Rapid Diagnosis of Medulloblastoma Molecular Subgroups

Published OnlineFirst February 16, 2011; DOI: 10.1158/1078-0432.CCR-10-2210

Clinical Cancer Imaging, Diagnosis, Prognosis Research

Ed C. Schwalbe1, Janet C. Lindsey1, Debbie Straughton1, Twala L. Hogg2, Michael Cole1, Hisham Megahed1, Sarra L. Ryan1, Meryl E. Lusher1, Michael D. Taylor4, Richard J. Gilbertson2, David W. Ellison3, Simon Bailey1, and Steven C. Clifford1

Abstract Purpose: Microarray studies indicate medulloblastoma comprises distinct molecular disease subgroups, which offer potential for improved clinical management. Experimental Design: Minimal mRNA expression signatures diagnostic for the Wnt/Wingless (WNT) and Sonic Hedgehog (SHH) subgroups were developed, validated, and used to assign subgroup affiliation in 173 tumors from four independent cohorts, alongside a systematic investigation of subgroup clinical and molecular characteristics. Results: WNT tumors [12% (21/173)] were diagnosed >5 years of age (peak, 10 years), displayed classic histology, CTNNB1 mutation (19/20), and associated chromosome 6 loss, and have previously been associated with favorable prognosis. SHH cases [24% (42/173)] predominated in infants (<3 years) and showed an age-dependent relationship to desmoplastic/nodular pathology; all infant desmoplastic/ nodular cases (previously associated with a good outcome) were SHH-positive, but these relationships broke down in noninfants. PTCH1 mutations were common [34% (11/32)], but PTCH1 exon1c hypermethylation, chromosome 9q and REN (KCTD11) genetic loss were not SHH associated, and SMO or SUFU mutation, PTCH1 exon1a or SUFU hypermethylation did not play a role, indicating novel activating mechanisms in the majority of SHH cases. SHH tumors were associated with an absence of COL1A2 methylation. WNT/SHH-independent medulloblastomas [64% (110/173)] showed all histolo- gies, peaked at 3 and 6 years, and were exclusively associated with chromosome 17p loss. Conclusions: Medulloblastoma subgroups are characterized by distinct genomic, epigenomic and clinicopathologic features, and clinical outcomes. Validated array-independent gene expression assays for the rapid assessment of subgroup affiliation in small biopsies provide a basis for their routine clinical application, in strategies including molecular disease-risk stratification and delivery of targeted therapeu- tics. Clin Cancer Res; 17(7); 1883–94. 2011 AACR.

Introduction cases with metastatic disease at diagnosis or incomplete surgical resection), current treatments only cure around Medulloblastoma, a primitive neuro-ectodermal tumor 40% to 60% of cases. In addition, many survivors exhibit of the cerebellum, is the most common malignant brain long-term therapy-associated late effects. The development tumor of childhood. Five-year overall survival rates have of novel targeted treatments, alongside refined patient increased over recent years to 70% to 80% for standard-risk stratification, will be essential to increase survival rates patients. However, for high-risk patients (infants <3 years, and reduce adverse sequelae (1). Improvements in understanding of the molecular basis of medulloblastoma will be fundamental to such advances. The constitutive activation of developmental signaling Authors' Affiliations: 1Northern Institute for Cancer Research, Newcastle pathways plays a key role in medulloblastoma pathogen- University, Newcastle upon Tyne, UK; Departments of 2Developmental 3 esis, and pathway components represent the major muta- Neurobiology and Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee; 4Division of Neurosurgery, Arthur and Sonia Labatt tional targets identified in the disease to date. The Sonic Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Hedgehog (SHH) pathway plays an essential role in normal Ontario, Canada cerebellar development, is activated by PTCH1 mutation in Note: Supplementary data for this article are available at Clinical Cancer around 10% of human primary medulloblastomas, and Research Online (http://clincancerres.aacrjournals.org/). promotes medulloblastoma development in mouse models Corresponding Author: Steven C. Clifford, Northern Institute for Cancer Research, Newcastle University, Sir James Spence Institute Level 5, Royal of the disease (2–4). Similarly, mutations in components of Victoria Infirmary, Newcastle upon Tyne NE1 4LP, UK. Phone: 44-191- the canonical Wnt/Wingless (WNT) signaling pathway have 2821319; Fax: 44-191-2821326; E-mail: [email protected] been described in up to 20% of cases (5–7). Importantly, doi: 10.1158/1078-0432.CCR-10-2210 these pathways appear to have therapeutic significance; 2011 American Association for Cancer Research. WNT-active cases are associated with a favorable prognosis

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Translational Relevance strategies and the identification of patients who could benefit from SHH and WNT targeted molecular therapeu- Understanding the molecular basis of medulloblas- tics; and (ii) provide a basis for biological investigations to toma will be essential to improve clinical outcomes further understand disease molecular pathogenesis and its through guidance of molecular risk-adapted adjuvant therapeutic applications. However, subgroup identification therapies and delivery of molecularly targeted agents. has so far relied on advanced genomics (i.e., microarray) Expression microarray studies indicate the existence of technologies, which are relatively expensive and have used discrete medulloblastoma molecular subgroups asso- different gene expression data collection and analysis ciated with activation of specific developmental signal- methodologies. The development of robust biomarkers ing pathways (i.e., SHH, WNT). However, any and assays for subgroup detection, which can be routinely translational utility will require definition of subgroup tested in clinical material across multiple treatment centers clinical and molecular correlates in large cohorts, along- and are informative in small tumor biopsies, will therefore side biomarkers and assays for routine subgroup assign- be essential for their future study and any clinical applica- ment before adjuvant therapy selection. We report tion. Moreover, studies in modestly sized cohorts have development of diagnostic gene expression signatures, limited comprehensive investigation of subgroup clinical which can be applied rapidly and cost-effectively in characteristics and their molecular basis. small biopsies, using array-independent methods, to In this study, we report the development and validation of assign subgroup status. Using these signatures in 173 minimal mRNA expression signatures, which are robust for medulloblastomas, we demonstrate that disease sub- the routine diagnosis of the SHH and WNT subgroups in groups are robust and reproducible and have distinct clinical material and can be applied rapidly and cost- clinical, molecular, and outcome characteristics of effectively using array-independent methodologies. Using therapeutic importance. These findings provide strong these signatures, we show that equivalent disease subgroups rationale and methodologies to support subgroup are detected in 4 independent medulloblastoma cohorts, assessment as a basis for future medulloblastoma clin- independent of gene expression assay used. We then use ical trials and biological investigations. these signatures to assign subgroup affiliation in this large combined cohort of 173 medulloblastomas and use these data as the basis for comprehensive investigations to define the clinical and molecular characteristics of each subgroup, (>90% overall survival; refs 8 and 9), whereas small mole- and their utility for improved disease management. cule inhibitors of the SHH pathway show preclinical and early-clinical activity against the disease (10, 11). Materials and Methods Recent array-based genome-wide genomic and transcriptomic investigations in medulloblastoma have identified Tissue samples distinct molecular disease subgroups, which are distin- A representative cohort of 55 medulloblastoma samples guished by their gene expression profiles, and display was analyzed, comprising 39 classic, 5 large cell/anaplastic related clinical disease features. Two disease groupings, (LCA), and 11 tumors of the DN histopathologic subtype characterized respectively by activation and mutation of (22), 21 female and 34 male cases, 11 infants (3 years), the WNT and SHH signaling pathways, are consistently 41 children (>3–15 years), and 3 adults (16 years). DNA supported by these studies (12, 13). The WNT subgroup is (n ¼ 55) and RNA (n ¼ 39) were extracted from these snap- best documented, and is distinguished by nuclear b-catenin frozen tissues using standard methods. A panel of consti- immunostaining, CTNNB1 mutations, and chromosome 6 tutional DNA samples from 100 normal individuals was loss (5, 13–15), alongside its associated favorable prog- obtained from the North Cumbria Community Genetics nosis (8, 9). The SHH subgroup is, however, less well Project in the United Kingdom. Research Ethics Committee characterized; PTCH1 mutations are only identified in a approval has been obtained for the collection, storage, and subset of SHH cases, indicating a role for other activating biological study of all material. mechanisms and correlates. A series of putative mechanisms of SHH activation [e.g., PTCH1 hypermethylation, Mutation screening SUFU/SMO mutation, and REN (KCDT11) genetic loss] All coding exons of the PTCH1 and SMO genes were PCR have been reported in medulloblastoma (16–19), but any amplified using the primers and conditions shown in relationships to the SHH subgroup remain to be estab- Supplementary Table S1. Mutation screening methods for lished. Moreover, SHH subgroup clinical features require the SUFU gene have been described previously (23). Muta- clarification; SHH activation has been associated with the tion screening was performed by analysis of PCR products desmoplastic/nodular (DN) disease histologic variant in for heteroduplex formation, before and after "spiking" with some studies, but not others, and associations with infant equal amounts of control wild-type DNA using denaturing cases have also been postulated (12, 17, 20, 21). high performance liquid chromatography (Wave DNA Identification of these medulloblastoma molecular sub- Fragment Analysis System, Transgenomic) according to groups has significant potential to (i) improve clinical the manufacturer’s instructions. Products detected as con- management, through molecular disease-risk stratification taining a heteroduplex were sequenced directly on an ABI

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377 sequencer (Applied Biosystems). In reported studies (group A) or SHH (group B) subgroups, defined by Kool including our own, denaturing high-performance liquid and colleagues (12), which were significant in both data chromatography has been reported to identify >90% of sets. Previous work had validated 3 SHH-associated genes sequence variants (23). Mutation analysis of the CTNNB1 (GLI1, PTCH2, and SFRP1) and 2 WNT-associated genes and APC genes was performed as previously described (8). (DKK2, WIF1) by quantitative reverse transcriptase real- time PCR (13) and these genes were also considered for Analysis of promoter methylation status inclusion in the signatures. Putative signature genes were Two promoter-associated CpG islands of the PTCH1 assessed for assay suitability, and assays were subsequently gene, spanning exons 1a and 1c (16), were identified performed using 50 ng total RNA per replicate, according to and characterized using the Emboss CpGPlot website manufacturer’s instructions. To eliminate the possibility of (http://www.ebi.ac.uk/emboss/cpgplot/): 1a methylation detecting genomic DNA, PCR products were designed status was determined by methylation-specific PCR across exon boundaries; products were also designed where (MSP), and 1c by bisulphite sequencing using previously possible to overlap the Affymetrix probes from which they published primers (16). A CpG island of the SUFU gene were derived. For genes with multiple transcripts, ampli- was also identified, and its methylation status analyzed by cons were designed that detected all known transcripts. Test MSP (24). COL1A2 methylation status was assessed by gene expression was normalized to 28S rRNA, and results bisulphite sequencing, and has previously been reported displayed are means based on independent assessment in (25). Bisulphite treatment, methylated, and unmethylated triplicate. The presence of SHH or WNT expression signa- controls have been described previously (23). Primers and tures was used to identify sample subgroup membership conditions for analysis of PTCH1 CpG island 1a are shown (see next section). Signature gene selection is summarized in Supplementary Table S2. Methylation status was desig- in Supplementary Table S3, and PCR primer sequences are nated as methylated or unmethylated, as previously listed in Supplementary Table S4. described (26). For loci assessed by MSP, any sample showing a visible PCR product using primers specific for Integration and analysis of combined gene expression the methylated sequence was classed as showing evidence data sets of methylation (i.e., methylated). For loci assessed by An additional medulloblastoma expression microarray bisulphite sequencing, the relative peak intensities at each data set, reported by Fattet and colleagues (15), was CpG residue were determined. Samples in which the normalized as described earlier. All 3 publicly available methylated peak represented >25% of the total peak height data sets have linked clinical (age at diagnosis, pathology in greater than 25% of the analyzed CpG sites were classed subtype, and gender) and PTCH1/CTNNB1 mutational as showing evidence of methylation (i.e., methylated). data available [except PTCH1 mutation data not available for the Fattet and colleagues (15) data set]. Expression Loss of hetererozygosity analysis data of signature genes from all 3 data sets, together with Loss of hetererozygosity (LOH) of chromosome regions our own, were integrated. First, our GeXP expression data 9q22.3 and the p-arm of chromosome 17 was analyzed were log2 transformed as for the expression microarray using polymorphic microsatellite markers D9S1689, data and, before joining, all data sets were scaled sepa- D9S1816, D9S287, D9S1809, D9S1786, D9S1851, and rately, on a per-gene basis, to give a mean of 0, with D17S2196, D17S936, D17S969, D17S974, D17S1866, variance of 1. Unsupervised hierarchical clustering, sup- D17S1308, respectively (www.ncbi.nlm.nih.gov/genome/ ported by principal component analysis (PCA), was used sts), as previously described (27). LOH of chromosome 6 to assign subgroup membership status (Fig. 1), and these was analyzed using previously described markers and data were used in conjunction with stacked bar plots methods (14). (Fig. 2) and silhouette plots (ref. 31; Supplementary Fig. S1) to assess performance of the signatures. There A multigene mRNA expression signature to identify wassomesampleoverlapbetweenstudies:11samples SHH and WNT pathway activated tumors were assessed by expression array by Thompson and A 13-gene multiplex mRNA expression assay (GeXP; colleagues (13) and GeXP; 3 samples were assessed by Beckman Coulter; ref. 28) was developed and used to test expression array by Kool and colleagues (12) and GeXP, tumors for membership of the SHH or WNT medulloblas- which enabled the comparison of subgroup assignment toma subgroups in 39 cases. Detailed clinical and patho- in individual samples using our signatures, when evalu- logic data for individual cases are summarized in Table 1. ated using different gene expression assays. Clinical and Two previously reported independent medulloblastoma genomic correlates were also combined for the joined expression microarray data sets (12, 13) were used to cohort. Duplicate analyses were removed from all corre- design SHH and WNT subgroup signatures. Data were lative investigations. respectively downloaded from the St Jude Research website (http: / /www.stjuderesearch.org / site / data / medulloblas- Statistical analysis toma) and the Gene Expression Omnibus (GEO; ref. 29). Fisher’s exact and c2 tests were used to identify relation- Data were rma normalized (30) using R and Bioconductor. ships between expression signatures and molecular and Using t tests, we identified probes differential for the WNT clinical features of the disease in the primary investigative

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Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2011 American Association for Cancer Research. 1886 lnCne e;1()Arl1 2011 1, April 17(7) Res; Cancer Clin al. et Schwalbe Downloaded from Published OnlineFirstFebruary16,2011;DOI:10.1158/1078-0432.CCR-10-2210 clincancerres.aacrjournals.org Table 1. Associations between molecular subgroups and medulloblastoma genomic, epigenomic, and clinical disease features

SHH WNT WNT-/SHH-independent Corrected 'p ' value 'p ' value 22 13 19 43 14 32 45 17 5 55 54 31 2 10 7 3 52 38 9 53 51 4 30 26 46 42 16 12 27 40 41 28 21 24 44 20 25 11 50 Molecular SHH -- subgroupa WNT --

PTCH FS FS FS FS FS 9.4 x 10-5 * 1.5 x 10-3 * SUFU MS ns ns Genetic SMO ns ns mutationb CTNNB1 MS MS MS 5.6 x 10-9 * 9.0 x 10-8 * APC ns ns on September 29, 2021. © 2011American Association for Cancer PTCH1a ns ns Research. Hyper- PTCH1c 0.03 * ns c methylation SUFU ns ns COL1A2 0.01 * ns

6 9.3 x 10-9 * 1.5 x 10-7 * Chromosomal 9q 0.09 ** ns lossd 17p (KCDT11 ) 0.06 ** ns

Age 1.0 2.6 2.9 3.5 4.8 11.5 14.2 16.7 19.0 9.0 10.0 10.3 2.5 2.5 2.6 2.9 3.0 3.3 4.1 4.3 4.6 4.6 5.0 5.1 5.4 5.5 5.7 6.3 6.8 7.5 8.5 8.6 9.8 10.0 10.2 12.5 12.6 15.4 17.0 ns ns Pathology 0.003 * 0.05* Clinical features Sex F MMFFM F MM F MM MMF M FFMMF MMMMMF MMMFFFM F MMFF ns ns M Stage ns ns

NOTE. Cases are shown arranged by signature status (SHH, WNT, WNT-/SHH-independent) as determined in Fig. 1, and then by ascending age, amolecular subgroup; bmutation status; chypermethylation status (black, methylated; white, unmethylated); and dchromosomal loss (black, allelic; white, no loss). Age is shown in years and categorized into infants (3 years, black) and noninfants (>3 years, white). Pathology variant is indicated by a white square (classic), black square (DN) or gray square (LCA). Gender is indicated by black squares (F, female) and white squares (M, male). M stage 0/1 is indicated by white squares and M stage 2/3 by black squares. Raw P and P values corrected for c P <

lnclCne Research Cancer Clinical multiple hypothesis testing are shown for relationships between molecular/clinical correlates and subgroup membership ( 2 tests, Bonferroni correction). Significant (* 0.05) and marginally significant (**P ¼ 0.051–0.10) associations are marked. Diagonally hatched boxes indicate unavailable data. Abbreviations: FS, frameshift; MS, missense mutation; ns, not significant. Published OnlineFirst February 16, 2011; DOI: 10.1158/1078-0432.CCR-10-2210

Medulloblastoma Molecular Subgroups

Figure 1. Diagnostic WNT and SHH subgroup gene expression signatures recognize equivalent molecular subgroups across multiple medulloblastoma cohorts. Data from 4 independent data sets are shown [A, primary investigation cohort; B, Kool and colleagues (n ¼ 62; ref. 12); C, Thompson and colleagues (n ¼ 46; ref. 13); and D, Fattet and colleagues (n ¼ 40; ref. 15)]. WNT and SHH subgroup expression signature positivity demonstrates close concordance with (i) underlying molecular defects in the respective pathways and (ii) discrete sample clusters identified on independent clustering of the most variable probes in each array data set (Supplementary Fig. S2). Each panel (A–D) shows hierarchical clustering of signature genes (WNT subgroup, red; SHH subgroup, blue; WNT-/SHH- independent, gray). Mutational information for CTNNB1 and PTCH1 is shown (colored boxes, mutation; gray checked boxes, missing data). Array clusters which show concordance with the detected SHH and WNT subgroup signatures (derived by clustering the most variable probes of each whole array data set) are shown [purple, SHH subgroup; orange, WNT subgroup (Supplementary Fig. S2)]. Biplots show PCA for each signature geneset. Arrows show projections of expression axes for each gene (SHH signature genes, blue; WNT signature genes, red; WNT-/SHH- independent cases, gray).

(GeXP) cohort (n ¼ 39) and in the combined cohort (n ¼ associated biomarkers, and clinical relevance. The 8- 173). Bonferroni corrections for multiple hypothesis test- gene SHH (BCHE, GLI1, ITIH2, MICAL1, PDLIM3, ing were applied where appropriate. Additional patient age PTCH2, RAB33A,andSFRP1) and 5-gene WNT data were kindly provided by Dr. M. Kool (Academic (CCDC46, DKK2, PYGL, TNC, and WIF1) subgroup Medical Centre, Amsterdam, the Netherlands). signatures developed were initially assessed by GeXP assay in our primary sample cohort (n ¼ 39) and were Results unequivocal for all samples, independent of data analysis method used (stacked bar plots, PCA, or unsuper- A diagnostic multigene mRNA expression signature vised hierarchical clustering; Figs. 1 and 2). Validation of assay for SHH and WNT medulloblastoma subgroups these signatures in 3 independent medulloblastoma We first developed and validated multigene mRNA expression microarray data sets [Thompson and collea- expression signatures to facilitate routine identification gues (n ¼ 46; ref. 13); Kool and colleagues (n ¼ 62; of the SHH and WNT medulloblastoma molecular dis- ref. 12); and Fattet and colleagues (n ¼ 40;ref.15)] ease subgroups and assessment of their molecular basis, showed the signatures could be successfully interrogated

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Figure 2. Identification of SHH and WNT subgroup medulloblastomas using diagnostic expression signatures. Data are illustrated from the 4 independent data sets using stacked bar plots [A, primary investigation cohort; B, Kool and colleagues (n ¼ 62; ref. 12); C, Thompson and colleagues (n ¼ 46; ref. 13); and D, Fattet and colleagues (n ¼ 40; ref. 15)]. WNT signature genes (red) and SHH signature genes (blue) in combination clearly define subgroup membership. Vertical dashed lines delineate sample groups positive for WNT and SHH signatures by hierarchical clustering and PCA analysis in Fig. 1. Right-hand panel indicates stacked order of genes for each signature. Before generation of bar plots, expression data from each cohort were scaled on a per gene basis to a mean of zero and a variance of 1. Samples expressing all or most signature genes at above average levels will show bars of greater positive magnitude.

by hierarchical cluster analysis and PCA, were diagnostic Integration of cohorts and gene expression data sets: in all cases, independent of cohort or gene expression molecular subgroup incidence assay used (Fig. 1), and showed close consistency with Based on our validated gene expression signatures and stacked bar plot data (Fig. 2). Signature positivity was data analysis methods, the molecular subgroup data from concordant with the disease subgroups apparent after all 4 cohorts could be combined for meta-analysis with the independent clustering of the most variable probes mutational and clinical data which were consistently within each entire array data set, and correctly classified reported for all studies. A total cohort of 173 cases was disease subgroup (i.e., as SHH, WNT, or WNT/SHH assessed in this analysis (Supplementary Table S5). Clinical independent) in 99% (146/148) of cases overall demographics available for the combined cohort were (Fig. 1). Data sets similarly showed complete concor- consistent with previously reported groupwide estimates dance in subgroup assignment, using all analytical for medulloblastoma (1); male:female ratio (1.42:1), methods,ofthe14cases(4SHH,2WNT,and8 histology [115 classic (68%), 39 (23%) DN, 16 (9%) WNT/SHH independent cases) where signatures were LCA, 3 cases with data not available; pathologic classifica- assessed in parallel by microarray and GeXP assays. tions were those reported in the original publications

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(12, 13, 15)], and age at diagnosis [median 6.45, range 0.3– PTCH1 exon 1c CpG island were observed in 12 of 27 of 35.3 years, 34 (20%) infant, 138 (80%) noninfant cases, 1 tumors successfully analyzed (44%; Table 1), suggesting a case with data not available]. An overall incidence of 21 potential role for this epigenetic mechanism. (12%) WNT and 42 (24%) SHH subgroup cases was Unlike PTCH1 mutations, other defects identified observed. Subclassification of the remainder of cases based [PTCH1 exon 1c methylation, SUFU missense mutation, on their transcriptomic patterns has not produced consis- and 17p allelic loss (REN; KCTD11)] were not associated tent findings in previous studies (12, 13). Remaining with the SHH subgroup (Table 1), indicating that any role WNT-/SHH-independent medulloblastomas were there- these mechanisms may play in medulloblastoma develop- fore classified as a single group, representing 110 (64%) ment is independent of the SHH pathway. cases. Genomic biomarkers of SHH and WNT pathway Genetic and epigenetic mechanisms of SHH and WNT activation activation We next investigated selected medulloblastoma chro- Genetic mutations in PTCH1 and CTNNB1 were exclu- mosomal abnormalities (chromosome 6, 9q, and 17p sively detected in SHH and WNT subgroup cases, respec- loss) and epigenomic defects (COL1A2 status) of biolo- tively, across all 4 cohorts, where data were available gical and/or prognostic significance (1, 25), for their [combined cohort, P ¼ 5.3 10 8 and P ¼ 0(c2 test), associations with the SHH and WNT disease subgroups, respectively; Fig. 1], further validating the fidelity of the and each other, to assess any utility as biomarkers of gene expression signatures developed. CTNNB1 mutation pathway activation. was the primary mechanism and correlate of WNT pathway Chromosome 6 loss, CTNNB1 mutation, and the activation; in 20 cases where combined expression and absence of chromosome 9 and 17 abnormalities, were mutation data were available, all except 1 WNT subgroup observed in all WNT cases in our primary cohort (Table 1), case (19/20; 95%) had concurrent CTNNB1 mutation. consistent with previous findings (12–15). Across the Consistent with this, no APC mutations were found in combined cohort, evidence of loss of an entire copy of the 55 cases tested within our primary cohort. chromosome loss 6 was associated with 88% (14/16) of PTCH1 mutation was a major mechanism of SHH path- WNT cases with available data (P < 3 10 16, Fisher’s way activation; 34% (11/32) of SHH subgroup cases inves- exact); however, this relationship was not exclusive. Chro- tigated harbored a PTCH1 mutation (Fig. 1). We therefore mosome 6 loss was also detected in occasional non-WNT next undertook a systematic investigation of alternative cases (2/145). Eight of 35 tumors tested (23%) showed genetic mechanisms of pathway activation in the remain- evidence of genetic loss at the 9q22.3 region surrounding ing majority of SHH cases in our primary cohort. Muta- the PTCH1 locus in our primary cohort (Table 1). Four of 8 tional analysis encompassed all pathway genes in which were in the SHH subgroup and 2 of 4 tumors with PTCH1 mutations have previously been reported; the complete mutations showed LOH of 9q22.3; however neither asso- coding sequences of PTCH1 [including exon 1B, which ciation reached significance (P ¼ 0.09 and 0.22 respec- has been shown to code for protein (32), but has not been tively, Fisher’s exact test). A significant inverse association analyzed in previous studies (3, 20, 21)], SUFU and between 17p loss and membership of the SHH and WNT SMO [which has only been previously been assessed for subgroups was observed; 17p losses were exclusively mutation at specific residues (18)]. In addition to PTCH1 observed in WNT-/SHH-independent cases [17p LOH in truncating mutations (n ¼ 5), only 1 further potentially 0/12 SHH or WNT cases vs. 9/25 WNT-/SHH-independent pathogenic missense SUFU mutation was identified, with cases (P ¼ 0.02, Fisher’s exact test)]. In addition, COL1A2 no evidence of SMO mutation found (Supplementary hypermethylation was detected in 76% (25/33) of cases; an Table S6). Additional nonpathogenic variants discovered absence of COL1A2 methylation was significantly asso- (e.g., polymorphisms or intronic changes) are summarized ciated with the SHH subgroup (P ¼ 0.01, c2 test), but this in Supplementary Table S7. Allelic loss of chromosome relationship was not maintained when a correction for 17p, targeting REN (KCTD11) at 17p13.2, has also been multiple hypothesis testing was applied (Table 1). previously associated with SHH activation in medulloblastoma (19, 33), and was observed in 24% (9/37) of cases. Medulloblastoma molecular subgroups display Epigenetic mechanisms of pathway activation were addi- distinct clinical features tionally investigated as an alternative to genetic determi- Analysis of the medulloblastoma molecular subgroups nants. Two predicted promoter-associated CpG islands defined by our gene expression signatures, in all 173 cases spanning exons 1a and 1c of PTCH1, and a promoter- of the combined cohort, revealed marked differences in associated CpG island within SUFU, were identified and their clinical disease features. WNT-/SHH-independent investigated for evidence of DNA hypermethylation, which tumors made up the majority of cases, and had their peak may lead to epigenetic transcriptional silencing. No evi- incidence in the 3- to 6-year age group, but were extremely dence of DNA methylation of the PTCH1 exon 1a-asso- rare in the first 2 years of life (Fig. 3). In contrast, SHH ciated or the SUFU CpG island was seen in any tumor subgroup tumors had their major peak of incidence in analyzed (n ¼ 39; Table 1), indicating no major role in infancy [50% (21/42) of SHH cases were 3 years of age]. disease development. Mixed patterns of methylation of the The SHH subgroup tumors represented the majority of

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A. WNT WNT/SHH ind n=109 B. SHH A WNT n=21 SHH n=42 100% 17% (n=20) (n=7) LCA CLAS 60% 24% CLAS DN (n(n=25) 25) (n=10) Case density C

C. WNT/SHH independent

8% n 13% Age (y) (9)( =9) (n=14) B LCA DN 45 40 CLAS 35 79% 30 (n=85) 25 F 20 15 Figure 4. Molecular subgroups show relationships to medulloblastoma 10 histologic variants. Subgroup and histologic information was available for 5 170 of 173 cases (Supplementary Table S5). A, WNT subgroup (n ¼ 20); 0 B, SHH subgroup (n ¼ 42); C, WNT-/SHH-independent cases (n ¼ 108; 0–3 3–6 6–9 9–12 12–15 15–18 >18 P ¼ 3.1 1011, c2 test). White, classic (CLAS); gray, LCA; black DN Age range (y) histologic variants. C 16 had a bi-modal age distribution with major and minor 14 peaks at 10 and 20 years, respectively. 12 Significant differences were also observed in the dis- 10 tribution of medulloblastoma histologic subtypes F 8 between the molecular subgroups (P ¼ 3.1 10 11, c2 6 test; Figs. 4 and 5). WNT subgroup cases exclusively 4 displayed classic histology (P ¼ 0.0003, Fisher’s exact 2 test), and WNT-/SHH-independent tumors were also pre- 0 dominantly of the classic subtype, but DN and LCA cases 0–1 1–2 2–3 3–4 4–5 5–6 were also observed. Consistent with previous studies (3, Age range (y) 12, 13, 21), SHH cases were overall strongly associated with DN histology (P ¼ 1.1 10 9,Fisher’sexacttest). Figure 3. Medulloblastoma molecular subgroups show distinct age of However, this relationship was not absolute and LCA and incidence distributions. Data for the WNT (gray), SHH (black), and WNT-/ classic cases were also observed in the SHH group. Most SHH-independent (hatched) subgroups are shown, based on a combined notably, examination of this large cohort revealed the cohort of 173 medulloblastomas. A, density plots show subgroup- dependent ages of incidence. Case density represents the smoothed relationship between SHH activation and DN pathology frequency of incidence within each of the 3 subgroups. Gray dotted line is to be age dependent (Figs. 4 and 5); DN cases made up plotted at 3 years of age. B, bar plots show age distribution of data set. the majority of infant (3 years old) SHH subgroup cases; C, bar plot shows age distribution of cases aged 6 years at diagnosis. F, all DN cases in this infant group displayed SHH activa- frequency. tion. DN pathology may therefore serve as a surrogate marker of SHH activation in the infant group. In contrast, there were almost equal proportions of DN, LCA, and the infant clinical group [62% (21/34) of cases 3 years of classic cases in SHH-expressing noninfant cases, and the age], but were less common in noninfant children [>3–15 majority of noninfant DN tumors were not SHH activated years; 15% (16/127)], and had a further increased inci- (P ¼ 8.6 10 5, Fisher’s exact test). No significant dence in adult cases [16 years (45%; 5/11P); overall P ¼ evidence for differences in metastatic stage [WNT 6% 5.8 10 9, c2 test]. Almost all cases <2 years of age [92% (1/16) M stage 2/3, SHH 16% (5/32), and WNT-/SHH- (11/12)] were SHH-positive. WNT subgroup cases were not independent 24% (20/82)] was observed between the observed in infants (minimum age observed, 5 years) and different expression subgroups (P ¼ 0.20, c2 test).

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lysis platforms for subgroup assignment provides a strong A1. ≤3 years, SHH B1. ≤3 years, DN basis for their clinical application; these methods are fea- sible for investigation in real time across multiple treatment 5% centers during clinical treatment and in future clinical (1) 100% trials. 19% (n=16) (n=4) We have shown that the disease subgroups recognized by CLAS these signatures are equivalent and consistently identified DN 76% SHH in 4 independent medulloblastoma cohorts, allowing their (n=16) n=21 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 charac- A2. >3 years, SHH B2. >3 years, DN teristics of these molecular disease subgroups. Our systematic investigation of specific medulloblastoma genetic and epigenetic defects in this study has 29% informed their roles as determinants or correlates of the (n=6) 39% molecular subgroups identified. Consistent with previous LCA 43% non- SHH (n=9) studies (12–14), CTNNB1 mutations were identified as the DN n ( =9) 61% SHH primary pathway-activating event present in almost all CLAS (n=14) WNT subgroup tumors, with chromosome 6 losses also 29% affecting the majority of these cases. PTCH1 mutation was (n=6) the major mechanistic correlate of SHH activation, identified in 34% of SHH cases. SHH-associated PTCH1 mutations were detected both in conjunction with chromosome Figure 5. Associations between SHH subgroup medulloblastomas and DN 9q loss, and in the heterozygous state, indicating disrup- histology are age-dependent. A, histologic variants [white, classic tion of a single PTCH1 allele can be sufficient to cause SHH (CLAS); gray, LCA; black DN] show significantly different distributions P ¼ c2 pathway disruption in medulloblastoma. An absence of ( 0.05; test) in SHH subgroup cases arising in infants [ 3 years at COL1A2 diagnosis (n ¼ 21); A1] and noninfants [>3 years (n ¼ 21); A2]. B, infant hypermethylation was also significantly asso- (n ¼ 16; B1) and noninfant (n ¼ 23; B2) DN medulloblastomas show ciated with SHH subgroup medulloblastomas, most significantly different relationships to the SHH subgroup (P ¼ 8.6 10 5; strongly in infant cases. Notably, a number of the pre- Fisher's exact test; SHH subgroup, white; non-SHH, black). viously reported determinants of SHH activation that we examined [PTCH1 exon 1c methylation (ref. 16), SUFU missense mutation (ref. 17), and 17p REN (KCTD11; ref. Discussion 19) allelic loss] were not specifically associated with the SHH subgroup, indicating any role they may play in The identification of distinct medulloblastoma molecu- medulloblastoma is SHH-independent. It is similarly lar subgroups (12, 13, 15) offers significant potential to unclear whether the mixed methylation patterns observed improve our understanding of disease biology and clinical for PTCH1 exon 1c in this study have functional signifi- management. Here, we report the development and valida- cance, as this was not assessed. In addition, other SHH tion of minimal diagnostic gene expression signatures pathway defects examined (PTCH1 exon 1a hypermethyla- which can be routinely applied to identify the SHH, tion), including events previously reported in medulloblas- WNT, and WNT-/SHH-independent medulloblastoma dis- toma [SMO mutations (ref. 18) or SUFU truncating ease subgroups. These gene expression signatures are robust mutations (ref. 17)] were not observed at all, suggesting and informative for subgroup identification in RNA their roles are either less common than previously thought extracted from snap-frozen tumor material, and using or are restricted to limited tumor subsets less well repre- different gene expression assays. In particular, the GeXP sented in our mutation screening cohort. This is likely the multiplex expression assay reported offers a number of case for SUFU mutations, which appear to be associated significant advantages over microarray methodologies for with germline inheritance and have their peak incidence in the routine assignment of subgroup affiliation; analysis can infants (17, 23, 34). Further mechanisms of pathway be undertaken straightforwardly, rapidly (in 1 working day, activation remain to be identified in the majority of compared with 2–3 days for a microarray experiment) and SHH cases. Chromosome 17 defects were the only events cost-effectively (approximately one tenth of microarray significantly correlated with the most common WNT-/ analysis costs) and, importantly, can be performed on SHH-independent subgroup, suggesting a role for chromo- small amounts of total RNA (150 ng, compared with some 17 genes in these cases. This disease subgroup, 500 ng to 5 mg for a typical microarray expression analysis), however, remains the least well characterized at the mole- a common limitation when only small amounts of biopsy cular level. Subdivision of this group has been proposed on material are available. This removal of the need for rela- the basis of its transcriptomic and genomic patterns; how- tively time-consuming, complex, and expensive array ana- ever, unlike the SHH and WNT groups, inconsistent results

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have been reported from different studies (12, 13), and the stacked bar plot expression score >8 (for WNT expression identification of specific genes and pathways associated signature positive cases) or >4 (SHH cases)]. For such cases, with the pathogenesis of this subgroup will be critical to additional markers of pathway activation (CTNNB1, future advances in our understanding of its molecular basis PTCH1 mutation) could aid definitive subgroup assign- and any underlying heterogeneity. ment, and it is notable that 1 of these 2 samples also The significant associations observed between medullo- harbored a PTCH1 mutation, further supporting SHH blastoma molecular subgroups and specific gene, pathway, subgroup membership. Silhouette analysis supported the and chromosomal defects (i) strongly support the existence robust assignment of subgroup status using our signatures of molecularly distinct SHH and WNT subgroups, (ii) (Supplementary Fig. S1) but, consistent with the other inform the contributory mechanisms involved in their analytical methods applied, did not support the subgroup pathogenesis, and (iii) provide potential biomarkers for assignment made for 3 of the 4 discrepant cases described subgroup identification. When assessed for suitability as earlier, further highlighting difficulties in their classifica- primary biomarkers, only CTNNB1 mutations, which were tion. Thus, in diagnostic applications, a "nonclassified" specifically observed in all but 1 WNT subgroup case, have designation could be reserved for these rare cases which do sufficient sensitivity and specificity to have utility. Nuclear not classify consistently across all analyses performed on localization of b-catenin has also been widely reported as a the signature data, particularly where subgroup assignment positive marker of WNT pathway activation (5, 8, 14), may impact clinical or therapeutic decisions. although its relationship to our WNT expression signature The combination of molecular and clinicopathologic and CTNNB1 mutations could not be investigated in this data from 4 independent cohorts for meta-analysis, total- study due to tissue limitations. Similarly, COL1A2 status ing 173 cases, has facilitated clear and significant insights to may have utility in the identification of SHH subgroup the clinical features of the medulloblastoma molecular infant desmoplastic medulloblastomas [this study and subgroups, which have either not been apparent or not (25)], particularly in cases where biopsy limitations do shown statistical significance in individual analyses of the not allow assessment of the DN pathologic variant. The smaller component cohorts reported to date (12, 13, 15). remainder of gene and chromosomal defects investigated The SHH (24% of cases), WNT (12%), and WNT-/SHH- were not suitable as primary biomarkers for positive sub- independent (64%) groups show different age distribu- group discrimination, as a result of their (i) nonexclusivity, tions and relationships to disease histopathology. SHH (ii) limitation to subsets of subgroup cases, or (iii) inverse subgroup tumors peak in infancy and are intimately cor- correlation with pathway activation. In comparison, gene related with DN pathology in this group, to the extent that expression signatures positively identified all subgroup DN pathology may be considered as a surrogate marker for cases and provide an accurate diagnostic test for subgroup SHH activation in medulloblastomas arising in infants <3 membership. The genomic markers examined may there- years old at diagnosis, although classic and LCA cases also fore provide useful secondary or confirmatory markers, constitute a minority of SHH subgroup cases in this age when used in conjunction with these signatures. It is not group. This relationship breaks down in noninfants (3 presently clear whether the expression signatures reported years at diagnosis), where SHH tumors are less common, translate into subgroup-specific protein expression. Protein and shows equivalent proportions of DN, classic, and LCA expression markers, which are testable by immunohisto- disease; SHH-independent DN cases are also commonly chemistry, may have utility for routine subgroup assign- observed in this age group. These data strongly indicate that ment in the diagnostic setting, however careful (i) SHH subgroup and (ii) DN tumors, arising in the infant investigation and validation of their sensitivity, robustness, and noninfant age groups, have different biological and and reflection of expression array subgroup data, will be clinical characteristics, and that SHH-positive DN tumors essential before their application. of infancy represent a unique disease subgroup associated The observation of 2 of 148 cases which were not with a favorable clinical behavior (35–37), and a charac- consistently classified using the presently reported signa- teristic molecular pathogenesis [COL1A2 unmethylated tures and their respective array data sets (Fig. 1) indicates (ref. 25)] and mutational spectrum [SUFU (refs. 17, potential difficulties in the robust classification of a small 34)]. Conversely, WNT tumors display classic pathology subset (<2%) of cases. Inspection of the microarray expres- and occur in noninfants. Notably, both the SHH and WNT sion data for these 2 cases revealed markedly reduced subgroups show at least 2 different incidence peaks in their expression of pathway signature genes relative to the other age distribution (both have second peaks in adults), sug- pathway positive cases in their respective cohorts, despite gesting additional clinical and molecular heterogeneity their subgroup positivity using our signatures (refs. 12, 13, within these groups. The inclusion of cohort-wide central 15; Fig. 2). In addition, the stacked bar plot analyses pathology review in future studies may aid the further revealed 2 further SHH-positive samples (T27, K452; refinement of the associations observed. Fig. 2) which, although clustered consistently between The lack of association between M stage and molecular signature and array on hierarchical cluster and PCA ana- subgroups (P ¼ 0.20) is in disagreement with the previous lysis, could be classified as indeterminate based on the study by Kool and colleagues (12), who reported metastatic application of quantitative criteria to the individual expres- tumors being less common in WNT and SHH pathway– sion data in stacked bar plot analysis [i.e., cumulative activated medulloblastomas. This could be due to the

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Medulloblastoma Molecular Subgroups

different measurement criteria for metastasis between the larly targeted SHH inhibitors are also currently under studies (this study compared M0/1 vs. M2/3, whereas Kool preclinical and clinical development, and have shown early and colleagues compared M0 vs. M1/M2/M3). Alterna- evidence of activity in medulloblastoma (10, 11). The tively, the increased numbers in this study (130 vs. 58 ability to accurately diagnose the SHH molecular subgroup with M stage data in the Kool and colleagues study) may will thus be important for the targeted delivery of these have enabled a more accurate characterization of the rela- novel agents, and our findings have identified SHH-posi- tionship between signaling pathway activation and metas- tive subgroups of medulloblastomas which would be pre- tasis, and future large clinically controlled studies which dicted to benefit most from SHH inhibition strategies. include central review of metastatic status should be infor- However, the SHH pathway plays a key role in normal, mative in this regard. including cerebellar, development, and its transient inhibi- The identification of medulloblastoma molecular sub- tion in young mice causes permanent defects in growth groups has significant prognostic and predictive potential plate formation and bone structure (39). In view of such to improve therapeutic delivery and disease outlook in the potential toxicities, we recommend caution in the applica- clinical setting and could represent a first step in the tion of our data, particularly in the infant age group where molecular diagnostic triage of medulloblastomas to guide SHH subgroup tumors predominate. treatment decisions. In addition to distinct clinical features, molecular subgroups also appear to have characteristic Disclosure of Potential Conflicts of Interest clinical behaviors; the favorable prognosis of WNT subtype medulloblastomas is now established in multiple clinical No potential conflicts of interest were disclosed. cohorts (8, 9, 15, 38) and will form the basis of treatment reductions in forthcoming international molecularly dri- Acknowledgments ven clinical trials (1). Combined data from this and other studies indicate SHH-positive DN tumors arising in infants Medulloblastomas investigated in this study include samples provided by the UK Children’s Cancer and Leukaemia Group (CCLG) as part of CCLG- represent a similarly favorable prognosis subgroup with a approved biological study BS-2007–04. This study was conducted with distinct molecular basis (25, 35–37). The nonavailability of ethics committee approval from Newcastle/North Tyneside REC (study outcome data for the 4 cohorts assessed in our meta- reference 07/Q0905/71). analysis have limited any direct assessment of survival associations in these cohorts. Moreover, their retrospective, Grant Support heterogeneously treated nature would likely confound This work was supported by grants from The Katie Trust (to S.C. Clifford), such analyses. Robust assessment of the prognostic impact The Samantha Dickson Brain Tumour Trust (to S.C. Clifford), and Cancer of the medulloblastoma molecular subgroups will there- Research UK (to S.C. Clifford and D.W. Ellison). The costs of publication of this article were defrayed in part by the fore now be essential, ideally within the context of ade- payment of page charges. This article must therefore be hereby marked quately powered, centrally reviewed, and uniformly treated advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate clinical trials cohorts, to determine their utility to direct the this fact. selection of adjuvant therapy. The molecular signatures we Received August 17, 2010; revised January 6, 2011; accepted January 10, report will have significant utility in this regard. Molecu- 2011; published OnlineFirst February 16, 2011.

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1894 Clin Cancer Res; 17(7) April 1, 2011 Clinical Cancer Research

Rapid Diagnosis of Medulloblastoma Molecular Subgroups

Ed C. Schwalbe, Janet C. Lindsey, Debbie Straughton, et al.

Clin Cancer Res Published OnlineFirst February 16, 2011.

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