Sporadic Medulloblastomas Research Articles

BIOL-08. RADIATION DRIVES METASTASIS IN MEDULLOBLASTOMA THROUGH AN INFLAMMATORY PROCESS

Abstract Current treatment for medulloblastoma is comprised of surgery, radiotherapy (RT) and/or chemotherapy. Although the primary tumor is controlled with this approach, recurrent metastatic disease is ubiquitously fatal. However, little is known about the biology of metastatic recurrences in medulloblastoma. In the present study, we observe across 3 non-overlapping cohorts of infant medulloblastoma that patients recur locally without focal RT and metastatically after focal RT. Using 3 independent murine flank xenograft medulloblastoma models, we found that the incidence of leptomeningeal metastasis was significantly increased in mice that received RT compared to sham controls. We have also found a significant increase in viable circulating tumor cells after RT. Our multi-omics (bulk RNA-seq, scRNA-seq and proteo/phosphoproteomics) approach using immunocompetent sporadic medulloblastoma models treated with RT showed activation of the innate inflammatory response through the overexpression of chemokines, cytokines and activation of immune cell types. In situ analysis of protein expression after RT showed a gradual increase in the abundance of macrophages, neutrophils and dendritic cells after RT, suggesting increased permeability and cell trafficking across the blood brain barrier. Applying live intravital microscopy using a low molecular weight vascular dye (150kDa), we observed a striking increase in blood vessel permeability in RT vs sham-treated brain tumors. Our observations support a model where RT drives metastasis through inflammation. To test this, we applied lipopolysaccharide (LPS), an inflammation inducing treatment, to a sporadic murine medulloblastoma model which significantly increased the metastatic burden. We then treated a xenograft model with dexamethasone in combination with RT, which resulted in total abrogation of metastatic dissemination compared to RT alone. Collectively, our findings suggest that while external beam irradiation is an effective and essential treatment for medulloblastoma, it may facilitate metastatic dissemination through an inflammation-induced process. These findings can help inform potential approaches to prevent disseminated relapsed disease.

Medulloblastoma and Cowden syndrome: Further evidence of an association.

Cowden syndrome (CS) is an autosomal dominant hamartoma and tumor predisposition syndrome caused by heterozygous pathogenic germline variants in PTEN in most affected individuals. Major features include macrocrania, multiple facial tricholemmomas, acral and oral keratoses and papillomas, as well as mammary, non-medullary thyroid, renal, and endometrial carcinomas. Lhermitte-Duclos disease (LDD), or dysplastic gangliocytoma of the cerebellum, is the typical brain tumor associated with CS; the lifetime risk for LDD in CS patients has been estimated to be as high as 30%. In contrast, medulloblastoma is much rarer in CS, with only 4 reported cases in the literature. We report a 5th such patient. All 5 patients were diagnosed between 1 and 2 years of age and not all showed the pathognomonic clinical stigmata of CS at the time of their medulloblastoma diagnosis. Where detailed information was available, the medulloblastoma was of the SHH-subtype, in keeping with the observation that in sporadic medulloblastomas, PTEN-alterations are usually encountered in the SHH-subtype. Medulloblastomas can be associated with several tumor-predisposition syndromes and of the 4 medulloblastoma subtypes, SHH-medulloblastomas in children have the highest prevalence of predisposing germline variants (approx. 40%). CS should be added to the list of SHH-medulloblastoma-associated syndromes. Germline analysis of PTEN should be performed in infants with SHH-medulloblastomas, regardless of their clinical phenotype, especially if they do not carry pathogenic germline variants in PTEN or PTEN, the most commonly altered predisposing genes in this age-group. In addition, these cases show that CS has a biphasic brain tumor distribution, both in regards to the age of onset and the tumor type: a small number of CS patients develop a medulloblastoma in infancy while many more develop LDD in adulthood.

Medulloblastomas associated with an APC germline pathogenic variant share the good prognosis of CTNNB1-mutated medulloblastomas.

Medulloblastomas may occur in a predisposition context, including familial adenomatosis polyposis. Medulloblastomas related to a germline pathogenic variant of adenomatous polyposis coli (APC) remain rare and poorly described. Their similarities with sporadic WNT medulloblastomas still require description. We performed a multicentric retrospective review of 12 patients treated between 1988 and 2018 for medulloblastoma with an identified or highly suspected (personal or familial history) APC germline pathogenic variant. We report personal and familial history APC gene pathogenic variants whenever available: clinical and histologic characteristics of the medulloblastoma, treatments, and long-term outcome, including second tumor and late sequelae. Medulloblastomas associated with APC pathogenic variants are mainly classic (11/11 patients, 1 not available), nonmetastatic (10/12 patients) medulloblastomas, with nuclear immunoreactivity for ß-catenin (9/9 tested cases). Ten of 11 assessable patients are disease free with a median follow-up of 10.7 years (range, 1-28 y). Secondary tumors included desmoid tumors in 7 patients (9 tumors), 1 thyroid carcinoma, 2 pilomatricomas, 1 osteoma, 1 vertebral hemangioma, and 1 malignant triton in the radiation field, which caused the only cancer-related death in our series. Medulloblastomas associated with an APC pathogenic variant have an overall favorable outcome, even for metastatic tumors. Yet, long-term survival is clouded by second tumor occurrence; treatment may play some role in some of these second malignancies. Our findings raise the question of applying a de-escalation therapeutic protocol to treat patients with APC germline pathogenic variants given the excellent outcome, and reduced intensity of craniospinal irradiation may be further evaluated.

MEMRI-based imaging pipeline for guiding preclinical studies in mouse models of sporadic medulloblastoma.

Genetically engineered mouse models of sporadic cancers are critical for studying tumor biology and for preclinical testing of therapeutics. We present an MRI-based pipeline designed to produce high resolution, quantitative information about tumor progression and response to novel therapies in mouse models of medulloblastoma (MB). Sporadic MB was modeled in mice by inducing expression of an activated form of the Smoothened gene (aSmo) in a small number of cerebellar granule cell precursors. aSmo mice were imaged and analyzed at defined time-points using a 3D manganese-enhanced MRI-based pipeline optimized for high-throughput. A semi-automated segmentation protocol was established that estimates tumor volume in a time-frame compatible with a high-throughput pipeline. Both an empirical, volume-based classifier and a linear discriminant analysis-based classifier were tested to distinguish progressing from nonprogressing lesions at early stages of tumorigenesis. Tumor centroids measured at early stages revealed that there is a very specific location of the probable origin of the aSmo MB tumors. The efficacy of the manganese-enhanced MRI pipeline was demonstrated with a small-scale experimental drug trial designed to reduce the number of tumor associated macrophages and microglia. Our results revealed a high level of heterogeneity between tumors within and between aSmo MB models, indicating that meaningful studies of sporadic tumor progression and response to therapy could not be conducted without an imaging-based pipeline approach.

Nanog-driven cell-reprogramming and self-renewal maintenance in Ptch1+/\u2212 granule cell precursors after radiation injury

Medulloblastoma (MB) is the most common pediatric brain tumor, comprising four distinct molecular variants, one of which characterized by activation of the Sonic Hedgehog (SHH) pathway, driving 25–30% of sporadic MB. SHH-dependent MBs arise from granule cell precursors (GCPs), are fatal in 40–70% of cases and radioresistance strongly contributes to poor prognosis and tumor recurrence. Patched1 heterozygous (Ptch1+/−) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene, the Shh receptor and negative regulator of the pathway, are uniquely susceptible to MB development after radiation damage in neonatal cerebellum. Here, we irradiated ex-vivo GCPs isolated from cerebella of neonatal WT and Ptch1+/− mice. Our results highlight a less differentiated status of Ptch1-mutated cells after irradiation, influencing DNA damage response. Increased expression levels of pluripotency genes Nanog, Oct4 and Sal4, together with greater clonogenic potential, clearly suggest that radiation induces expansion of the stem-like cell compartment through cell-reprogramming and self-renewal maintenance, and that this mechanism is strongly dependent on Nanog. These results contribute to clarify the molecular mechanisms that control radiation-induced Shh-mediated tumorigenesis and may suggest Nanog as a potential target to inhibit for adjuvant radiotherapy in treatment of SHH-dependent MB.

Immunohistochemical localization of β-catenin in medulloblastoma

ObjectivesThe aim of this study was to explore the relationship between immunohistochemical expression of β-catenin and the relevant clinicopathological features of medulloblastoma in Egyptian patients.BackgroundMedulloblastoma is a small round blue cell malignancy of the cerebellum and is a major cause of morbidity and mortality in pediatric oncology. Identification of the signaling pathways involved in the pathogenesis of medulloblastoma represents a key challenge for medulloblastoma management. The wingless (WNT)-signaling pathway has been reported to be responsible for 15% of sporadic medulloblastoma. β-Catenin represents the downstream effector of the WNT pathway. Activation of the WNT-signaling pathway results in stabilization of β-catenin and its translocation from the cytoplasm to the nucleus where it regulates the related genes.Patients and methodsThis retrospective study was conducted on 49 tissue specimens of medulloblastoma patients for evaluation of immunohistochemical expression of β-catenin.ResultsNone of the studied medulloblastoma patients showed nuclear localization of β-catenin. However, 29 patients exhibited cytoplasmic staining and 20 patients were absolutely negative. No statistically significant difference was found when comparing patients with cytoplasmic β-catenin expression and negative patients with the studied parameters. In addition, the H-score value of patients with cytoplasmic β-catenin did not show significant relationship with the studied clinicopathologic parameters.ConclusionIn view of the absence of nuclear localization of β-catenin, we could conclude that β-catenin does not play a role in the pathogenesis in all evaluated medulloblastoma patients. However, a large-scale study is recommended to elicit the exact role of the WNT-signaling pathway through dysregulation of components other than β-catenin.

High Frequency of Germline SUFU Mutations in Children With Desmoplastic/Nodular Medulloblastoma Younger Than 3 Years of Age

Germline mutations of the SUFU gene have been shown to be associated with genetic predisposition to medulloblastoma, mainly in families with multiple cases of medulloblastoma and/or in patients with symptoms similar to those of Gorlin syndrome. To evaluate the contribution of these mutations to the genesis of sporadic medulloblastomas, we screened a series of unselected patients with medulloblastoma for germline SUFU mutations. A complete mutational analysis of the SUFU gene was performed on genomic DNA in all 131 consecutive patients treated for medulloblastoma in the pediatrics department of the Institut Gustave Roussy between 1972 and 2009 and for whom a blood sample was available. We identified eight germline mutations of the SUFU gene: one large genomic duplication and seven point mutations. Mutations were identified in three of three individuals with medulloblastoma with extensive nodularity, four of 20 with desmoplastic/nodular medulloblastomas, and one of 108 with other subtypes. All eight patients were younger than 3 years of age at diagnosis. The mutations were inherited from the healthy father in four of six patient cases in which the parents accepted genetic testing; de novo mutations accounted for the other two patient cases. Associated events were macrocrania in six patients, hypertelorism in three patients, and multiple basal cell carcinomas in the radiation field after age 18 years in one patient. These data indicate that germline SUFU mutations were responsible for a high proportion of desmoplastic medulloblastoma in children younger than 3 years of age. Genetic testing should be offered to all children diagnosed with sonic hedgehog-driven medulloblastoma at a young age.

Abstract CN06-04: The hedgehog signaling pathway in cancer.

Abstract CN06-04: The hedgehog signaling pathway in cancer.

Reply to J.C. Lindsey et al

After the first report by Tabori et al and our article published in Journal of Clinical Oncology, the potential impact of TP53 mutation status in sporadic medulloblastoma has been controversially discussed in the field of neurooncology. The latest report by Lindsey et al, conducted in another completely independent cohort investigating TP53 and CTNNB1 mutation status in 50 patients with primary medulloblastoma supports our recently published data. In our study investigating 310 patients with primary medulloblastoma, TP53 mutations were detected in all major cytogenetic and transcriptomic subgroups of medulloblastoma but were clearly overrepresented among Wnt/Wingless (WNT) -driven tumors characterized by mutations in the CTNNB1 gene. This finding was confirmed by Lindsey et al in their cohort, given that three of five tumors harboring a TP53 mutation belonged to the WNT-subgroup. Inaddition,wefoundanoverrepresentationinMYCN-amplifiedmedulloblastoma that remains to be confirmed in independent cohorts. The results of a recent study by Gibson et al indicate that, in CTNNB1-mutated mice, the deletion of TP53 might be necessary for the accumulation of further mutations and therefore the development of medulloblastoma. Notably, these tumors anatomically resemble human WNT-subgroup medulloblastoma. Taken together, these data provide the first in vivo evidence of a cooperative role of concomitant mutations of CTNNB1 and TP53 in WNTmedulloblastoma tumorigenesis. Our cohort included two patients with Li-Fraumeni syndrome (LFS) as confirmed by the detection of TP53 germline mutation; one of the patients had not previously received a diagnosis of LFS. Interestingly, Lindsey et al also report one patient with a newly identified TP53 germline mutation (LFS) who died about one year after diagnosis. Accordingly, undiagnosed patients with hidden LFS might constitute a significant proportion of supposedly sporadic medulloblastoma with TP53 mutation. Concerning the patient outcome, the observations of Lindsey et al are in line with our results, showing that TP53 mutation is not associated with a poor patient outcome, in contrast with the report by Tabori et al. To evaluate the value of TP53 mutation status as a prognostic marker in medulloblastoma, it is essential to take into account that molecular characteristics of the tumor might determine the patient’s outcome (ie, WNT-driven tumors being associated with a favorable prognosis), that the clinical course of patients with LFS may differ from that of patients with medulloblastoma harboring a sporadic TP53 mutation, and that the impact of the TP53 mutation status on prognosis might depend on the different therapy regimen applied. Thus, it would be preferable to separately evaluate—ideally prospectively—the effect of TP53 mutation status on prognosis in the different trial arms within a randomized multicenter clinical trial. In conclusion, the study by Lindsey et al supports our findings that TP53 mutations occur in all molecular subgroups and are not per se an adverse prognostic marker across all subgroups of medulloblastoma. This supports the notion that medulloblastoma comprises distinct entities with specific genetic alterations that are decisive for the patient’s outcome.

Heterogeneity of familial medulloblastoma and contribution of germline PTCH1 and SUFU mutations to sporadic medulloblastoma

PTCH1 and SUFU are both regulators of the sonic hedgehog signalling pathway. Germline inactivating mutations in both genes are associated with multisystem phenotypes including medulloblastoma. Somatic inactivating mutations in PTCH1 and SUFU each occur in approximately 10% of medulloblastomas. Recently, SUFU mutations were reported in familial medulloblastoma pedigrees without additional phenotypic features. We sought to further investigate the contribution of germline PTCH1 and SUFU mutations to familial and sporadic medulloblastoma. We performed full-gene mutational analysis of both PTCH1 and SUFU in three familial medulloblastoma pedigrees and 83 individuals with sporadic non-familial medulloblastoma. We identified no mutations in PTCH1 or SUFU in the three familial medulloblastoma pedigrees. We identified no PTCH1 mutations and two SUFU mutations that cause premature protein truncating in the series of sporadic non-familial medulloblastomas. The SUFU mutations were identified in two of the 16 individuals with desmoplastic medulloblastomas. These data indicate that familial medulloblastoma is a genetically heterogeneous disorder with at least one further susceptibility gene to be discovered. Furthermore, although both PTCH1 and SUFU play a key role in the sonic hedgehog signalling pathway, PTCH1 does not make an appreciable contribution to non-familial sporadic medulloblastoma, whereas inactivating germline mutations of SUFU cause ~2-3% of sporadic medulloblastomas and>10% of desmoplastic medulloblastomas.

TP53 Mutation Is Frequently Associated With CTNNB1 Mutation or MYCN Amplification and Is Compatible With Long-Term Survival in Medulloblastoma

The role of TP53 mutations in the tumorigenesis of sporadic medulloblastoma (MB) and the value of TP53 mutation status as a prognostic marker are not yet definitely elucidated. A recent report identified TP53 mutations in MB as an adverse prognostic marker. Hence, the current study was conducted to validate the prognostic role of TP53 mutation in MB and to understand its contribution to tumorigenesis. A comprehensive genetic analysis of 310 MB samples was performed by screening for TP53 mutations and further relating the TP53 mutation status to p53 immunostaining, cytogenetic aberrations, and clinical variables. Mutation analysis of TP53 revealed mutations in 21 (6.8%) of 310 samples. Germline TP53 mutations were found in two patients with a history suggestive of a hereditary cancer syndrome. TP53 mutation status was not associated with unfavorable prognosis (P = .63) and was not linked to 17p allelic loss but was over-represented in the prognostically favorable WNT subgroup of MB as defined by CTNNB1 mutation (seven of 35 TP53-mutated tumors v 14 of 271 TP53 wild-type tumors; P = .005) and in tumors carrying high-level MYCN amplification (seven of 21 TP53-mutated tumors v 14 of 282 TP53 wild-type tumors; P = .001). The contradictory results in the recent literature concerning the prognostic value of TP53 mutation might be explained by different frequencies of WNT MBs, different frequencies of patients with Li-Fraumeni syndrome, and different cumulative doses of alkylating drugs applied in these studies.

Abstract 1976: The discovery of mechanisms of resistance to SMO antagonists and the therapeutic implications

Abstract Smoothened (Smo) is a G-protein coupled receptor (GPCR)-like molecule that activates the Hedgehog (Hh) signal transduction pathway. In the resting state, the 12-pass transmembrane protein Patched (Ptch) inhibits Smo activity. When Ptch inhibition is attenuated, Smo signals via a cytosolic complex of proteins leading to activation of the Gli family of transcription factors. Genetic activation of the Hh pathway at or upstream of Smo is linked to tumorigenesis in several cancers. In particular, somatic mutations in Ptch and Smo leading to constitutive pathway activation are found in sporadic medulloblastoma (MB) and basal cell carcinoma (BCC). Evidence suggests that antagonists of Smo may abrogate the tumorigenic phenotype engendered by Ptch inactivation. NVP-LDE225 is a potent and selective orally available Smo antagonist that robustly inhibits Smo-dependent signaling in vitro and in vivo. NVP-LDE225 exerted dose-related anti-tumor activity in vivo in several genetically defined MB models that are driven by mutations in Ptch leading to near complete tumor regression and Hh pathway inhibition. However, following long-term continuous dosing of NVP-LDE225 in medulloblastoma allograft models, evidence of resistance to NVP-LDE225 was observed. Here, we describe our efforts to proactively identify mechanisms of resistance to targeted therapy of Smo. Genome-wide DNA- and RNA-profiling of resistant tumors revealed distinct resistance mechanisms allowing tumors to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, was identified as one mechanism leading to restoration of pathway signaling despite adequate drug exposure. Additional mining of the gene expression data for pathway signatures that are selectively deregulated in resistant tumors identified increased phosphatidylinositol-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTor inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1976.

Abstract 7: Snail1 promotes proliferation and induces N-Myc in medulloblastoma cells

Abstract Activation of the Sonic Hedgehog (SHH) pathway and increased expression of the Gli1 transcription factor have been implicated in the tumorigenesis of both sporadic and syndromic medulloblastoma, a malignant pediatric brain tumor that arises from cerebellar progenitor cells. While SHH pathway activity is required for normal neural stem cell proliferation, molecular mechanisms of SHH pathway dysregulation and transformation of neural precursor cells remain poorly defined. In epithelial cells, Snail1 was found to be induced by Gli1 expression and is believed to promote Gli1-mediated transformation (Li et al. 2006), however the role of Snail1 in neural cells is not well understood. We have identified Snail1 as a putative target of Gli1 in neural stem cells. We hypothesize that Snail1 participates in transduction of SHH/Gli1 signaling in the nervous system and plays a role in proliferation of neuronal progenitor cells in the cerebellum. Mice haploinsufficient for Patched (Ptc) have constitutive activation of the SHH pathway and a subset develop medulloblastoma. Quantitative RT-PCR analyses of mRNA isolated from early postnatal untransformed cerebella show that Snail1 mRNA is increased in vivo in Ptc+/- mice versus wild-type (WT) siblings.. Enforced expression of Gli1 in cultured WT murine granule precursor cells (GPCs) induced Snail1 mRNA.. Enforced expression of Snail1 was also sufficient to induce proliferation of GCPs in the absence of SHH/Gli1 exposure. Given that activation of SHH/Gli1-pathway induces proliferation of GPCs and is associated with medulloblastoma formation, we hypothesized that Snail1 may play a role in the proliferation and transformation of neural progenitor cells. We have found that the stem-like cells generated from medulloblastomas in Ptc+/- mice express higher levels of Snail mRNA than tumor parenchyma. Quantitative RT-PCR analysis demonstrates that Snail1 mRNA is expressed at baseline and is dramatically induced in human medulloblastoma cell lines after exposure to SHH. Enforced expression of Snail1 further increased proliferation of medulloblastoma cells as demonstrated by EdU incorporation. Analysis of potential Snail1 targets in human medulloblastoma cells reveals that β-catenin and N-Myc protein are induced by Snail1 overexpression. N-Myc is induced by SHH in GPCs (Kenney et al. 2003) and is known to induce proliferation in GPCs during both normal cerebellar development and medulloblastoma formation. Together these results give further insight into the mechanism of SHH-induced proliferation of neural progenitor and suggest that Snail1 may contribute to malignant transformation of GCPs through N-Myc. These data establish Snail1 as a target of SHH/Gli1 pathway activity in both normal and neoplastic cells derived from cerebellum. Ongoing experiments will further elucidate how this novel proliferative process affects transformation of neural cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 7.

Abstract A44: Predictive biomarkers of efficacy to the hedgehog pathway inhibitor, GDC-0449, in advanced basal cell carcinoma and medulloblastoma in phase I studies

Abstract The Hedgehog (Hh) signaling pathway has been directly implicated in the pathogenesis of basal cell carcinoma (BCC) and medulloblastoma (MB). Constitutive Hh signaling, most often due to underlying loss of function mutations in the inhibitory receptor Patched1 (PTCH1), has been demonstrated in a majority of common localized BCCs and approximately 25–30% of sporadic MB cases. In this study, we examined molecular correlatives of clinical efficacy in locally-advanced / metastatic BCC and MB patients treated with GDC-0449, an inhibitor of the Hh pathway that targets the serpentine receptor Smoothened (SMO). An analysis of Hh pathway transcriptional target gene expression in archival tissue biopsies provided evidence for active Hh signaling in patients exhibiting clinical benefit (objective response or disease stabilization) on therapy. This was further supported by the identification of either loss of function or activating mutations in PTCH1 or SMO, respectively. We were unable to provide evidence for active Hh signaling in 2 patients with progressive disease as best response, potentially underlying the reason for lack of efficacy in these patients. In addition to providing molecular support for Hh pathway activity in patients exhibiting treatment benefit, this is the first evidence for Hh pathway dysregulation in the previously uncharacterized, advanced BCC setting. Furthermore, the results begin to support the utility of transcriptional-based tests as a strategy to potentially select the subset of MB patients with Hh-driven disease that would likely benefit on GDC-0449. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A44.

Heterozygous germ-line mutations in the NBN gene predispose to medulloblastoma in pediatric patients

The NBN (NBS1) gene belongs to a group of double-strand break repair genes. Mutations in any of these genes cause genome instability syndromes and contribute to carcinogenesis. NBN gene mutations cause increased tumor risk in Nijmegen breakage syndrome (NBS) homozygotes as well as in NBN heterozygotes. NBS patients develop different types of malignancies; among solid tumors, medulloblastoma (MB), an embryonal tumor of the cerebellum, has been reported most frequently. The majority of medulloblastomas occur sporadically, some of them manifest within familial cancer syndromes. Several signaling pathways are known to be engaged in hereditary and sporadic MB. The aim of our study was to identify mutations in selected exons of the NBN gene and to determine the frequency of the most common NBN gene mutations in pediatric patients with different types of medulloblastoma. We screened a total of 104 patients with MB and identified 7 heterozygous carriers (6.7%) of two different germ-line mutations of NBN gene; all of them had classic MB. Our results indicate that heterozygous carriers of the germ-line NBN gene mutations (c.511A>G and c.657_661del5) may exhibit increased susceptibility to developing MB. The risk of medulloblastoma is estimated to be 3.0 (for c.511A>G) and 4.86 (for c.657_661del5) times higher than in the general Polish population (p<0.05). These results suggest that heterozygous NBN germ-line mutations may contribute to the etiology of medulloblastoma.

P18Ink4c and p53 Act as Tumor Suppressors in Cyclin D1–Driven Primitive Neuroectodermal Tumor

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Mutations in the Nijmegen Breakage Syndrome Gene in Medulloblastomas

Cerebellar medulloblastoma is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutations is a rare autosomal recessive disease with clinical features that include microcephaly, mental and growth retardation, immunodeficiency, increased radiosensitivity, and predisposition to cancer. There may be functional interactions between NBS1 and the TP53 pathways. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of sporadic medulloblastomas. Forty-two cases of medulloblastomas were screened for mutations in the NBS1 gene (all 16 exons) and the TP53 gene (exons 5-8) by single-stranded conformational polymorphism followed by direct DNA sequencing. Seven of 42 (17%) medulloblastomas carried a total of 15 NBS1 mutations. Of these, 10 were missense point mutations and 5 were intronic splicing mutations. None of these were reported previously as germ-line mutations in NBS patients. No NBS1 mutations were detected in peritumoral brain tissues available in two patients. Of 5 medulloblastomas with TP53 mutations, 4 (80%) contained NBS1 mutations, and there was a significant association between TP53 mutations and NBS1 mutations (P = 0.001). We provide evidence of medulloblastomas characterized by NBS1 mutations typically associated with mutational inactivation of the TP53 gene.

Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF‐dependent transcription in medulloblastomas

Medulloblastomas (MBs) represent the most common malignant brain tumors in children. Most MBs develop sporadically in the cerebellum, but their incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations in the APC tumor suppressor gene. APC is part of a multiprotein complex involved in the Wnt signaling pathway that controls the stability of beta-catenin, the central effector in this cascade. Previous genetic studies in MBs have identified mutations in genes coding for beta-catenin and its partners, APC and AXIN1, which cause activation of Wnt signaling. The pathway is negatively controlled by the tumor suppressor AXIN2 (Conductin), a scaffold protein of this signaling complex. To investigate whether alterations in AXIN2 may also be involved in the pathogenesis of sporadic MBs, we performed a mutational screening of the AXIN2 gene in 116 MB biopsy samples and 11 MB cell lines using single-strand conformation polymorphism and sequencing analysis. One MB displayed a somatic, tumor-specific 2 bp insertion in exon 5, leading to carboxy-terminal truncation of the AXIN2 protein. This tumor biopsy showed nuclear accumulation of beta-catenin protein, indicating an activation of Wnt signaling. In 2 further MB biopsies, mutations were identified in exon 5 (Glu408Lys) and exon 8 (Ser738Phe) of the AXIN2 gene, which are due to predicted germline mutations and rare polymorphisms. mRNA expression analysis in 22 MBs revealed reduced expression of AXIN2 mRNA compared to 8 fetal cerebellar tissues. Promoter hypermethylation could be ruled out as a major cause for transcriptional silencing by bisulfite sequencing. To study the functional role of AXIN2 in MBs, wild-type AXIN2 was overexpressed in MB cell lines in which the Wnt signaling pathway was activated by Wnt-3a. In this assay, AXIN2 inhibited Wnt signaling demonstrated in luciferase reporter assays. In contrast, overexpression of mutated AXIN2 with a deleted C-terminal DIX-domain resulted in an activation of the Wnt signaling pathway. These findings indicate that mutations of AXIN2 can lead to an oncogenic activation of the Wnt pathway in MBs.

Array-based profiling of reference-independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma

Existing microarray-based approaches for screening of DNA methylation are hampered by a number of shortcomings, such as the introduction of bias by DNA copy-number imbalances in the test genome and negligence of tissue-specific methylation patterns. We developed a method designated array-based profiling of reference-independent methylation status (aPRIMES) that allows the detection of direct methylation status rather than relative methylation. Array-PRIMES is based on the differential restriction and competitive hybridization of methylated and unmethylated DNA by methylation-specific and methylation-sensitive restriction enzymes, respectively. We demonstrate the accuracy of aPRIMES in detecting the methylation status of CpG islands for different states of methylation. Application of aPRIMES to the DNA from desmoplastic medulloblastomas of monozygotic twins showed strikingly similar methylation profiles. Additional analysis of 18 sporadic medulloblastomas revealed an overall correlation between highly methylated tumors and poor clinical outcome and identified ZIC2 as a frequently methylated gene in pediatric medulloblastoma.

Intracellular Distribution of β-Catenin in Human Medulloblastoma Cell Lines with Different Degree of Neuronal Differentiation

Gene mutations impairing the functions of the WNT signaling transduction pathway have been found in approximately 15% of human sporadic medulloblastomas. To understand the functional role of the WNT pathway in medulloblastoma, we have investigated the intracellular distribution of β-catenin in a series of 17 human medulloblastomas to correlate such expression with neuronal differentiation and in cultured cell models following functional silencing of the APC gene by small-interference RNA (siRNA). Transient siRNA transfection resulted in a 50% reduction of the APC gene product levels in both DAOY and D283MED cell lines. In the former, less-differentiated cell line, β-catenin levels remained unchanged or were slightly reduced, but β-catenin translocated in the nucleus following APC gene siRNA silencing. In contrast, in the more differentiated D283MED cells, β-catenin levels increased about twofold while mainly maintaining the cytoplasmic and cell membrane localization. Cytoplasmic/nuclear localization of β-catenin was present in 12 of 17 cases of medulloblastoma with a prevalent distribution in the classic, 6/7 cases, and large cell/anaplastic variant, 4/4 cases. The nodular/desmoplastic lesions showed strongly positivity in the cell membrane mainly of intranodular cells with advanced neuronal differentiation. These observations support an important functional role of WNT/β-catenin pathway in neuronal differentiation in medulloblastoma.