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Department of Experimental Oncology and Molecular Medicine Unit of Molecular Therapies XXVIII^ Riunione Nazionale MITO “Il lato oscuro di Venere”. Mesagne (BR) January 19-20 2017 miRNAs come biomarcatori nel carcinoma ovarico Delia Mezzanzanica Biomarker definition A biomarker is ‘ a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathologic processes, or pharmacologic responses to a therapeutic intervention’. Biomarkers Definitions Working Group (National Institutes of Health 2001) Biomarkers can be specific cells, molecules, genes, gene products, enzyme or hormone evaluated for: disease prevention, diagnostic ad prognostic assessment, drug target identification and drug response. Biomarkers can be divided into integral, integrated and exploratory ENGOT OV-16 NOVA (Niraparib maintenance in Pt-sensitive recurrent EOC) trial included: Integral biomarker, detection of germline BRCA mutation (gBRCAmut) before randomization for treatment with a PARP inhibitor vs placebo. Integrated biomarker, HRD status in the non gBRCAmut arm to assess the treatment efficacy in HRD-positive population in terms of progression free survival. Exploratory biomarker, in non-gBRCAmut HRD-positive cohort, impact of somatic BRCA mutation on progression free survival. GCIG Brainstorming TR meeting, Lisbon 2016 microRNAs are non-coding RNA that regulate many biological processes by controlling gene expression. miRNAs are transcribed by RNA polymerase II into long pri-miRNAs that are cleaved by DROSHA to form precursor miRNA (pre-miRNA). After nuclear export to the cytoplasm pre-miRNAs are directly processed by DICER and cleaved to generate a 22 nt miRNA duplex. The miRNA guide strand (highlighted in red), is incorporated into RISC. miRNAs mediate gene silencing via mRNA target cleavage and degradation or translational repression, depending on the complementarity between miRNA and targeted mRNA. Adams BD et al. Curr Biol 2014 Role of miRNAs Each miRNA can bind to and regulate multiple mRNAs. Aberrant expression of a single miRNA can affect translation of multiple genes within a cell, leading to profound phenotypic responses. miRNA effects on target genes are tissue specific. miRNA expression signatures are associated with tumor type, tumor grade and clinical outcomes, therefore miRNA could be potential candidates as: diagnostic biomarkers prognostic biomarkers therapeutic targets or therapeutic tools. Potential minimal invasive, diagnostic, and prognostic marker. miRNAs can be found circulating in peripheral blood and body fluids, they are very stable, bound to protein complexes or incorporated into micro vesicles (exosomes) resistant to degradation by RNAses. Therapeutic potential of miRNAs miRNAs can function as either oncogenes (targeting oncosuppressive genes) or tumor suppressor (targeting oncogenic genes). miRNA mimics (miRNA replacement therapy) to restore loss-of-function inhibition of the upregulated oncomiRs using antisense miRs (miRNA inhibition therapy) In hepatocellular carcinoma, miR-34 was recognized to be frequently downregulated: phase I clinical trial in patients with hepatocellular carcinoma or liver metastatic cancer with MRX34, a miR-34 mimic. The miR-34 family has been shown to be significantly downregulated also in OC. Biospecimens Biospecimens them self have become objects of investigation for translational research and precision medicine. Cancer research being genomics, proteomics, metabolomics depends on biospecimens, and finding the right targets for detection, therapy and prevention relies on the high quality of patient-derived specimen. GCIG Brainstorming TR meeting, Lisbon 2016 miRNAs and ovarian cancer Two of the most frequently identified, deregulated miRNAs in OC are the miR-200 and the let-7 families. The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) is involved in EMT regulation. No definitive conclusions on the prognostic impact of the miR-200 family in OC have been drawn due to diverging results. However the majority of the studies show that high miR-200 expression is linked to a favorable prognosis. The human let-7 family (Let-7a, Let-7b, Let-7c, Let-7d, Let-7e, Let-7f, Let-7g, Let-7i, miR-98, and miR-202) is known to suppress multiple oncogenes in OC and to inhibit cell cycle activators, is reported to be frequently downregulated in OC and therefore is believed to function as a tumor suppressor. miRNAs and ovarian cancer Several miRNA families with a prognostic role in the neo-adjuvant chemotherapy setting of HGSOC (analysis on primary tumor and on sample at interval surgery). Integrated analysis of miRNA and gene expression profiles in stage I EOC allowed identification of a prognostic pathway (16 miRNAs and 10 genes) associated with overall survival and progression-free survival Importance of future longitudinal management of relapsed EOC. analyses to improve the clinical Our effort: profile for miRNA and GE synchronous primary tumors, secondary localizations and relapses. A. Definition of a miRNA-based predictor of EOC early relapse : a team work Surgeons Tumor samples collection during surgical procedures Pathologists Pathological evaluation and sample selection Oncologists Follow up and clinical data collection Researchers RNA extraction/quality control, Profiling, Data analysis Profiling data Clinical data Data matrix generation and analysis Case materials Three chemo-naive case materials were used for this study: -OC179 FFPE material, derived from the MITO-2 clinical trial (carboplatin/taxol vs carboplatin/caelyx) -OC263 Frozen (and FFPE) material, collected at INT-MI and CRO-Aviano -OC452 TCGA public dataset collection 894 EOC cases: the largest collection so far analyzed A miRNA-based predictor of EOC early relapse- Discovery Phase published type of material n° samples profiling miRNA platform n° of miRNA on array MITO2 OC179 Present study FFPE 179 INT-MI Agilent miRBASE 17 1512 INT-MI Illumina v2 miR-BASE 12 public dataset Agilent miR-Base 10 OC263 INT_CRO ID OC130 Bagnoli&DeCecco frozen/FFPE Oncotarget 2011 TRAINING SET 130 OC133 Present study frozen 133 TCGA OC452 Nature 2011 frozen 452 1146 VALIDATION (including SET 1 putative) 723 VALIDATION SET 2 Data preprocessing - filtering for exclusion of miRNA not detectable in all samples - reannotation on miRBase 21.0 - exclusion of viral miRNAs, putative miRNA, not specific or discontinued probeset 385 unique miRNA shared among all studies following reannotation A miRNA-based predictor of EOC early relapse- Discovery Phase Development of the model TRAINING SET (OC179 MITO2 case material) Clinical end-point: time to progression /relapse 1. Calculate the prognostic index (risk-score) - Calculation of the prognostic risk score Selection of the features entering into the model: semi supervised method, using the relative expression of the selected 385 miRNAs and their impact (weight) on progression free survival. - Internal validation (10-fold cross validation approach) Reiterative process of classification to define the risk-score 2. Patient stratification A new sample is predicted as high (low) risk if its prognostic risk score is larger than (smaller than or equal to) the calculated prognostic index median value obtained in cross-validation. Low risk we developed a model named MiROvaR containing 35 unique miRNAs for the identification of patients at high risk of relapse/progression High risk hsa-miR-890 hsa-miR-513a-5p hsa-miR-513b-5p hsa-miR-135b-5p hsa-miR-141-3p hsa-miR-200c-3p hsa-miR-429 hsa-miR-200a-3p hsa-miR-200b-3p hsa-miR-592 hsa-miR-508-3p hsa-miR-514a-3p hsa-miR-509-3p hsa-miR-509-5p hsa-miR-506-3p hsa-miR-507 hsa-miR-143-5p hsa-miR-486-5p hsa-miR-195-3p hsa-miR-23a-5p hsa-miR-193b-5p hsa-miR-574-5p hsa-miR-99b-5p hsa-miR-151a-3p Hsa-iR-30d-5p hsa-miR-423-5p hsa-miR-30b-3p hsa-miR-330-3p hsa-miR-452-5p hsa-miR-100-3p hsa-miR-193a-5p hsa-miR-29c-5p hsa-miR-29a-5p hsa-miR-484 hsa-miR-769-5p A miRNA-based predictor of EOC early relapse- Discovery Phase Validation of the model MiROvaR stratified patients in High and Low risk of relapse with significantly different time to relapse TRAINING SET – OC179 MiROvaR score 1.0 Progression FreeSurvival VALIDATION SET 1 – OC263 LOW HIGH 0.8 VALIDATION SET 2- TCGA 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.2 0.0 0.0 log-rank p= 0.00068 0.0 0 20 40 60 80 100 120 HR 3.16 (95%CI:2.33-4.29) log-rank p<0.0001 0 Risk prediction Low High N° patients 90 89 Events 52 72 Median PFS 38 18 95%CI 24-nyr 15-22 20 40 60 80 100 Risk prediction Low High N° patients 122 141 Events 73 122 Median PFS 34 12 HR 1.39 (95%CI:1.11-1.74) log-rank p=0.0045 0.4 120 0 time (months) time (months) 0.6 95%CI 26-45 10-13 Risk prediction Low High 20 40 N° patients 169 283 60 Events 115 212 The miRNA classifier maintained its independent prognostic impact after adjustment for the two strongest prognostic clinical variables for EOC: Residual Disease after primary surgery and FIGO stage. MiROvaR Adjusted HR: 3.09 (95%CI: 2.24-4.28), pval <0.0001 1.41 (95%CI: 1.11-1.79), pval=0.0047 80 time (months) OC2634 TCGA 100 Median PFS 19 15 120 95%CI 17-27 14-18 A miRNA-based predictor of EOC early relapse- Discovery Phase Low risk High risk hsa-miR-890 hsa-miR-513a-5p hsa-miR-513b-5p hsa-miR-135b-5p hsa-miR-141-3p hsa-miR-200c-3p hsa-miR-429 hsa-miR-200a- 3p hsa-miR-200b-3p hsa-miR-592 hsa-miR-508-3p hsa-miR-514a- 3p hsa-miR-509-3p hsa-miR-509-5p hsa-miR-506-3p hsa-miR-507 hsa-miR-143-5p hsa-miR-486-5p hsa-miR-195-3p hsa-miR-23a-5p hsa-miR-193b-5p hsa-miR-574-5p hsa-miR-99b-5p hsa-miR-151a-3p Hsa-iR- 30d-5p hsa-miR-423-5p hsa-miR-30b-3p hsa-miR-330-3p hsa-miR-452-5p hsa-miR-100-3p hsa-miR-193a-5p hsa-miR-29c-5p hsa-miR-29a-5p hsa-miR-484 hsa-miR-769-5p MiROvaR contains 35 unique miRNAs with individually different relevance and individually different impact on patients’ prognosis. Among the 16 miRNAs that gave 100% of cross-validation support to the classifier, 13/16 were individually associated to favorable prognosis 3/16 were individually associated to poor prognosis Maintenance/loss of potentially oncosuppressive miRNAs has a greater impact on EOC prognosis than expression/loss of potentially oncogenic miRNAs. 4/5 miR-200 family members are included in the classifier as main contributors with potentially oncosuppressive role miR-506 family members are included in the classifier as main contributors with potentially oncosuppressive role, confirming our previous data. Bagnoli&DeCecco et al, Oncotarget 2011 Liu et al , JNCI 2015 Sun et al J Pathol 2015 Unique id hsa-miR-193a-5p hsa-miR-508-3p hsa-miR-509-5p hsa-miR-514a-3p hsa-miR-506-3p hsa-miR-507 hsa-miR-509-3p hsa-miR-592 hsa-miR-29c-5p hsa-miR-513b-5p hsa-miR-513a-5p hsa-miR-200c-3p hsa-miR-141-3p hsa-miR-200b-3p hsa-miR-423-5p hsa-miR-486-5p hsa-miR-200a-3p hsa-miR-23a-5p hsa-miR-330-3p hsa-miR-30b-3p hsa-miR-484 hsa-miR-769-5p hsa-miR-135b-5p hsa-miR-100-3p hsa-miR-99b-5p hsa-miR-143-5p hsa-miR-429 hsa-miR-151a-3p hsa-miR-574-5p hsa-miR-452-5p hsa-miR-29a-5p hsa-miR-195-3p hsa-miR-890 hsa-miR-30d-5p hsa-miR-193b-5p p-value % CV Support Hazard Ratio 0,0000177 100 1,977 0,0000311 100 0,747 0,0000474 100 0,684 0,0000478 100 0,811 0,0000507 100 0,635 0,0000572 100 0,588 0,0000713 100 0,783 0,0001548 100 0,255 0,0007134 100 1,595 0,0007233 100 0,817 0,0007357 100 0,766 0,0015449 100 0,793 0,0016807 100 0,819 0,0026893 100 0,786 0,002895 90 1,765 0,0029908 90 1,345 0,0031706 100 0,808 0,0052072 80 1,641 0,0060584 80 1,856 0,0064133 100 1,983 0,0078602 80 1,6 0,008215 70 1,762 0,008942 80 0,851 0,0089818 90 1,958 0,0093801 70 1,35 0,0095842 80 1,674 0,0122341 60 0,835 0,013404 60 1,363 0,0161045 50 1,283 0,0174535 60 1,276 0,0179111 50 1,765 0,0186502 40 1,629 0,0231142 40 0,085 0,0233194 40 1,253 0,0240755 60 1,506 miRNA-506 in EOC miR-506 expression is associated with a better response to therapy and longer PFS and OS miR-506 downregulation promotes an aggressive phenotype Ectopic expression of miR-506: inhibited cell proliferation promoted senescence via direct targeting CDK4 and CDK6 suppresses the CDK4/6-FOXM1 signaling pathway, which is activated in the majority of ovarian carcinomas Inhibits EMT, cell migration and invasion by targeting SNAI2 simultaneously suppressed Vimentin and N-cadherin increases sensitivity to cisplatin and olaparib by targeting RAD51 to suppress HR-mediated repair of double-strand breaks MiROvaR a strong predictor of EOC risk of progression/relapse Relevance of EMT processes in EOC aggressiveness To stratify patients according to risk of progression regardless of clinical-pathological characteristics of tumor at presentation Suitable in several clinical settings to contribute to a more precise patients’ selection - refine selection of high-risk subgroup of patients who may experience an OS advantage after Bevacizumab treatment (ICON7 trial) - selection of stage I-II patients likely to relapse and who can really benefit from chemotherapy Next steps: toward a clinical grade assay - TEST VALIDATION PHASE (external validation, selection of method, number of features) - EVALUATION FOR CLINICAL UTILITY (prospective clinical trial) Acknowledgements Department of Experimental Oncology and Molecular Medicine Unit of Molecular Therapies Functional Genomics Delia Mezzanzanica Silvana Canevari Paola Alberti Roberta Nicoletti Loris De Cecco Edoardo Marchesi Dept of Gynaecologic Oncology Dept of Pathology Francesco Raspagliesi Domenica Lorusso Antonino Ditto Maria Luisa Carcangiu Barbara Valeri Wei Zhang Anil Sood Sandro Pignata Francesco Perrone Daniela Califano Simona Losito Massimo di Maio Gennaro Chiappetta Giosué Scognamiglio Erika Cecchin Giuseppe Toffoli Roberto Sorio Vincenzo Canzonieri Gustavo Baldassarre Funding Thank you for your attention