Large Cell/anaplastic Research Articles

Abstract 1426: MiRNA expression profiling of the murine GTML medulloblastoma model reveals similarities with human tumors and identifies novel candidate miRNAs

Abstract MicroRNAs (miRNAs) are small molecules which regulate gene expression post-transcriptionally and aberrant expression of many miRNAs is associated with different forms of cancer, including the childhood brain tumor medulloblastoma. Amplification and over-expression of either MYC or MYCN transcription factors are particularly frequent in some of the most clinically aggressive subtypes of this disease. The GTML transgenic mouse is a recently established model for highly aggressive subtypes of this tumor, in which over-expression of MYCN is targeted to the central nervous system utilizing a glutamate transporter (GLT1). Tumors exhibit two distinguishable histopathological phenotypes characteristic of specific human medulloblastoma subtypes: classic and large cell/anaplastic (LCA). Here, we evaluate the deregulation of miRNAs in tumors derived from GTML mice in order to assess the contribution of miRNAs to medulloblastoma pathogenesis. The expression of 591 miRNAs was analyzed using TaqMan low density arrays (Applied Biosystems) in 8 cerebella (controls) and 15 tumors exhibiting either LCA (n=12), and classic (n=3) phenotype. Cluster analyses performed on the overall expression of miRNAs showed a clear segregation between tumors and normal cerebella, but no definitive difference between tumor LCA and classic histopathology. We identified 37 miRNAs that were differentially expressed between tumors and controls (>2-fold; p-value< 0.05 after Bonferroni correction for multiple comparisons). Interestingly 25 of these miRNAs were conserved between mouse and human genomes, and 8 were previously associated with human medulloblastoma. Among 17 up-regulated miRNAs, four were members of the miR-17∼92a polycistronic cluster (a cluster of oncomiRs up-regulated by MYCN). We also identified miR-182, miR-96, miR-183 and miR-183*, all members of the same cluster, as up-regulated. Interestingly over-expression of miR-182 and miR-183 has been previously reported within the Wnt subtype of human medulloblastoma. Among 20 miRNAs under-expressed in the mouse tumors, miR-135a and miR-135b are conserved in human and predicted to target MYCC. Moreover miR-135a is deleted in human classic medulloblastoma with high frequency (33%). The association of miR-135b with human medulloblastoma has been previously suggested, although the role of this miRNA in medulloblastoma is controversial, since either deletion or amplification of the relevant genetic locus occurs in human tumors. Our study represents the first miRNA profiling of the recently established GTML mouse model of aggressive MYCN-driven medulloblastoma. The degree of similarity in miRNA expression observed between murine and human tumors indicates that this model is a suitable tool for the assessment of the biologic role and suitability of miRNAs as therapeutic targets in human medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1426. doi:1538-7445.AM2012-1426

Abstract 3444: Enforced expression of MycN and C-Myc induces different medulloblastoma subtypes

Abstract Medulloblastoma (MB), a tumor of the cerebellum, is the most common malignant brain tumor of childhood. MBs are classified histologically and molecularly into four distinct major subgroups, two of which correspond to the deregulation of major developmental pathways, Sonic Hedgehog (SHH) and WNT. Among the two other subgroups, one correlates with high levels of C-MYC expression. Using orthotopic transplantation in the cerebellum or cortex of naïve recipient mice, we showed that enforced expression of MycN in cerebellar granule neuron progenitors (CGNPs) isolated from the cerebellum of postnatal (P) day 6-7 Cdkn2c-/-, Trp53-/-, Atoh1-GFP mice induced MBs with a SHH “signature”. Interestingly, overexpression of C-Myc in the same CGNPs induced MBs with Large Cell Anaplastic (LCA) features and mRNA and microRNA profiles distinct from the SHH subgroup. In vitro analysis revealed that tumor cells were resistant to Shh antagonists, such as cyclopamine and BMP4. Ongoing experiments are focusing on understanding the differences between MycN and C-Myc function in CGNPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3444. doi:10.1158/1538-7445.AM2011-3444

Survival and Prognostic Factors of Early Childhood Medulloblastoma: An International Meta-Analysis

To assess the prognostic role of clinical parameters and histology in early childhood medulloblastoma. Clinical and histologic data from 270 children younger than age 5 years diagnosed with medulloblastoma between March 1987 and July 2004 and treated within prospective trials of five national study groups were centrally analyzed. Two hundred sixty children with medulloblastoma and specified histologic subtype were eligible for analysis (median age, 1.89 years; median follow-up, 8.0 years). Rates for 8-year event-free survival (EFS) and overall survival (OS) were 55% and 76%, respectively, in 108 children with desmoplastic/nodular medulloblastoma (DNMB) or medulloblastoma with extensive nodularity (MBEN); 27% and 42%, respectively, in 145 children with classic medulloblastoma (CMB); and 14% and 14%, respectively, in seven children with large-cell/anaplastic (LC/A) medulloblastoma (P < .001). Histology (DNMB/MBEN: hazard ratio [HR], 0.44; 95% CI, 0.31 to 0.64; LC/A medulloblastoma: HR, 2.27; 95% CI, 0.95 to 5.54; P < .001 compared with CMB), incomplete resection and metastases (M0R1: HR, 1.86; 95% CI, 1.29 to 2.80; M+: HR, 2.28; 95% CI, 1.50 to 3.46; P < .001 compared with M0R0), and national group were independent prognostic factors for EFS, and OS. The HRs for OS ranged from 0.14 for localized M0 and DNMB/MBEN to 13.67 for metastatic LC/A medulloblastoma in different national groups. Our results confirm the high frequency of desmoplastic variants of medulloblastomas in early childhood and histopathology as a strong independent prognostic factor. A controlled de-escalation of treatment may be appropriate for young children with DNMB and MBEN in future clinical trials.

Definition of Disease-Risk Stratification Groups in Childhood Medulloblastoma Using Combined Clinical, Pathologic, and Molecular Variables

Medulloblastomas are heterogeneous and include relatively good-prognosis tumors characterized by Wnt pathway activation, as well as those that cannot be successfully treated with conventional therapy. Developing a practical therapeutic stratification that allows accurate identification of disease risk offers the potential to individualize adjuvant therapy and to minimize long-term adverse effects in a subgroup of survivors. Using formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry, fluorescent in situ hybridization, and direct sequencing to identify tumors with a Wnt pathway signature and those harboring copy number abnormalities (CNAs) of potential prognostic significance (MYC/MYCN amplification, CNAs of chromosome 6 and 17), we evaluated clinical, pathologic, and molecular outcome indicators and stratification models in a cohort (n = 207) of patients with medulloblastoma 3 to 16 years of age from the International Society of Pediatric Oncology CNS9102 (PNET3) trial. Metastatic disease and large-cell/anaplastic (LC/A) phenotype were the clinicopathologic variables associated with poor progression-free survival (PFS). Nuclear immunoreactivity for β-catenin, CTNNB1 mutation, and monosomy 6 all identified a group of good-prognosis patients. MYC amplification was associated with poor outcome, but other CNAs were not. Low-risk medulloblastomas were defined as β-catenin nucleopositive tumors without metastasis at presentation, LC/A phenotype, or MYC amplification. High-risk medulloblastomas were defined as tumors with metastatic disease, LC/A phenotype, or MYC amplification. Low-risk, standard-risk, and high-risk categories of medulloblastoma had significantly (P < .0001) different outcomes. Integrating assays of molecular biomarkers undertaken on routinely collected diagnostic FFPE tissue into stratification schemes for medulloblastoma alongside clinical and pathologic outcome indicators can refine current definition of disease risk and guide adjuvant therapy.

Pleiotropic role forMYCNin medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Large cell/anaplastic medulloblastoma: Outcome according to myc status, histopathological, and clinical risk factors

To evaluate the prognostic impact of large cell/anaplastic (LC/A) histology together with molecular and clinical risk factors in childhood medulloblastoma. Three consecutive prospective medulloblastoma trials were screened for patients with the histological diagnosis of LC/A medulloblastoma. Tumors were considered as LC/A if they displayed areas of severe cytological anaplasia or a significant or predominant large cell component. Histology was centrally confirmed. Genomic DNA amplification of c-myc and n-myc, and mRNA expression of c-myc and trkC were analyzed. Twenty-eight patients with LC/A medulloblastoma with a median age of 6.1 years (1.4-16.5 years) and a median follow-up of 4.5 years were identified (5% of all medulloblastoma). Four-year event-free (EFS) and overall survival (OS) were 58% and 67%. Young age and metastases (n = 13, 4-year EFS 31% vs. 82% in 15 children >4 years and without metastases, P = 0.001), large cell histology (n = 9, 4-year EFS 22% vs. 75%, P = 0.005) and c-myc amplification (n = 9, 4-year EFS 22% vs. 89%, P < 0.0001) were negative prognostic factors. C-myc amplification was highly correlated with young age (P < 0.001), metastases (P = 0.002) and large cell histology (P = 0.007). Outcome of 12 patients with severely anaplastic tumors without these risk factors was not impaired (4-year EFS 86%). In a subgroup of patients without clinical and molecular risk factors outcome was favorable despite severely anaplastic histology. In contrast, c-myc amplification and large-cell histology were associated with an inferior outcome. Intensified treatment strategies should be considered for children with LC/A medulloblastoma and these characteristics.

Low-level copy gain versus amplification of myc oncogenes in medulloblastoma: utility in predicting prognosis and survival

Medulloblastoma (MB) is a malignant embryonal tumor of the cerebellum. Amplification of c-myc or N-myc is infrequently identified and, when present, is often associated with the large cell/anaplastic (LC/A) phenotype. The frequency of low-level copy gain of myc oncogenes and its relationship to prognosis of MB has not been explored. Archival cases of MB were histologically reviewed and classified into 3 major subtypes: classic, nodular, and LC/A. Using quantitative real-time polymerase chain reaction (PCR), the authors analyzed 58 cases with a pure histological subtype for the copy number (CN) of myc (c-myc and N-myc) oncogenes. Cases with > 5-fold CN were further analyzed using the fluorescent in situ hybridization (FISH) assay. Kaplan-Meier survival analysis was performed. A > 5-fold myc CN was noted in 5 (20.8%) of 24 LC/A, 1 (5.3%) of 19 classic, and 2 (13.3%) of 15 nodular subtypes. In a significant number of tumors (14 [56%] of 24 LC/A, 13 [68%] of 19 classic, and 10 [67%] of 15 nodular MBs) the CN was > 2-fold but < 5-fold. High-level amplification, defined as > 10-fold CN, was only seen in the LC/A subtype (5 cases), although moderate amplification (> 5-fold but < 10-fold) could be detected in other histological subtypes. Fluorescence in situ hybridization readily detected most cases corresponding to tumors with > 5-fold amplicon CN by quantitative real-time PCR, and could detect all 5 cases with > 10-fold CN by quantitative real-time PCR. The group of patients with > 5-fold myc amplicon CN showed significantly shorter survival than those with < 5-fold CN (p = 0.045), independent of histological subtype. Since FISH could easily detect most cases in the moderate-to-high myc gene amplification (> 5-fold CN) group, the FISH assay has utility in detecting subsets of MB with poorer prognosis.

Medulloblastoma: histopathologic and molecular markers of anaplasia and biologic behavior

Large cell/anaplastic (LC/A) medulloblastoma (MB) is a recently recognized variant of medulloblastoma known to be associated with an advanced stage and a poor prognosis. Although Eberhart et al. suggested histopathologic grading of medulloblastoma in 2002, no consensus has been reached in terms of determining the criteria of an LC/A variant, and its biological behavior continues to be the subject of debate. We retrospectively analyzed 74 cases (range 0.25-15 years) of MB clinicopathologically using the criteria established by Eberhart et al. The LC/A variant was identified in 16 cases (22% of MB cases), five of which showed a poor outcome. Most LC/A variant cases revealed synaptophysin immunoexpression (75%), but no epidermal growth factor receptor (EGFR) expression. Expression of synaptophysin, NeuN, GFAP, p53, c-erbB2, and EGFR did not differ in LC/A and non-LC/A variants. Seven of the 74 cases of medulloblastoma showed erbB2 amplification by FISH, four of which were LC/A variants. N-myc amplification was observed in only one LC/A variant, but no c-myc amplification was found. In patients younger than 10 years, the LC/A variant showed a significantly poorer outcome than the non-LC/A variant (P = 0.02), while no difference was found in older patients. Multivariate analysis revealed only metastasis on MRI and p53 expression, but not anaplasia as unfavorable prognostic factors. Our study suggests that prognostic implications of anaplasia in medulloblastoma are uncertain, and that the reproducibility of the histopathologic criteria of the LC/A variant should be reassessed before they can be applied in practical use.