The identification of distinct medulloblastoma molecular subgroups(12
) offers significant potential to improve our understanding of disease biology and clinical management. Here, we report the development and validation of minimal diagnostic gene expression signatures which can be routinely applied to identify the SHH, WNT and WNT/SHH-independent medulloblastoma disease subgroups. These gene expression signatures are robust and informative for subgroup identification in RNA extracted from snap-frozen tumour material, and using different gene expression assays. In particular, the GeXP multiplex expression assay reported offers a number of significant advantages over microarray methodologies for the routine assignment of subgroup affiliation; analysis can be undertaken straightforwardly, rapidly (in one working day, compared to 2-3 days for a microarray experiment) and cost-effectively (approximately one-tenth of microarray analysis costs) and, importantly, can be performed on small amounts of total RNA (150 ng, compared with 500ng to 5μg for a typical microarray expression analysis), a common limitation when only small amounts of biopsy material are available. This removal of the need for relatively time-consuming, complex and expensive array analysis platforms for subgroup assignment provides a strong basis for their clinical application; these methods are feasible for investigation in real-time across multiple treatment centres during clinical treatment and in future clinical trials.
We have shown that the disease subgroups recognised by these signatures are equivalent and consistently identified in four independent medulloblastoma cohorts, allowing their assembly into a large combined cohort. Coupled with an extensive analysis of our novel primary cohort, this has allowed significant insights into the underlying molecular mechanisms, associated biomarkers, and clinical characteristics of these molecular disease subgroups.
Our systematic investigation of specific medulloblastoma genetic and epigenetic defects in this study has informed their roles as determinants or correlates of the molecular subgroups identified. Consistent with previous studies(12
mutations were identified as the primary pathway activating event present in almost all WNT subgroup tumours, with chromosome 6 losses also affecting the majority of these cases. PTCH1
mutation was the major mechanistic correlate of SHH activation, identified in ~34% of SHH cases. SHH-associated PTCH1
mutations were detected both in conjunction with chromosome 9q loss, and in the heterozygous state, indicating disruption of a single PTCH1
allele can be sufficient to cause SHH pathway disruption in medulloblastoma. An absence of COL1A2
hypermethylation was also significantly associated with SHH subgroup medulloblastomas, most strongly in infant cases. Notably, a number of the previously reported determinants of SHH activation that we examined (PTCH1
exon 1c methylation(16
) and 17p (REN
) allelic loss) were not specifically associated with the SHH subgroup, indicating any role they may play in medulloblastoma is SHH-independent. It is similarly unclear whether the mixed methylation patterns observed for PTCH1
exon 1c in the present study have functional significance, as this was not assessed. Additionally, other SHH pathway defects examined (PTCH1
exon 1a hypermethylation), including events previously reported in medulloblastoma (SMO
) or SUFU
)) were not observed at all, suggesting their roles are either less common than previously thought, or are restricted to limited tumour subsets less well represented in our mutation screening cohort. This is likely the case for SUFU
mutations, which appear to be associated with germline inheritance and have their peak incidence in infants(17
). Further mechanisms of pathway activation remain to be identified in the majority of SHH cases. Chromosome 17 defects were the only events significantly correlated with the most common WNT/SHH-independent subgroup, suggesting a role for chromosome 17 genes in these cases. This disease subgroup however remains the least well characterised at the molecular level. Sub-division of this group has been proposed on the basis of its transcriptomic and genomic patterns however, unlike the SHH and WNT groups, inconsistent results have been reported from different studies(12
), and the identification of specific genes and pathways associated with its pathogenesis will be critical to future advances in our understanding of its molecular basis and any underlying heterogeneity.
The significant associations observed between medulloblastoma molecular subgroups and specific gene, pathway and chromosomal defects (i) strongly support the existence of molecularly distinct SHH and WNT subgroups, (ii) inform the contributory mechanisms involved in their pathogenesis, and (iii) provide potential biomarkers for subgroup identification. When assessed for suitability as primary biomarkers, only CTNNB1
mutations, which were specifically observed in all but one WNT subgroup case, have sufficient sensitivity and specificity to have utility. Nuclear localisation of β-catenin has also been widely reported as a positive marker of WNT pathway activation(5
), although its relationship to our WNT expression signature and CTNNB1
mutations could not be investigated in the present study due to tissue limitations. Likewise, COL1A2
status may have utility in the identification of SHH subgroup infant desmoplastic medulloblastomas (this study and (25
)), particularly in cases where biopsy limitations do not allow assessment of the DN pathological variant. The remainder of gene and chromosomal defects investigated were not suitable as primary biomarkers for positive subgroup discrimination, as a result of either their (i) non-exclusivity, (ii) limitation to sub-sets of subgroup cases, or (iii) inverse correlation with pathway activation. In comparison, gene expression signatures positively identified all subgroup cases and provide an accurate diagnostic test for subgroup membership. The genomic markers examined may therefore provide useful secondary or confirmatory markers, when used in conjunction with these signatures. It is not presently clear whether the expression signatures reported translate into subgroup-specific protein expression. Protein expression markers, which are testable by immunohistochemistry, may have utility for routine subgroup assignment in the diagnostic setting, however careful investigation and validation of their sensitivity, robustness and reflection of expression array subgroup data, will be essential prior to their application.
The observation of 2/148 cases which were not consistently classified using the presently reported signatures and their respective array datasets () indicates potential difficulties in the robust classification of a small subset (<2%) of cases. Inspection of the microarray expression data for these 2 cases revealed markedly reduced expression of pathway signature genes relative to the other pathway positive cases in their respective cohorts, despite their subgroup positivity using our signatures (12
) (). Additionally, the stacked bar plot analyses revealed 2 further SHH-positive samples (T27, K452; ) which, although clustered consistently between signature and array on hierarchical cluster and PCA analysis, could be classified as indeterminate based on the application of quantitative criteria to the individual expression data in stacked bar plot analysis (i.e. cumulative stacked bar plot expression score >8 (for WNT expression signature positive cases) or >4 (SHH cases)). For such cases, additional markers of pathway activation (CTNNB1, PTCH1
mutation) could aid definitive subgroup assignment, and it is notable that 1 of these 2 samples also harboured a PTCH1
mutation, further supporting SHH subgroup membership. Silhouette analysis supported the robust assignment of subgroup status using our signatures (Supplementary Figure 1
) but, consistent with the other analytical methods applied, did not support the subgroup assignment made for 3 of the 4 discrepant cases described above, further highlighting difficulties in their classification. Thus, in diagnostic applications, a ‘non-classified’ designation could be reserved for these rare cases which do not classify consistently across all analyses performed on the signature data, particularly where subgroup assignment may impact clinical or therapeutic decisions.
The combination of molecular and clinico-pathological data from four independent cohorts for meta-analysis, totalling 173 cases, has facilitated clear and significant insights to the clinical features of the medulloblastoma molecular subgroups, which have either not been apparent or not shown statistical significance in individual analyses of the smaller component cohorts reported to date(12
). The SHH (24% of cases), WNT (12%) and WNT/SHH-independent (64%) groups show different age distributions and relationships to disease histopathology. SHH subgroup tumours peak in infancy and are intimately correlated with DN pathology in this group, to the extent that DN pathology may be considered as a surrogate marker for SHH activation in medulloblastomas arising in infants <3 years old at diagnosis, although classic and LCA cases also constitute a minority of SHH subgroup cases in this age group. This relationship breaks down in non-infants (≥3 years at diagnosis), where SHH tumours are less common, and show equivalent proportions of DN, classic and LCA disease; SHH-independent DN cases are also commonly observed in this age group. These data strongly indicate that (i) SHH subgroup and (ii) DN tumours, arising in the infant and non-infant age groups, have different biological and clinical characteristics, and that SHH-positive DN tumours of infancy represent a unique disease subgroup associated with a favourable clinical behaviour(35
), and a characteristic molecular pathogenesis (COL1A2
)) and mutational spectrum (SUFU
)). Conversely, WNT tumours display classic pathology and occur in non-infants. Notably, both the SHH and WNT subgroups show at least two different incidence peaks in their age distribution (both have second peaks in adults), suggesting additional clinical and molecular heterogeneity within these groups. The inclusion of cohort-wide central pathology review in future studies may aid the further refinement of the associations observed.
The lack of association between M stage and molecular subgroups (p
=0.20) is in disagreement with the previous study by Kool et al
), who reported metastatic tumours being less common in WNT and SHH pathway activated medulloblastomas. This could be due to the different measurement criteria for metastasis between the studies (the present study compared M0/1 versus M2/3, while Kool et al
compared M0 versus M1/M2/M3). Alternatively, the increased numbers in this study (130 versus
58 with M stage data in the Kool et al
study) may have enabled a more accurate characterisation of the relationship between signalling pathway activation and metastasis, and future large clinically controlled studies which include central review of metastatic status should be informative in this regard.
The identification of medulloblastoma molecular subgroups has significant prognostic and predictive potential to improve therapeutic delivery and disease outlook in the clinical setting, and could represent a first step in the molecular diagnostic triage of medulloblastomas, to guide treatment decisions. In addition to distinct clinical features, molecular subgroups also appear to have characteristic clinical behaviours; the favourable prognosis of WNT subtype medulloblastomas, is now established in multiple clinical cohorts(8
), and will form the basis of treatment reductions in forthcoming international molecularly-driven clinical trials(1
). Combined data from this and other studies indicate SHH-positive DN tumours arising in infants represent a similarly favourable prognosis subgroup with a distinct molecular basis(25
). The non-availability of outcome data for the four cohorts assessed in our meta-analysis have limited any direct assessment of survival associations in these cohorts. Moreover, their retrospective, heterogeneously-treated nature would likely confound such analyses. Robust assessment of the prognostic impact of the medulloblastoma molecular subgroups will therefore now be essential, ideally within the context of adequately powered, centrally-reviewed and uniformly-treated clinical trials cohorts, to determine their utility to direct the selection of adjuvant therapy. The molecular signatures we report will have significant utility in this regard. Molecularly targeted SHH inhibitors are also currently under pre-clinical and clinical development, and have shown early evidence of activity in medulloblastoma(10
). The ability to accurately diagnose the SHH molecular subgroup will thus be important for the targeted delivery of these novel agents, and our findings have identified SHH-positive subgroups of medulloblastomas which would be predicted to benefit most from SHH inhibition strategies. However, the SHH pathway plays a key role in normal, including cerebellar, development, and its transient inhibition in young mice causes permanent defects in growth plate formation and bone structure(39
). In view of such potential toxicities, our data recommend caution in their application, particularly in the infant age group where SHH subgroup tumours predominate.
Statement of translational relevance
Understanding the molecular basis of medulloblastoma will be essential to improve clinical outcomes, through guidance of molecular risk-adapted adjuvant therapies, and delivery of molecularly targeted agents. Expression microarray studies indicate the existence of discrete medulloblastoma molecular subgroups associated with activation of specific developmental signalling pathways (i.e. SHH, WNT). However, any translational utility will require definition of subgroup clinical and molecular correlates in large cohorts, alongside biomarkers and assays for routine subgroup assignment prior to adjuvant therapy selection. We report development of diagnostic gene expression signatures, which can be applied rapidly and cost-effectively in small biopsies, using array-independent methods, to assign sub-group status. Using these signatures in 173 medulloblastomas, we demonstrate disease subgroups are robust and reproducible, and have distinct clinical, molecular and outcome characteristics of therapeutic importance. These findings provide strong rationale and methodologies to support subgroup assessment as a basis for future medulloblastoma clinical trials and biological studies investigations.