Molecular Subgroups Of Medulloblastoma Research Articles

Radiomic Features from Multi-Modal MRI Combined with Clinical Parameters Predict Molecular Subgroups in Patients with Medulloblastoma

Background: The 2016 WHO Classification of Central Nervous System tumors has included four molecular subgroups under medulloblastoma (MB) as sonic hedgehog (SHH), wingless (WNT), Grade 3 and Group 4. We aimed to develop machine learning models for predicting MB molecular subgroups based on multimodal magnetic resonance imaging (MRI) radiomics, tumor locations and clinical factors. Methods: 122 MB patients were enrolled retrospectively. After selecting robust, nonredundant and relevant features from 5529 extracted radiomics features, a random forest model was constructed based on a training cohort (n = 92) and evaluated on a testing cohort (n = 30). By combining the radiographic features and clinical parameters, two combined prediction models were also built. Findings: The subgroup can be classified using an 11-feature radiomics model with a high area under curve (AUC) of 0.8264 for WNT and modest AUCs of 0.6683, 0.6004 and 0.6979 for SHH, Group 3 and Group 4 in testing cohort, respectively. Incorporating location and hydrocephalus into the radiomics model resulted in improved AUCs of 0.8403 and 0.8317 for WNT and SHH, respectively. After adding gender and age, the AUCs for WNT and SHH were further improved to 0.9097 and 0.8654, while the accuracies were 70% and 86.67% for Group 3 and Group 4, respectively. Interpretation: The prediction performance was excellent for WNT and SHH, while that for Group 3 and Group 4 needs further improvements. Machine learning algorithms offer potentials to non-invasively predict molecular subgroups of MB. Funding Statement: This research was supported by the National Natural Science Foundation of China (No. 81702465, 61571432 and U1804172), the Science and Technology Program of Henan Province (No. 182102310113, 192102310123 and 192102310050). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: The Human Scientific Ethics Committee of the First Affiliated Hospital of Zhengzhou University has approved the protocol of this study (No. 2019-KY-176).

Preoperative Neutrophil to Lymphocyte Ratio and Platelet to Lymphocyte Ratio are Associated with the Prognosis of Group 3 and Group 4 Medulloblastoma

Inflammation and immunoreaction markers were correlated with the survival of patients in many tumors. However, there were no reports investigating the relationships between preoperative hematological markers and the prognosis of medulloblastoma (MB) patients based on the molecular subgroups (WNT, SHH, Group 3, and Group 4). A total 144 MB patients were enrolled in the study. The differences of preoperative hematological markers among molecular subgroups of MB were compared by One-way ANOVA method. Kaplan-Meier method was used to calculate the curves of progression free survival (PFS) and overall survival (OS). The comparison of survival rates in different groups were conducted by the Log-rank test. Multivariate analysis was used to evaluate independent prognostic factors. Increased preoperative NLR (neutrophil-to-lymphocyte ratio, PFS, P = 0.004, OS, P < 0.001) and PLR (platelet-to-lymphocyte ratio, PFS, P = 0.028, OS, P = 0.003) predicted poor prognosis in patients with MB, while preoperative MLR (monocyte-to-lymphocyte ratio), MPV (mean platelet volume), PDW (platelet distribution width), and AGR (albumin-to-globulin ratio) were revealed no predictive value on the prognosis of patients with MB. Furthermore, high preoperative NLR and PLR predicted unfavorable prognosis in childhood MB patients. However, preoperative NLR and PLR were not associated with the prognosis in adult MB patients. Multivariate analysis demonstrated preoperative NLR (PFS, P = 0.029, OS, P = 0.005) and PLR (PFS, P = 0.023, OS, P = 0.005) were the independent prognostic factors in MB patients. Emphatically, the levels of preoperative NLR and PLR in Group 3 MB were significantly higher than those in WNT MB. High preoperative NLR was associated with unfavorable OS in Group 3 (P = 0.032) and Group 4 (P = 0.027) tumors. Similarly, increased preoperative PLR predicted poor PFS (P = 0.012) and OS (P = 0.009) in Group 4 tumors. Preoperative NLR and PLR were the potential prognostic markers for MB patients. Preoperative NLR and PLR were significantly associated with the survival of Group 3 and Group 4 tumors.

Simplified Molecular Subtyping of Medulloblastoma for Reduced Cost and Improved Turnaround Time.

Molecular subtyping of medulloblastoma (MB) has become increasingly important for prognosis and management. Typically this involves detailed molecular genetic testing which may not be available in all centers. The purpose of the present study was to find a simplified approach to assign molecular subtypes of MB for routine use in centers with more limited resources. The molecular subtypes of MBs from 32 Thai patients, aged 0.5 to 35 years, were first determined by NanoString. These results were then compared with those obtained using a combination of limited immunohistochemistry (IHC) (β-catenin, GAB-1, YAP-1, p75-NGFR, OTX2) and CTNNTB exon 3 mutation analysis. By NanoString assay, there were 6 MBs (19%) in the wingless (WNT) group, 8 (25%) in the sonic hedgehog (SHH) group, 7 (22%) in group 3, and 11 (34%) in group 4. Although β-catenin immunostaining missed 4/6 WNT MBs, CTNNTB mutation analysis confirmed all WNT MB cases with amplifiable DNA. The IHC panel correctly assigned all the other molecular subtypes, except for 1 MB in group 4. Thus, our protocol was able to correctly categorized 31/32 cases or 97% of cases. Our study is the first to report molecular subtypes of MB in Southeast Asia. We found that molecular subgroups of MBs can be reliably assigned using a limited IHC panel of β-catenin, GAB-1, YAP-1, p75-NGFR, OTX2, together with CTNNTB exon 3 mutation analysis. This simplified approach incurs lower cost and faster turnaround time compared with more elaborate molecular methodologies and should be beneficial to centers with reduced laboratory resources.

Downregulation of miR-204 expression defines a highly aggressive subset of Group 3/Group 4 medulloblastomas

Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells.

C-Myc activation promotes cofilin-mediated F-actin cytoskeleton remodeling and telomere homeostasis as a response to oxidant-based DNA damage in medulloblastoma cells

Medulloblastoma (MB) is a common and highly aggressive pediatric brain tumor of a heterogeneous nature. According to transcriptome-based profiling, four molecular subgroups of MB have been revealed, namely WNT, SHH, Group 3 and Group 4. High MYC mRNA expression and MYC gene amplification in MB have been considered as indicators of poor prognosis. However, the role of c-Myc in MB biology is still not well established. In the present study, the effects of c-Myc activation in UW228-MycER MB cell line were investigated using 4-hydroxytamoxifen (4-OHT) induction system. Upon 4-OHT stimulation, an increase in metabolic activity, large-cell/anaplastic (LC/A) phenotype and oxidative stress-mediated DNA damage were observed. However, 53BP1 foci were not implicated in DNA damage response. Instead, cofilin nuclear translocation, changes in F-actin cytoskeleton and the levels of cytoskeletal proteins were shown. Moreover, the telomere length was found to be unaffected that may be associated with the upregulation of TRF proteins. Transcription of nascent RNA (synthesis of new rRNA) and the expression of RNA polymerase I-specific transcription initiation factor RRN3/TIF-IA were also elevated. Moreover, increased levels of DNMT2, a modulator of stress responses, were observed. A small fraction of cells responded differently as oncogene-induced senescence was also noticed. We postulate that c-Myc-mediated modulation of genetic stability of MB cells may trigger cellular heterogeneity and affect adaptive responses to changing environment.

Pilot study of DNA methylation-derived neutrophil-to-lymphocyte ratio and survival in pediatric medulloblastoma

IntroductionMethylation-derived neutrophil-to-lymphocyte ratio (mdNLR) has been identified as a potential prognostic biomarker of outcomes in various cancers. We evaluated the prognostic value of blood-derived mdNLR within a retrospective cohort of pediatric medulloblastoma patients. Materials and methodsDNA methylation was measured in archival peripheral blood samples collected on 56 pediatric medulloblastoma patients. Hazard ratios (HR) and 95% confidence intervals (CI) for the association between mdNLR and survival were evaluated using Cox proportional hazard models. ResultsCompared to patients who were alive at last follow-up (n = 43), the mean mdNLR value was slightly higher in deceased patients (n = 13) (12.3 vs. 5.2,P = 0.163). Elevated log-transformed mdNLR was suggestively associated with an increased likelihood of death in unadjusted models (HR=1.43, 95%CI: 0.92–2.22) and significantly associated with mortality in adjusted models (HR=1.61, 95%CI: 1.01–2.58). DiscussionFuture work is warranted to investigate the relationship between mdNLR outcomes in specific pediatric medulloblastoma molecular subgroups.

Reproducibility of the NanoString 22-gene molecular subgroup assay for improved prognostic prediction of medulloblastoma.

Medulloblastoma is the most frequent malignant brain tumor in children. Four medulloblastoma molecular subgroups, MBSHH , MBWNT , MBGRP3 and MBGRP4 , have been identified by integrated high-throughput platforms. Recently, a 22-gene panel NanoString-based assay was developed for medulloblastoma molecular subgrouping, but the robustness of this assay has not been widely evaluated. Mutations in the gene for human telomerase reverse transcriptase (hTERT) have been found in medulloblastomas and are associated with distinct molecular subtypes. This study aimed to implement the 22-gene panel in a Brazilian context, and to associate the molecular profile with patients' clinical-pathological features. Formalin-fixed, paraffin-embedded (FFPE) medulloblastoma samples (n = 104) from three Brazilian centers were evaluated. Expression profiling of the 22-gene panel was performed by NanoString and a Canadian series (n = 240) was applied for training phase. hTERT mutations were analyzed by PCR followed by direct Sanger sequencing and the molecular profile was associated with patients' clinicopathological features. Overall, 65% of the patients were male, average age at diagnosis was 18 years and 7% of the patients presented metastasis at diagnosis. The molecular classification was attained in 100% of the cases, with the following frequencies: MBSHH (n = 51), MBWNT (n = 19), MBGRP4 (n = 19) and MBGRP3 (n = 15). The MBSHH and MBGRP3 subgroups were associated with older and younger patients, respectively. The MBGRP4 subgroup exhibited the lowest 5-year cancer-specific overall survival (OS), yet in the multivariate analysis, only metastasis at diagnosis and surgical resection were associated with OS. hTERT mutations were detected in 29% of the cases and were associated with older patients, increased hTERT expression and MBSHH subgroup. The 22-gene panel provides a reproducible assay for molecular subgrouping of medulloblastoma FFPE samples in a routine setting and is well-suited for future clinical trials.

Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection.

BackgroundCytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB.MethodsUsing D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB.ResultsWe found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%.ConclusionsWe report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci.

Abstract 5874: Targeting DDX3 in medulloblastoma by RK-33

Abstract Introduction: Medulloblastoma is the most common form of pediatric brain cancer. Current treatments of surgical resection, craniospinal irradiation, and chemotherapy increase survival but around 40% of patients experience disease recurrence. Classically, four molecular subgroups of medulloblastoma have been identified: Wnt; Sonic hedgehog (SHH); Group 3; and Group 4. More recent work has subtyped up to seven molecular subgroups. The RNA helicase DDX3 is involved in many biological activities such as transcription, mRNA translation, splicing, and nuclear export. About 8% of medulloblastoma cases involve mutations in DDX3, of which 11% involve the pediatric Wnt and SHH subtypes. In addition, coinciding DDX3 and CTNNB1 mutations associated with dysregulated Wnt /β-catenin signaling in Wnt-type medulloblastomas have also been reported. Aims and Methods: The goal of this work was to study whether RK-33, a small molecule inhibitor of DDX3, would be effective against medulloblastoma as a single agent and in combination with radiotherapy. We studied DDX3 expression in medulloblastoma cell lines DAOY and UW228 and human tissue microarrays by immunohistochemistry, western blotting, and qRT-PCR. We targeted DDX3 by siRNA and by RK-33 and observed proliferation and viability changes by MTS assays. Flow cytometric analysis was used to determine effect of RK-33 on cell cycle and apoptosis. TOP/FOP promoter assay was utilized to study effect on TCF promoter activity. We used colony formation assays to study effect of combining radiation and RK-33 treatment on cells. DAOY tumors were generated in nude mice and treated with combination therapy to study effects in vivo. Results: High DDX3 expression was observed in 55% of pediatric and 66% of adult medulloblastomas. DDX3 knockdown resulted in reduced cellular proliferation. Both the medulloblastoma cell lines were sensitive to RK-33, with IC50 values of 2.5 µM and 3.5 µM, respectively and resulted in decreased levels of DDX3 expression, with no observable toxicity. RK-33 resulted in a G1 phase cell cycle arrest before the onset of apoptosis. DDX3 knockdown resulted in a significant decrease of TCF activity and reduced transcription of Axin2, CCND1, MYC, and Survivin. A combination of RK-33 and radiation resulted in a significantly higher inhibitory effect than using single treatment of either RK-33 or radiation. We conclude that inhibiting DDX3 via the small molecule inhibitor RK-33 is effective in reducing medulloblastoma tumors and that the effect is synergistic with radiation treatment. Citation Format: Saritha Tantravedi, Farhad Vesuna, Allison Martin, Michael Lim, Charles G. Eberhart G. Eberhart, Paul J. van Diest, Venu Raman. Targeting DDX3 in medulloblastoma by RK-33 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5874.

Abstract 148: Canonical Wnt activation as a therapeutic strategy in pediatric medulloblastoma

Abstract Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current molecular subgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumor through selective activation of the canonical Wnt pathway. Initial characterization of patient-derived Wnt and non-Wnt MB lines demonstrated a significant reduction in in vitro self-renewal and proliferative capacity of Wnt MBs. This was further validated by RNA-seq, which identified a marked reduction in the expression of stem cell self-renewal genes Bmi1 and Sox2 in Wnt MBs compared to non-Wnt MBs. Further, Wnt MB-derived xenografts maintained a significant increase in overall survival compared to non-Wnt MB xengrafts, further highlighting the protective nature of activated Wnt signaling in MB. Activated Wnt signaling by way of small molecule Wnt agonists in treatment-refractory MBs resulted in decreased in vitro self-renewal and expression of self-renewal genes, Bmi1 and Sox2. In order to validate the therapy-sensitive nature of Wnt-activated cells, we developed stable patient-derived lines containing a 7XTOPFlash reporter for endogenous Wnt signaling. Rare subclonal Wnt-active cells demonstrated a reduced self-renewal and tumor-initiating capacity through in vivo limiting dilution assays when compared to bulk Wnt-inactive cells. The therapeutic relevance of these findings were demonstrated with an in vivo survival advantage in those mice with orthotopic injections of cells with endogenous Wnt activity when compared to xenografts generated from Wnt-inactive cells. To develop a rationale clinical therapeutic, we used a novel substrate-competitive peptide inhibitor for GSK. Treatment with our peptide inhibitor showed a significant reduction in tumor burden with a corresponding increase in survival of patient-derived tumors that were otherwise treatment-resistant. The clinical utility of our findings is further supported by our analysis of integrated genomics data from 763 primary MBs, in which a validated Wnt gene signature was found to predict improved survivorship among children with poor-outcome and metastatic MBs. Our work establishes activated Wnt signaling as a novel treatment paradigm in childhood MB, identifies a rationale therapeutic approach for recurrent MB, and provides evidence for the context-specific tumor suppressive function of the canonical Wnt pathway. Citation Format: Sheila Kumari Singh, Branavan Manoranjan, Anna Dvorkin-Gheva, Chitra Venugopal, Steven Moreira, Michelle Kameda-Smith, Minomi Subapanditha, Ashley Adile, David Bakhshinyan, Neil Savage, Blake Yarascavitch, Olufemi Ajani, Adam Fleming, Bradley Doble. Canonical Wnt activation as a therapeutic strategy in pediatric medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 148.

Abstract A33: Molecular classification of Brazilian medulloblastoma by a NanoString gene panel

Abstract Background: Medulloblastoma is the most frequent malignant brain tumor in children. Currently, four distinct medulloblastoma molecular subgroups have been identified: SHH, WNT, Group 3, and Group 4. The nCounter® technology (NanoString) is a high-throughput platform, highly sensitive, robust and useful for analyses of highly degraded samples, such as formalin-fixed, paraffin-embedded (FFPE) samples. Recently, a gene panel of 22 genes employing the nCounter® technology was developed to distinguish meduloblastoma molecular subgroups. Aim: To apply and to validate a gene panel for classification of Brazilian medulloblastoma molecular subgroups using the nCounter® technology. Methods: We evaluated 47 tumor samples (FFPE) from patients diagnosed with medulloblastoma from Barretos Cancer Hospital, São Paulo, Brazil. Clinicopathologic features from all patients were collected and overall (OS) survival and 5-year OS were calculated. Gene expression profiling was carried out using the nCounter Elements™ custom panel (NanoString), and the custom CodeSet was composed of distinctive genes for each medulloblastoma molecular subgroup (WNT, SHH, Group 3, Group 4) plus housekeeping genes. Heatmap clustering was employed for initial classification of medulloblastoma molecular subgroups and the PAM method was applied for class prediction. hTERT hotspot mutations were screened by Sanger sequencing. Statistical analyses: Kappa index, Fisher's exact test, Kaplan-Meier method and log-rank test; level of significance: 95%. Results: Forty-seven patients diagnosed with medulloblastoma were analyzed (average age = 14.5 years). The median OS was 66.5 months and the 5-year OS was 54%. After heatmap clustering, we distinguished 44 tumors: 21 SHH, 7 WNT, 5 Group 3, 11 Group 4, and 3 of them were not distinguished. After class prediction by PAM method, we classified 44 tumors: 22 SHH, 6 WNT, 5 Group 3, 11 Group 4, and 3 of them were not classified. Among the 3 cases which were not distinguished by heatmap clustering, 2 of them were predicted as SHH and 1 remained unclassified and 2 cases distinguished by heatmap clustering as WNT and SHH subgroups were unclassified by PAM method prediction. Classifications by heatmap clustering and PAM method were highly concordant according to kappa index. WNT group presented the lowest 5-year OS and the Group 3 presented the highest 5-year OS. hTERT hotspot mutation (C228A) was mostly found in the SHH subgroup. Conclusion: We validated in a Brazilian cohort a modern approach for classification of medulloblastoma molecular subgroups. The nCounter® technology associated with PAM method is helpful to distinguish the medulloblastoma molecular subgroups and prospectively can be applied as a forthcoming diagnostic approach. Citation Format: Leticia Ferro Leal, Adriane Feijo Evangelista, Flavia Escremim de Paula, Adriana Cruvinel Carloni, Gisele Caravina de Almeida, Bruna Mancano, Rui Manuel Reis. Molecular classification of Brazilian medulloblastoma by a NanoString gene panel [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A33.

Distinctive localization and MRI features correlate of molecular subgroups in adult medulloblastoma.

Medulloblastoma (MB) is recognized as comprising four molecular subgroups with distinct transcriptional profiles, clinical features, and outcomes. Previous studies demonstrate that pediatric MBs present with subgroup-specific MRI manifestations. We hypothesized that combination of anatomical localization and conventional features based on MR imaging can predict these subgroups in adult MBs. MR Imaging manifestations of 125 adult patients with MB were analyzed retrospectively based on pre-operative MRI scans. MB molecular subgroups were evaluated by the expression profiling array and immunohistochemistry. A pediatric MB cohort of 60 patients were analyzed for comparison with data of adult patients. Multiple logistic regression analysis revealed that tumor location (P < 0.0001) and pattern of enhancement (P = 0.0048) were significantly correlated with molecular subgroups in adult MBs. Ninety-two percent of adult MBs were correctly predicted by using logistic regression model based on the anatomical localization patterns and pattern of enhancement. Exclusively intra-cerebellar growth, localization in the rostral cerebellum, and no brainstem contact were specific to adult SHH-MBs. Group 4-MBs in adult were characterized by minimal/no enhancement compared with other two subgroups. Infant SHH-MBs represented significant different localization patterns compared with SHH tumors in children and adults. We identified that molecular subgroups of adult MBs could be well predicted by tumor localization patterns and enhancement pattern. Our study also provided important evidence that MB subgroups in adult possibly derived from different cellular origins.

Abstract 5831: Activated Wnt signaling for the treatment of recurrent medulloblastoma

Abstract Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current molecular subgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumor through selective targeting by small molecules and transgenic patient-derived lines containing a stabilized beta-catenin mutant. Activated Wnt signaling by way of Wnt agonists in treatment-refractory MBs resulted in decreased in vitro self-renewal and promoted differentiation. Comparative gene expression profiling of control and transgenic lines containing a stabilized beta-catenin mutant demonstrated a reduction in stem cell self-renewal genes following beta-catenin overexpression, including Sox2 and Bmi1. In order to validate the therapy-sensitive nature of Wnt-activated cells, we developed stable patient-derived lines containing a 7XTOPFlash reporter for endogenous Wnt signaling. Rare subclonal Wnt-active cells demonstrated a reduced self-renewal and tumor-initiating capacity through in vivo limiting dilution assays when compared to bulk Wnt-inactive cells. The therapeutic relevance of these findings were demonstrated with an in vivo survival advantage in mice with orthotopic injections of cells containing a stabilized beta-catenin mutant representative of constitutively active Wnt signaling or endogenous Wnt-active cells. Xenografts generated from Wnt-activated tumors were smaller in size, maintained a lower rate of proliferation, and reduction in MB self-renewal genes. To further illustrate the clinical utility of activated Wnt signaling, we modified the Children’s Oncology Group therapy protocol for childhood MB so that xenografts may receive chemo/radiotherapy. Tumors generated from Wnt-active xenografts were much more radiosensitive and displayed a significant reduction in spinal metastasis when compared to mice receiving standard therapy without Wnt activation. To develop a rationale clinical therapeutic, we developed unique agonist antibodies that target the Wnt co-receptor LRP5. Treatment with LRP5 antibodies showed a significant reduction in tumor burden and increase in survival of patient-derived tumors that were otherwise treatment-resistant. Our work establishes for the first time activated Wnt signaling as a novel treatment paradigm in childhood MB, identifies a rationale therapeutic approach for recurrent MB, and provides evidence for the context-specific tumor suppressive function of the canonical Wnt pathway. Note: This abstract was not presented at the meeting. Citation Format: Branavan Manoranjan, Chitra Venugopal, Zvezdan Pavlovic, David Bakhshinyan, Michelle Kameda-Smith, Minomi Subapanditha, Sujeivan Mahendram, Jason Moffat, Bradley W. Doble, Sheila Singh. Activated Wnt signaling for the treatment of recurrent medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5831. doi:10.1158/1538-7445.AM2017-5831

Differences in the Prognostic Value of Tumor Extent of Resection among the Molecular Subgroups of Medulloblastoma: A Single Centre Study of 113 Cases

Medulloblastoma (MB) is one of the most common pediatrics malignant tumors of the central nervous system. Studies in America and Europe have demonstrated significant differences in the prognostic value of medulloblastoma extent among molecular subgroups of medulloblastoma. However, studies focusing on the Chinese population are still lacking. A total of 113 patients with medulloblastoma who underwent surgical resection in Huashan Hospital between January 2002 and December 2013 were included in this study. Histological diagnoses were confirmed by 2 or more pathologists. Immunohistochemistry and CTNNB1 exon 3 mutation analysis were used to determine the different subgroups. Complete or incomplete resection was defined based on surgeons' reports and confirmed by postoperative computer tomography (CT). In this study, we included 113 patients with medulloblastoma (13 with WNT subgroup, 18 with SHH subgroup, and 82 with non-SHH/WNT subgroups) to assess their event-free and overall survival. We identified event-free survival and overall survival benefit for complete resection over incomplete resection. We found that for patients with NON-SHH/WNT medulloblastoma, incomplete resection was significantly associated with progression and overall survival compared with complete resection. To our best knowledge, the present study is the first to demonstrate the prognostic value of tumor extent of resection among the molecular subgroups of 113 medulloblastoma in the Chinese population. The prognostic benefit of the increased extent of resection for patients with medulloblastoma is attenuated after the molecular subgroups are taken into account. We still need further study to assess the benefit of surgical resection of small residual portions.

From One to Many: Further Refinement of Medulloblastoma Subtypes Offers Promise for Personalized Therapy

There is significant intertumoral heterogeneity within the four molecular subgroups of medulloblastoma. In this issue of Cancer Cell, Cavalli etal. apply similarity network fusion to gene expression and DNA methylation data to identify 12 medulloblastoma subtypes with distinct molecular and clinical profiles, making an important step toward molecularly tailored therapy.

MEDU-06. NOVEL MOLECULAR SUBGROUPS IMPROVE CLINICAL CLASSIFICATION AND OUTCOME PREDICTION FOR CHILDHOOD MEDULLOBLASTOMA

Background - International consensus recognises four medulloblastoma molecular subgroups - WNT (MBWNT), SHH (MBSHH), Group 3 (MBGrp3) and Group 4 (MBGrp4) - each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. Subgroups harbor distinct clinico-pathological and molecular features, underpin current disease sub-classification and initial subgroup-directed therapies are underway in clinical trials (i.e. reduced risk-adapted treatments for favorable-risk MBWNT patients; SMO inhibitors for MBSHH patients). However, significant biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. Methods - We undertook comprehensive molecular profiling and unsupervised class discovery (non-negative matrix factorization, t-SNE) of test and validation cohorts (n=704 in total), to identify consensus primary molecular subgroups within childhood medulloblastoma ( 3.0 years). Findings - Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified, characterized by distinct biological/clinical features. For instance, MBSHH comprised two age-dependent subgroups, while MBGrp3 and MBGrp4 each split into two subgroups with significantly different survival rates. Survival analysis identified secondary features predictive of outcome. Cross-validated subgroup-dependent models incorporating these novel subgroups along with secondary features and established disease risk-factors, outperformed current disease risk-stratification schemes. These schema stratified patients into four clinical risk-groups - favorable-risk (91% 5-year survival, 25% of patients), standard-risk (81%, 23%), high-risk (42%, 38%) and very high-risk (28%, 13%) - to be considered for treatment reduction, intensification or novel therapies respectively. Interpretation - The discovery of seven novel, clinically-significant, subgroups significantly improves disease risk-stratification and provides a new foundation for future research and clinical investigations.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study

SummaryBackgroundInternational consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions.MethodsIn this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent.FindingsSeven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82–100]); standard risk (50 [23%] patients; 81% survival [70–94]); high-risk (82 [38%] patients; 42% survival [31–56]); and very high-risk (29 [13%] patients; 28% survival [14–56]).InterpretationThe discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations.FundingCancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Differences in the prognostic value of tumor extent of resection among the molecular subgroups of medulloblastoma:A single centre study of 113 cases

Medulloblastoma (MB) is one of the most common pediatrics malignant tumors of the central nervous system.Studies in America and Europe have demonstrated significant differences in the prognostic value of medulloblastoma extent among molecular subgroups of medulloblastoma.However, studies focusing on the Chinese population are still lacking.A total of 113 patients with medulloblastoma who underwent surgical resection in Huashan Hospital between January 2002 and December 2013 were included in this study.Histological diagnoses were confirmed by 2 or more pathologists.Immunohistochemistry and CTNNB1 exon 3 mutation analysis were used to determine the different subgroups.Complete or incomplete resection was defined based on surgeons' reports and confirmed by postoperative computer tomography (CT).In this study, we included 113 patients with medulloblastoma (13 with WNT subgroup, 18 with SHH subgroup, and 82 with non-SHH/WNT subgroups) to assess their event-free and overall survival.We identified event-free survival and overall survival benefit for complete resection over incomplete resection.We found that for patients with NON-SHH/WNT medulloblastoma, incomplete resection was significantly associated with progression and overall survival compared with complete resection.To our best knowledge, the present study is the first to demonstrate the prognostic value of tumor extent of resection among the molecular subgroups of 113 medulloblastoma in the Chinese population.The prognostic benefit of the increased extent of resection for patients with medulloblastoma is attenuated after the molecular subgroups are taken into account.We still need further study to assess the benefit of surgical resection of small residual portions.

Определение молекулярно-генетических подгрупп медуллобластомы на основании анализа уровня экспрессии генов

Определение молекулярно-генетических подгрупп медуллобластомы на основании анализа уровня экспрессии генов

Clinicopathological analysis of UHRF1 expression in medulloblastoma tissues and its regulation on tumor cell proliferation.

Studies have showed the involvement of ubiquitin-like with PHD and RING finger domains 1 (UHRF1) in tumorigenesis and progression. This study focused on the relationships between UHRF1 and medulloblastoma (MB). Immunostaining and western blotting demonstrated differential expression of UHRF1 in MB tissues and no UHRF1 expression in normal cerebellum tissues. Univariate survival analysis revealed MB patients with high UHRF1 expression had significantly shorter OS and PFS than patients with low UHRF1 (OS p=0.009, PFS p=0.003). Multivariate analysis illustrated that UHRF1 expression level is an independent prognostic factor influencing the OS and PFS (OS p=0.038, PFS p=0.014). UHRF1 expression levels were significantly different among molecular subgroups of MB (p=0.003). Down-regulation of UHRF1 by RNAi inhibited proliferation and clonogenic ability of MB cell lines with cell cycle arrest in G1/G2-phase. Meanwhile, cells transfected with lenti-shUHRF1 showed increased p16 expression and location shift of CDK4 in MB cells. These findings indicate UHRF1 may promote cell proliferation and be a potential biomarker that can be used as a prognostic parameter and a therapeutic target for MB.