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PhD Thesis in Molecular Medicine

Biomolecular characterization of metastatic medulloblastoma and study of telomere lengthening control. PhD Thesis in Molecular Medicine. CANDIDATE SUPERVISOR Dott. Simone Minasi Prof. Francesca Romana Buttarelli. XXX CYCLE A.A. 2017-2018.

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PhD Thesis in Molecular Medicine

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  1. Biomolecular characterization of metastatic medulloblastoma and study of telomere lengthening control PhD Thesis in Molecular Medicine CANDIDATE SUPERVISOR Dott. Simone Minasi Prof. Francesca Romana Buttarelli XXX CYCLE A.A. 2017-2018

  2. Medulloblastoma Clinical features, treatment and classification • Medulloblastoma is the most common malignant brain tumor of childhood. • Itarises in the cerebellum. • 40 new pediatric cases per year in Italy. • Treatment consists of surgery, radiation and chemotherapy. At present, overallsurvivalisaround 60%. • Grade IV in WHO classification. • 3 histologicalvariants: Classic (80%), LC/A and D/N. Simone Minasi

  3. Medulloblastoma Leptomeningeal dissemination • Medulloblastoma can disseminate through the cerebrospinal fluid in the leptomeningeal space to coat brain and spinal cord. • Approximately 30% of children with metastasis at diagnosis. • Metastatic dissemination is considered as a marker of poor prognosis. • At present is not known which specific molecular alterations drive neoplastic cells to migrate and metastasize. Simone Minasi

  4. Medulloblastoma Molecular subgroups and biomarkers • Very heterogeneous disease. • 4 subgroups. • Various genetic, epigenetic and molecular features. • Correlation with prognosis and frequency of metastasis. Metastatic MDB in literature were not so far analyzed independently from the non-metastatic counterpart. From: Taylor MD et al., 2012. Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol. Simone Minasi

  5. Medulloblastoma Current risk stratification A clinical trial PNET 5 MB has been designed for children with MDB of low/standard risk (according to the risk-group definitions). No gold standard treatment has been defined for metastaticcases. Genetic/molecular information and risk stratification remain unknown for some medulloblastomas with metastasis at the onset. From: Ramaswamy V et al., 2016. Risk stratification of childhood medulloblastoma in the molecular era: the current consensus. Acta Neuropathol. Simone Minasi

  6. Medulloblastoma FSTL5 expression • FSTL5 high expression associated with group 3 and 4. • Undefined molecular mechanism. • Correlation with tumour size and local infiltration in hepatocarcinoma. From: Remke M et al., 2011. FSTL5 is a marker of poor prognosis in non-WNT/non-SHH medulloblastoma. JCO. FSTL5 gene expression was not previously analyzed in metastatic MDB. Simone Minasi

  7. Control of telomereselongation in medulloblastoma • Telomeres maintenance is enriched in some SHH and group 3 MDB. • Results suggest that pathways which control elongation of telomeres can have a role and can characterize some molecular subtypes of medulloblastoma. Telomeres elongation has been reported in pediatric gliomas and non-metastatic MDB, but it was not previously investigated in metastatic medulloblastomas. From: Cavalli FMG et al., 2017. Intertumoral Heterogeneity within Medulloblastoma Subgroups. Cancer Cell. Simone Minasi

  8. AIM OF THE STUDY • Study alterations involved in the process of invasion and cell survivor in 39 pediatric MDB with leptomeningeal dissemination selected from a cohort of 60. • Analyze several molecular biomarkers to define if metastatic medulloblastomas are molecularly similar to non-metastatic. • Identify specific molecular alterations involved in invasiveness and malignancy of metastatic medulloblastoma cells. • Study FSTL5 expression to evidence if over-expression could have a role in leptomeningeal dissemination and to better characterize groups 3/4. • Evaluate the prognostic relevance of molecular subgroups, to allow a better risk stratification of metastatic cases. • Study the activation of mechanisms for telomeres lengthening control, to figure out if telomeres elongation could have a role in metastatic medulloblastomas, compared to pHGG and non-metastatic MDB. Simone Minasi

  9. RESULTS Biomarkers analysis 5 WNT, 7 SHH, 9 Group 3 and 6 Group 4 MDB 42% of metastatic MDB have the biomolecular characteristics of group 3 and 4. 33% of cases show biomolecular features of group SHH and WNT. 25% of cases are Not Classifiable (NC) and do not show a definemolecularprofile. Simone Minasi

  10. RESULTS Prognostic relevance of molecular subgroups p val<0,05 WNT and NC groups associated with good outcome (OS>80%) SHH, Group 3 and 4 associated with poor outcome (OS<60%) WNT metastatic MDB can be included in standard risk group. Not Classifiable group is also associated with standard risk. Simone Minasi

  11. RESULTS FSTL5 expression analysis Non-metastatic MDB FSTL5 over-expression is never associated with SHH or WNT group. FSTL5 is over-expressed exclusively in group 3 and 4. Not Classifiable group never harboured the FSTL5 over-expression. Simone Minasi

  12. RESULTS Prognostic relevance of FSTL5 expression FSTL5 down-expression associated with good outcome (OS>90%) FSTL5 over-expression associated with bad outcome (OS<50%) p val<0,05 FSTL5 is an independent prognostic biomarker for metastatic MDB and its expression analysis can be a reliable tool for prognostic evaluation of metastatic cases. Simone Minasi

  13. 1st SUMMARY OF RESULTS Study the biomolecular features of metastatic medulloblastomas and the alterations involved in the processes of invasion and migration. • Distribution of metastatic cases into 4 molecular subgroups is highly similar to the distribution of non-metastatic MDB and reflects a high molecular heterogeneity. There is no evidence of a prevalence of a specific molecular subgroup. • Our molecular subgrouping system can define risk classes for metastatic MDB and highlights WNT-group risk class as standard risk (OS>80%). • FSTL5 over-expression is exclusive of group 3/4 and is associated with poor prognosis; therefore, FSTL5 can be used to better define molecular subgroups and outcome of metastatic patients. FSTL5 isnotinvolveddirectlyinmigration and metastatic spread. • A subgroup of metastatic MDB (25%) showing heterogeneous biomarkers and lacking a define molecular profile. NC group is not characterized by FSTL5 over-expression and is associated with standard risk. Simone Minasi

  14. Control of telomereselongation ALT and TERT pathways High increase of telomereslength Shortening of telomereslength Intermediate increase of telomeres length From: Sturm D et al., 2014. Telomere maintenance mechanisms in glioblastoma. Nature Reviews Cancer. Simone Minasi

  15. RESULTS TERT promoter and H3.3 sequencing analysis H3.3 mutational status analysis via pyrosequencing TERT promoter mutational status analysis via pyrosequencing WT WT C228T K27M C250T G34R TERT mutations in 7% of pHGG H3.3 mutations in 21% of pHGG TERT mutations characterized20% of MDB with metastasisat the onset H3.3 mutations in 0% of MDB with metastasis at the onset Simone Minasi

  16. RESULTS ATRX and UTSS analysis TERT promoter methylation in UTSS region via MS-PCR ATRX nuclear expression via IHC and RT-PCR UTSS hypermethylation in 28% of pHGG UTSS hypermethylationcharacterized60% of MDBwith metastasisat the onset ATRX loss in 21% of pHGG ATRX losscharacterized28% of MDB with metastasisat the onset . Simone Minasi

  17. RESULTS Telomeres length analysis via Telo-FISH Telomeres elongation characterized both metastatic MDB and pHGG with similar incidence, in order to escape from senescence and have a proliferative advantage. All subtypes of medulloblastoma show elongation of telomeres with similar frequency. Simone Minasi

  18. RESULTS ALT and TERT activation in metastatic MDB and pHGG Elongation Shortening ATRX nuclearloss associated with telomereselongation. ATRX and H3.3 G34R mutations associated with telomereselongation. TERT mutations associated with telomereselongation, only in association with TERT hypermethylation in UTSS region. H3.3 K27M mutations not always associated with telomereselongation. TERT mutations not associated with telomereselongation in UTSS not-hypermethylatedcases. TERT mutations not associated with telomereselongation in UTSS not-hypermethylatedcases. Simone Minasi

  19. 2nd SUMMARY OF RESULTS Study the activation of pathways involved in control telomeres elongation and senescence escape. • Metastatic MDB control elongation of telomeres both via telomerase reactivation (14%) and ALT mechanism (27%), induced by ATRX mutations. ALT and telomerase activation are mutually exclusive. • ALT pathway is activated with higher frequency (27%) in metastatic MDB compared to non-metastatic (<5%), highlighting the differences between metastatic and non-metastatic tumors in control of senescence escape. • Metastatic MDB show higher activation of telomerase (14%)compared to pHGG (0%). TERT promoter mutations correlate with telomeres elongation only in association with UTSS hypermethylation. • Ourfindingssuggestthattelomereselongation of tumor cells in metastatic MDB is a common process to escape from replicative senescence, for allmolecularsubtypes. Simone Minasi

  20. FUTURE IMPLICATIONS • Molecular characterization of pediatric medulloblastomas with metastasis at the onset could help in identifying patients for de-escalation of therapy in future clinical trial. • Molecular subtypes and biomarkers can help clinicians to identify “personalized treatment” options for metastatic patients, in order to reduce side effects in children. • Analysis of telomere lengthening in metastatic MDB may contribute to risk stratification of patients and future development of targeted therapies. Simone Minasi

  21. ACKNOWLEDGEMENTS Preclinical and clinical Neuro-pathology Lab Prof.ssa F.R. Buttarelli Dott.ssa C. Baldi Pathological Anatomy Unit Prof. F. Giangaspero Prof.ssa M. Antonelli Sig.ra A. Angeloni and C. Valli Pediatric Oncology Unit Prof.ssa A. Clerico Dott.ssa M. Paiano Pediatric Oncology Unit Prof. M. Massimino Istitute fur Neurophatologie Director Prof. T. Pietsch Dott. M. Gessi Fabrizio Procaccini ONLUS Simone Minasi

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  24. Control of telomeres elongation Hypermethylation of TERT promoter (UTSS region) UTSS hypermethylation (>30%): Associated with the most aggressive brain tumors. Increased TERT expression. Not associated with ALT. From: Castelo-Branco P et al., 2013. Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. Lancet Oncol. Simone Minasi

  25. RESULTS FSTL5 molecular mechanism FSLT5 over-expression leads to increase secretion of this protein binding Ca2+. FSTL5 have calcium ion storage activity, interacting selectively with Ca2+. Dysregulation of Ca2+ homeostasis is an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Remodel of Ca2+ homeostasis affects cell cycle regulators (p21 and Cdc25C) leading to tumor growth and neo-angiogenesis. Extracellular levels of Ca2+ promotes Ras and Rac1 activity, Pyk2 phosphorylation and activation of others Ca2+-dependent effectors involved in cell migration and invasion. We hypothesize that FSLT5 over-expression could affect tumor microenvironment via calcium dysregulation and could trigger to migration, invasiveness and malignancy of cells in high-risk subgroups of metastatic medulloblastoma. Simone Minasi

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