sequencing panel⁶ revealed a CTNNB1 p.G34R mutation, which is a recurrent hotspot mutation in exon 3 of the gene, encoding the β-catenin protein. This mutation, a hallmark of WNT-activated molecular subtype medulloblastomas,⁷ leads to decreased degradation of β-catenin protein.⁸ Also identified were PTCH1 p.E405* and p.L39Afs*51 mutations resulting in premature protein truncation and predicted loss of function, which are characteristic genetic alterations driving the SHH-activated molecular subtype of medulloblastoma.⁹ The first was present at an allelic frequency of 79%, suggesting a loss of heterozygosity event, notable because mutant PTCH1 alleles must exist hemizygously to activate the SHH pathway. The second was at a subclonal frequency of 8%. Methylation profiling (performed at Heidelberg University Hospital in Germany) was most consistent with WNT-activated medulloblastoma (calibrated class score, 0.99). Copy data from both the sequencing and the methylation profiling identified loss of chromosome 17p and gain of chromosome 17q, consistent with an isochromosome 17q (Figs 2A and 2C). Case 1 was lost to follow-up after the initial surgery.

FIG 1.

Micrographs of tumors in (A) case 1 and (B) case 2, revealing embryonal neoplasms with large, hyperchromatic nuclei consistent with the diagnosis of medulloblastoma.

TABLE 1.

Somatic Variants and Copy Number Changes in Case 1

FIG 2.

Methylation plots for (A) case 1 and (B) case 2, demonstrating in case 1 loss of chromosome 17p and gain of chromosome 17q (arrows), consistent with isochromosome 17q. (C) Clustering of both patients along other medulloblastomas (MB) on a reduced dimensionality plot with an enlarged figure inset of the WNT-activated medulloblastomas, consistent with WNT-activation as the initiating event. AD, adults; CHL, children; G3, group 3; G4, group 4; INF, infants; MB, medulloblastoma; SHH, sonic hedgehog.

CASE 2

A 29-year-old man presented with acute onset of severe frontal headaches, progressing to obtundation, and was found to have a large intraventricular hematoma centered in the fourth ventricle, at first concerning for possible ruptured aneurysm. The patient underwent emergent craniectomy, at which time a portion of hematoma, found to be adherent to the roof of the fourth ventricle, was sent for permanent pathology. Unexpectedly, on histologic examination, a minute fragment of neoplastic tissue was detected (Fig 1B). The specimen was particularly challenging to diagnose morphologically because of its scant nature, and definitive diagnosis was deferred to molecular analysis.

CONTEXT

• Key Objective

• We examine the characteristics of medulloblastomas with hybrid molecular subtypes.

• Knowledge Generated

• We studied two medulloblastomas with hybrid subclass features: one with WNT/SHH dual-activation and another with features of WNT, SHH, and isochromosome 17q—a tertiary feature conventionally thought to be exclusive to group 3 and group 4 (G3/G4) medulloblastomas. We further identified hybrid medulloblastomas that display canonical features of both WNT-activated and G3/G4 subgroups among published cases. All hybrid medulloblastomas reported to date have methylation profiles that cluster with the WNT-activated molecular subtype.

• Relevance

• We demonstrate the potential for secondary and even tertiary acquisition of other molecular subtypes in hybrid medulloblastomas. Our findings support the need for detailed genomic and epigenetic molecular characterization of these tumors.

Similar to the first patient, next-generation sequencing of the tumor using a 447-gene targeted exome sequencing assay¹⁰ demonstrated CTNNB1 p.S37F, a recurrent activating mutation in exon 3 characteristic of WNT-activated medulloblastoma (Table 2). Three PTCH1 inactivating nonsense mutations were also identified (p.W256*, p.S733*, p.R770*), 1 as clonal and 2 as subclonal events. These were present in the context of subclonal copy-neutral loss of heterozygosity at distal chromosome 9q, detected by SNP array (Oncoscan,Thermo Fisher Scientific, Inc, Waltham, MA) and encompassing the PTCH1 locus, further supporting loss of function of the gene. DNA methylation profiling demonstrated a profile most consistent with WNT-activated medulloblastoma, however, with a low calibrated class score (0.13, compared with 0.09 for G3/G4 and 0.06 for SHH subtypes; Figs 2B and 2C). Integrating all of these data, the tumor was diagnosed as medulloblastoma, WHO grade IV, with characteristics of both WNT- and SHH-pathway–activated molecular subgroups. Case 2 has not had a recurrence 20 months after surgery and craniospinal radiotherapy.

TABLE 2.

Somatic Variants and Copy Number Changes in Case 2

DISCUSSION

In summary, these 2 WNT/SHH-pathway dual-activated tumors combined with the 6 previously reported patients present several commonalities, as well as novel insights. First, our patients and the patients in those case reports previously published all had methylation profiles that led them to cluster most closely with WNT-activated molecular subtype medulloblastoma, suggesting that the initial oncogenic CTNNB1 mutations occurred in precursor lineage cells similar to other WNT tumors, and then SHH-pathway–activating events may have been acquired secondarily. This is further supported by the subclonal nature of many of the PTCH1 truncating mutations observed, as well as the classic histology found in case 1—WNT-activated molecular subtype medulloblastoma typically display classic histology, whereas SHH-activated molecular subtypes sometimes display desmoplastic nodular or extensive nodularity histology.¹¹ The morphologic subclass of case 2 could not be determined because of its scant nature. Both tumors displayed multiple PTCH1 alterations likely to abrogate protein function, including frameshift and nonsense truncating mutations, similar to 1 prior reported case containing PTCH1 mutations in the previous WNT/SHH dual-activated medulloblastoma case series; additionally, neither case contained TP53 mutations. Notably, case 1 was also accompanied by 2 truncating mutations in FBXW7 at hotspots reported in approximately 10% of SHH-activated medulloblastomas, further supporting a dual-activated molecular subtype. FBXW7 encodes a key tumor suppressor ubiquitination-proteasome component responsible for degrading MYC and Notch pathway constituents and putatively suggests more aggressive behavior.¹² A similar mutation was also found in one of the previously reported patients with dual-activated, PTCH1-mutated disease.

An important novel aspect of our report is the finding that there is additional overlap of genotype classes beyond just dual WNT- and SHH-activating characteristics. The isochromosome 17q in case 1 is a hallmark of G3/G4 medulloblastomas, but is not generally associated with WNT-activated medulloblastomas and has been hypothesized to be a poor prognostic factor in the setting of G3/G4 subtype tumors. In the initial report of dual-activated medulloblastomas, 1 other patient with clonal CTNNB1 p.G34V, monosomy 6, loss of 17p, and gain of 17q consistent with isochromosome 17q was reported. This patient, however, lacked an activating SHH-pathway mutation. In the context of these novel findings, rereview of the copy number alterations of a recent medulloblastoma single-cell transcriptomics article identified an additional likely case (SJ129) of a WNT methylation profile medulloblastoma with 17p loss and 17q gain.¹³ To our knowledge, these are the only other known patients with WNT-activated medulloblastoma displaying isochromosome 17q reported in the literature. All patients with medulloblastomas with hybrid molecular subtypes reported to date have demonstrated a WNT subgroup methylation profile. The attributes of all known hybrid medulloblastomas in the literature are summarized in Table 3.

TABLE 3.

Summary of Hybrid Medulloblastomas in the Published Literature

In terms of clinical characteristics, of the 7 reported clinically evaluable patients with WNT/SHH dual-activated medulloblastomas (5 previously reported and 2 in the present report), 5 have occurred in young adults (23 to 46 years old; 3 men and 2 women). The hybrid medulloblastomas, therefore, may differ from the typical WNT-activated molecular subtype tumors, which characteristically occur in the pediatric population.¹¹ Given that WNT-activated molecular subtype medulloblastomas have an excellent prognosis^14,15but SHH-activated molecular and G3/G4 subtype tumors have more guarded and variable outcomes, identification of additional patients is needed to further strengthen prognostic and treatment recommendations.¹ Additionally, the heterogeneity of molecular subtypes may have therapeutic implications, particularly in patients with secondary activation of the SHH pathway, for which inhibitors of smoothened (eg, vismodegib and sonidegib) have demonstrated antitumoral activity specifically for SHH-activated medulloblastomas in clinical trials.^16,17 Because none of the hybrid medulloblastomas appeared to have been treated with smoothened inhibitors, additional investigation into the mechanisms and ramifications of secondary SHH activation will help elucidate whether such hybrid medulloblastomas may also respond to new smoothened inhibitors.

Taken altogether, our findings contribute to a progressively more complex molecular classification of medulloblastomas that challenges the notion of mutually exclusive subtypes. This work further highlights the importance of integrating advanced molecular techniques into the diagnostic classification of medulloblastomas. Genomic characterization, via next-generation sequencing, and epigenetic evaluation, namely, through DNA methylation profiling, has dramatically improved our ability to characterize CNS tumors like medulloblastomas in a clinically relevant manner.

AUTHOR CONTRIBUTIONS

Conception and design: Jeffrey Helgager, Matija Snuderl, J. Bryan Iorgulescu, Keith L. Ligon

Financial support: Keith L. Ligon

Administrative support: Keith L. Ligon

Provision of study materials or patients: Jeffrey Helgager, Keith L. Ligon

Collection and assembly of data: Jeffrey Helgager, Peter Pytel, Eudocia Q. Lee, Matija Snuderl, J. Bryan Iorgulescu, Keith L. Ligon

Data analysis and interpretation: Jeffrey Helgager, Peter Pytel, Varshini Vasudevaraja, Matija Snuderl, J. Bryan Iorgulescu, Keith L. Ligon

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).

Eudocia Q. Lee

Honoraria: Medlink

Consulting or Advisory Role: Eli Lilly, Prime Oncology

Patents, Royalties, Other Intellectual Property: Royalties from Wolter Kluwer for UptoDate

Matija Snuderl

Stock and Other Ownership Interests: Cellceutix Corporation, Epizyme, Geron, Isis Pharmaceuticals, Acceleron Pharma, C2I

Honoraria: Illumina

Consulting or Advisory Role: C2I

Travel, Accommodations, Expenses: Silicon Biosytems, Illumina

Keith L. Ligon

Stock and Other Ownership Interests: Travera

Consulting or Advisory Role: Bristol-Myers Squibb, RareCyte, BroadBranch, Integragen

Research Funding: Plexxikon (Inst), Amgen (Inst), X4 Pharma (Inst), Tragara (Inst), Bristol-Myers Squibb (Inst), Eli Lilly (Inst)

Patents, Royalties, Other Intellectual Property: Molecular Diagnostics assay patent

No other potential conflicts of interest were reported.