Medulloblastoma, the most common malignant pediatric brain tumour, arises in the cerebellum, and disseminates through the cerebrospinal fluid (CSF) in the leptomeningeal space to coat the brain and spinal cord1. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and most children at the time of recurrence. Therefore, affected children are treated with radiation to the entire developing brain and spinal cord followed by high dose chemotherapy with ensuing deleterious effects on the developing nervous system 2. The mechanisms of CSF dissemination are poorly studied and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour 3,4. Here we show that in both mouse and human medulloblastoma, multiple metastases from a single patient are extremely similar to each other, but divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted sub-clone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could represent a major barrier to the development of effective targeted therapies.
Thirty percent of Ptch+/− mice develop non-disseminated medulloblastoma by eight months of age5. Recently, the Sleeping Beauty (SB) transposon system was shown to be an effective tool for functional genomics studies of solid tumour initiation and progression 6,7. We expressed the SB11-transposase in cerebellar progenitor cells in transgenic mice under the Math1 enhancer/promoter, but did not observe any tumours when bred to transgenic mice with a concatemer of the T2/Onc transposon (Fig. 1, Supplemental Fig. S1, S2)8. However, Ptch +/−/Math1-SB11/T2Onc mice showed increased penetrance of medulloblastoma (~100% (271 out of 279) compared to ~40% (54 out of 139) of controls, and decreased latency (8 months to 2.5 months) (Fig. 1, Supplemental Fig. S2). While Ptch +/− medulloblastomas are usually localized, the addition of SB transposition results in metastatic dissemination through the CSF pathways, identical to the pattern seen in human children (Fisher’s exact test, p=1.8e-07, odds ratio=5.2, Supplemental Table S1) (Fig. 1c, d, g, h, Supplemental Figure S2). As neither transposon, nor transposase alone had an effect on tumour incidence, latency, or dissemination, we conclude that SB-induced insertional mutagenesis drives medulloblastoma progression on the Ptch +/− background (Fig. 1i, Supplemental Fig. S2).
Humans with germline mutations in TP53 have Li-Fraumeni syndrome and are at increased risk to develop medulloblastoma. While no medulloblastomas were found in Tp53mut (Tp53 +/− or Tp53−/−) mice, 40% of Tp53mut/Math1-SB11/T2Onc mice developed disseminated medulloblastoma (Fig. 1e–h, j, Supplemental Fig. S2)9. Human medulloblastomas with TP53 mutations frequently have large cell/anaplastic histology. Tp53mut/Math1-SB11/T2Onc medulloblastomas exhibit large cells, nuclear atypia, and nuclear molding typical of large cell/anaplastic histology (Fig. 1f). We conclude that SB transposition can drive the initiation and progression of metastatic medulloblastoma on a Tp53mut background.
We used linker-mediated PCR and Roche 454 sequencing to identify the site of T2/Onc insertions in Ptch+/−/Math1-SB11/T2Onc, and Tp53mut/Math1-SB11/T2Onc primary medulloblastomas and their matched metastases. Genes that contained insertions statistically more frequently than the background rate were identified as gene-centric commonly inserted sites (gCISes)10. We identified 359 gCISes from 139 primary tumours on the Ptch background and 26 gCISes from 36 primary medulloblastomas on the Tp53 background (Supplemental Tables S2–S7, Supplemental Figures S3–S5). A large number of gCISes targeted candidate medulloblastoma oncogenes/tumour suppressor genes (Supplemental Table S8)11. Insertions in candidate tumour suppressor genes including EHMT1, CBP, and MXI1 are predicted to be loss of function (Fig. 1k,l,m), while insertions in putative medulloblastoma oncogenes are largely gain of function, as exemplified by MYST3 (Fig. 1n).
Many gCISes mapped to regions of amplification, focal hemizygous deletion, and homozygous deletion, which we recently reported in the genome of a large cohort of human medulloblastomas (Supplemental Table S8) 11. There is a high level of overlap between gCISes and known cancer genes (COSMIC database) (Supplemental Table S9,10), suggesting that many gCISes are bona fide driver genes in medulloblastoma (Fisher’s exact test p=0.0012)12. Similarly, many mouse gCIS/ human amplified genes are over-expressed in human Shh medulloblastomas (Supplemental Fig. S6). Conversely, mouse gCISes deleted in human medulloblastomas were frequently expressed at a lower level in human medulloblastomas (Supplemental Fig. S6). Expression of 6/7 gCISes studied by immunohistochemistry on a human medulloblastoma tissue microarray were associated with a significantly worse overall and progression free survival in human medulloblastoma (Supplemental Table 11, Supplemental Figures S7, S8) 13. We conclude that our SB-driven leptomeningeal disseminated medulloblastoma model resembles the human disease anatomically, pathologically and genetically and thus represents an accurate model of the human disease that can be used to identify candidate driver events and understand the pathogenesis of human medulloblastoma.
We compared the gCISes identified from Ptch+/−/Math1-SB11/T2Onc, and Tp53mut/Math1-SB11/T2Onc primary medulloblastomas and matched metastases (Supplemental Table S2). Strikingly, the overlap between primary tumour gCISes (pri-gCISes) from Ptch+/−/Math1-SB11/T2Onc tumours and those from the metastases (met-gCISes) from the same animals was only 9.3% of gCISes (Figure 2a). Similarly, the overlap in pri-gCISes from primary Tp53mut/Math1-SB11/T2Onc gCISes and the matching met-gCISes was only 8.9% (Figure 2b). Leptomeningeal metastases and the matched primary tumour share identical, highly clonal insertion sites (Fig. 2c). The chances of two (or three) unrelated tumours having SB insertions in exactly the same TA dinucleotide are extremely low. We conclude that leptomeningeal metastases and matched primary tumour arise from a common transformed progenitor cell, and have subsequently undergone genetic divergence. Sequencing also identified insertions that are highly clonal in the metastases, but not seen in the matched primary tumour (not shown). Endpoint PCR for these insertions in the matched primary/metastatic tumours show that the insertion is highly clonal in the metastase(s), and present in a very small subclone of the primary tumour (Fig. 2d, Supplemental Figure S9). These data are consistent with a model in which metastatic disease arises from a minor restricted subclone of the primary tumour. Dissemination could occur repeatedly from the same subclone of the primary tumour, which seeds the rest of the CNS, or could occur once followed by reseeding of the rest of the leptomeningeal space by the initial metastasis. Insertions that are restricted to a minor subclone of the primary tumour, but which are clonal in the metastases, could correspond to the ‘metastasis virulence’ genes, described previously, that offer a genetic advantage during dissemination, but not to the primary tumour 14. Another explanation of our data could be reseeding of the primary tumour by a metastatic clone that had acquired additional genetic events in the periphery. This latter hypothesis is mitigated by the presence in the same animal of highly clonal insertions in the metastasis that are completely absent from the primary tumour 15. As reseeding should be accompanied by contamination of the primary tumour with events found in the metastases, absence of these events in the matched primary tumour makes reseeding much less likely (Fig. 2e). We hypothesize that events found only in one metastasis represent progression events acquired post-metastasis, and which could lead to localized progression of metastatic disease as is sometimes seen in human children. We observed highly clonal insertions in the primary tumour, including known medulloblastoma oncogenes such as Notch2, or Tert, which are not found in the matching metastases (Fig. 2f). This pattern could be explained through remobilization of the SB transposon in the metastatic tumour; however, no signs of the DNA footprint left after SB remobilization at these loci were observed (Supplemental Fig. S10)16. We suggest that these events, which may constitute driver events in the primary tumour, have arisen in the primary tumour after the metastases have disseminated (post-dispersion events). Although these known oncogenes represent attractive targets for therapy, their utility as targets for therapy may be limited if the target is not also found in the leptomeningeal compartment of the disease. Our data from two separate mouse lines supports a model in which medulloblastoma disseminates early from a restricted subclone of the primary tumour, and where the primary tumour and the matched metastases then undergo differential clonal selection and evolution. Failure to account for the differences between the primary and leptomeningeal compartments could lead to the failure of targeted therapies. Failure to study the leptomeningeal disease could result in systematically overlooking critical targets for therapy in this compartment (Fig. 2e).
Examination of met-gCIS genes using GSEA demonstrates differences between the primary and metastatic disease, which importantly include enrichment for genes involved in the cytoskeleton among the metastases (Supplemental Table S12). Targets that are present in both compartments, and which are maintenance genes, will be optimal targets for therapy of both the primary and metastatic compartments, as exemplified by Pdgfra (Fig. 2c, Supplemental Tables S7, S9).
Pten, Akt2, Igf2, and Pik3r1 are all met-gCISes, implicating the PI3-kinase pathway in medulloblastoma progression. We injected the cerebella of Nestin-TVA mice 17 with either Shh virus alone, or Shh + Akt virus. Cerebellar injection of Shh alone resulted in medulloblastoma in 6/41 animals, compared to 20/42 animals injected with Shh + Akt (p=0.0018). Poignantly, while metastases were never seen with Shh virus alone (0/41), medulloblastoma metastases were seen in 9/42 animals injected with Shh + Akt (p=0.0024) (Supplemental Fig. S11). In vivo modeling validates PI3-kinase signaling and suggests that it can contribute to leptomeningeal dissemination of medulloblastoma.
Prior publications and clinical approaches to human medulloblastoma have largely assumed that the primary tumour and its matched metastases are highly similar 3,4. To test this assertion we formally reviewed all cases of medulloblastoma from the last decade at The Hospital for Sick Children, and identified 19 patients who had both bulk residual primary tumour post-surgery, and MRI visible metastases, both of which could be followed for response to treatment in the two compartments (Supplemental Fig. S12 and Supplemental Table S13). While it is possible that metastases might have received reduced radiotherapy than the primary tumor in a subset of patients, in 58% of overall cases (11/19) we observed a disparate response to therapy between the primary tumor and matched metastases (binomial test, p<2.2e-16). Identification of definitive differences in the clinical response to standard therapy between the primary and metastatic compartment awaits the completion of large, well controlled, prospective clinical trials.
We examined seven matched primary/metastatic medulloblastomas for copy number aberrations (Fig. 3, Supplemental Figures S13, S14, Supplemental Tables S14, S15). In each case, the primary tumour and the matched metastases share complicated genetic events that highly support descent from a common transformed progenitor cell. Similar to our mouse data, in each case we see clonal genetic events in the metastatic tumour(s) that are not present in the matched primary tumour (Fig. 3. Supplemental Fig. S14). We also observe genetic events in the primary tumour that are absent from the matched metastasis, consistent with a ‘post-dispersion event’ (Fig. 3, Supplemental Fig. S14). Examination of a case with multiple leptomeningeal metastases demonstrates a deletion of chromosome 1p in only 1/3 metastases (Fig. 3a). This pattern of genetic events that are present only in a subset of metastases could be a mechanism for the emergence of therapy resistant metastatic clones.
We performed interphase FISH for the known medulloblastoma oncogenes MYCN and MYC on a collection of 17 paraffin embedded primary/metastatic pairs of human medulloblastoma 18–20. MYCN was amplified in 3 primary medulloblastomas, but not in the matching metastases (Fig. 3b, Supplemental Fig. S15). Conversely, MYC was amplified in 2 primary tumours and their matching metastases (Fig. 3c). These data are consistent with MYCN amplification being a ‘post-dispersion’ event, similar to examples in SB driven mouse medulloblastoma, and highly suggest that anti-MYCN therapeutics could lack efficacy in the metastatic compartment of human medulloblastoma. The possibility that MYCN amplicons are ‘lost’ overtime in the metastases cannot be excluded.
We subsequently analyzed promoter CpG-methylation in these matched pairs and found that there was a great deal of discordance between the primary tumour and matched metastases (Fig. 3d, Supplemental Figures S13, S16, Supplemental Tables S16–17). Finally, we performed whole-exome sequencing on a limited set of matched primary/metastatic medulloblastomas, and found many single nucleotide variants (SNVs) that were restricted to one compartment or the other (Supplemental Fig. S13, Supplemental Table S18). Discordance of CNAs, promoter CpG methylation events, and SNVs between the primary tumour and its matched metastases supports a bicompartmental model for metastatic medulloblastoma. The mutational load in the human tumours (combination of CNAs, CpG methylation, and SNVs) compares favorably to the mutational load in our transposon driven mouse models (median number of gCISes=25 per tumour, Supplemental Table S19). Validation of individual CNAs restricted to the metastases, reveals that they can be detected in a very minor subclone of the primary tumour, in keeping with the relationship identified in the mouse model (Supplemental Fig. S17, Supplemental Table S20–21). Pathway analysis using DAVID to compare mouse gCISes with genes affected in human metastases identified only a single statistically significant shared signaling pathway – insulin signaling (p=0.027) (Supplemental Table S22). The known role for insulin receptor signaling in primary medulloblastoma21, and the data presented above on the role of Akt in metastatic medulloblastoma, might suggest prioritization of insulin signaling as a therapeutic target to be tested in clinical trials.
We performed unsupervised hierarchical clustering on CpG methylation data revealing that normal cerebellar controls cluster away from the medulloblastomas, while metastases clustered with their matching primary tumour (Fig. 4a). However, metastases cluster more closely to each other than they do to the matched primary tumour (z-test, p=0.0014, Supplemental Fig. S18). Unsupervised hierarchical clustering of CNA and exome SNV data reveals the same relationships (Fig. 4b,c). Evident within the exome data are many events that are shared only by patient matched metastases, as well as events restricted to the primary tumour, both of which are similar to the genetic patterns observed in the mouse. These three data sets support a model in which patient matched human medulloblastoma metastases are epigenetically, and genetically very similar to each other, but have substantially diverged from their primary tumour, resulting in two different disease compartments: primary and metastatic.
Our data from two different mouse models with support by initial data from human medulloblastoma suggests that leptomeningeal metastases of medulloblastoma from a single human/mouse are genetically similar to each other, but highly divergent from the matched primary tumour, consistent with a bicompartmental model of the disease. Our results are consistent with a model in which metastases arise from a restricted subclone of the primary tumour through a process of clonal selection in both humans and mice. That metastases might arise from a pre-existing minor subclone of the primary tumour through clonal selection was suggested more than three decades ago, but remains a controversial hypothesis whose truth may vary from one disease to another 22–25. Failure to account for the divergent molecular pathology of the metastatic compartment may result in selection of therapeutic targets present in the primary tumour (which is more amenable to surgical control) and not the metastases, which are the more frequent cause of death.