UNIVERSITY OF TORINO DEPARTMENT OF ONCOLOGY. Giorgio V. Scagliotti University of Torino Dipartment of Oncology

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1 Giorgio V. Scagliotti University of Torino Dipartment of Oncology

2 Molecular landscape of MM not fully characterized to allow personalized treatment Recurrent genetic alterations in MM: - NF % (HoD, HeD, M) (Takeda et al. 2012, Thurneysen et al. 2009, Bott et al. 2011) - BAP % (M) (Bott et al. 2011, Yoshikawa et al ) - CDKN2A % (HoD, HeD, M) (Bott et al. 2011, Takeda et al. 2012, Matsumoto et al. 2013, Cosmic database for mutation) - Chr loss in 1p, 3p, 9p, 22q (Jean et al. 2012) - Chr gain in 1q, 5p, 7p, 8q, and 17q (Jean et al. 2012) HoD homozygous deletion HeD heterozygous deletion M somatic mutation

3 CDKN2A 80%, NF2 60%, BAP1 21% (more smokers) Germline BAP1 mutations associated to mesothelioma, uveal melanoma, renal cell carcinoma and cholangiocarcinoma Bott. et al. Nature Genetics 2011, Testa JR Nature Genetics 2011

4 Merlin is acting as a TSG Sekido Y. et al. Cancer Sci. 2010; 101;1-6

5 Sekido Y. Cancer Sci. 2010; 101:1-6

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7 The NF2 tumor suppressor gene and its gene product, Merlin, are inactivated in 60% of mesothelioma tumors Merlin inhibits FAK phosphorylation and disrupts interaction of FAK with Src and PI3K FAK inhibitors could have therapeutic potential in Merlin null tumors Poulikakos et al, Oncogene 2006, 25:5960-8

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9 22 cases of MPM and matched blood samples 517 somatic mutations in 490 genes Guo G et al. Cancer Res 2015;75:

10 Discovery set (7 matched tumour/blood) Validation set (29 tumours) Sure Select Human All Exon capture Illumina HiSeq 2000 Variant calling (GATK) Gene based information Recurrent mutations in novel and known cancer genes Annotation with ANNOVAR Filtering for somatic variants Candidate driver selection + validation by Sanger sequencing Function based information Population frequency (dbsnp, 1000g, NHLBI, COSMIC) Pathway Analysis ORAL40.02:Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing- SK Lu

11 Characteristics Discovery cohort (n=7) Validation cohort (n=29) Age (years) Median 68 (60-80) 67 (52-82) 70 5 (71%) 18 (62%) > 70 2 (29%) 11 (38%) Sex Male 5 (71%) 23 (79%) Female 2 (29%) 6 (21%) Smoking Ever smoked 6 (86%) 17 (59%) Never smoked 1 (14%) 9 (31%) Not known 0 3 (10%) Asbestos exposure Yes 4 (57%) 22 (76%) No 2 (29%) 6 (21%) Not known 1 (14%) 1 (3%) Histological subtypes Epithelioid 6 (86%) 21 (72%) Biphasic 1 (14%) 8 (28%) Sarcomatoid 0 0 Tumour abundance (%) Mean Range ORAL40.02:Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing- SK Lu

12 Targeted regions sequenced to a mean depth of 100x for normal and 150x for tumours 1,226 somatic mutations in discovery set distributed across 1074 genes 706 damaging protein altering mutations Types of somatic mutations splice site nonframeshift deletion frameshift insertion frameshift deletion nonsense missense silent ORAL40.02:Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing- SK Lu

13 Discovery set Validation set Histology E E E E E E B E E E E E E E E E E E E E E E E E E E E E E B B B B B B B BAP1 V V V 25% NF2 V V 17% Chromatin remodelling genes JMJD1C V V V SETD2 V V ASH1L V SMYD3 V WHSC1L1 V SETD6 V DMAP1 V PHF21A V SETD1B V CDH8 V TBL1XR1 V Missense Non-sense INDELs Splicing V Sanger Validated ORAL40.02:Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing- SK Lu

14 Mutations: - 2 missense in FZD2-5 missense in LRP2-1 frameshift del in TBL1XR1 Gene expression profiling of 25 tumours vs 5 normal pleural tissues (unpublished data): - Significant up-regulation of WNT2B, WNT3, WNT9A, BMP4 involved in WNT signaling Created with NetworkAnalyst, Xia et al

15 Mutations: - 2 missense in CACNA1B - 1 missense in RASGRP4-1 missense in STK4 Gene expression data: - Significant up-regulation of EGFR, MET, PAK4, RAC3, LAMA5, LAMC2, ITGA3, ITGB4 involved in MAPK signaling Created with NetworkAnalyst, Xia et al ORAL40.02:Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing- SK Lu

16 J. Thorac. Oncol. 2014,Dec 15 Epub ahead of print

17 DNA extracted from 123 MPM tissue FFPE samples with clinical annotates. Amplicons NGS libraries for customized genomic analyses included all exons of BAP1/NF2 and 50 oncogenes included in Ion AmpliSeq Cancer Hotspot Panel v.2 Variations frequently identified in MPM patients (>=30 patients & at least 10% allelic frequency (AF)) that affect proteins stability or previously correlated to cancer by COSMIC database were evaluated also with Sanger sequencing for validation purposes. Statistical correlation between variations identified by NGS and clinical pathological features were evaluated with Fisher exact test stratifying patients by allelic frequency in three overlapping groups: rare (<=5% AF), sporadic (<=10% AF) and common (<=25% AF) events. Presented by: G. V. Scagliotti

18 Patients & Methods Presented by: G. V. Scagliotti

19 In the left panel are summarized the variations identified in the top 20 genes (>50 patients mutated with at least 10% of allelic frequency): in red the patients (pts) with at least a variation (intronic, synonymous, non synonymous and regulative) while in blue the patients with at least 1 non synonymous/regulative variation in the top 20 genes. The right panel shows the heatmap of patients with non synonymous/regulative variations. The blue block identifies variations while the color intensity is proportional at number of mutations observed in each patients. Presented by: G. V. Scagliotti

20 Cioce M et al. Cell Death & Dis. 2014;5:e1167

21 Cioce M et al. Cell Death & Dis. 2014;5:e1167

22 BAP1/NF2 panel: BAP1 variations was concentrated mainly in exon 13 Loci of BAP1 & NF2 are shown. The blue blocks represent the exons, while the red blocks indicated the regions amplified and sequenced in BAP1/NF2 custom panel. The red and yellow circles indicate the regions with at least 10 variations (red) or mutated patients (yellow). The size of circles is proportional to variations/patients counted for each regions. The variations identified in NF2 were distributed throughout the sequenced exons. Conversely, the BAP1 variations were enriched mainly in exons 13 and 17. No associations between more frequent NGS variations reported in both NF2 & BAP1 and clinical pathological characteristics were identified. Presented by: G. V. Scagliotti

23 Nuclear BAP1 protein expression was correlated to non synonymous variations detected by BAP1/NF2 custom panel. The lack of BAP1 protein expression significantly correlates with the detection of non synonymous variations identified with allelic frequency of 10 or 25% (Fisher test p value <0.01 for both 10 and 25% of allelic frequency). This data suggest that non synonymous variations identified could affect BAP1 protein stability. Presented by: G. V. Scagliotti

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25 In the top left of the figure is summarized the TP53 locus: exons are labelled in blue and amplified regions in red. In left bottom part the genotypes of 3 patients identified by NGS are shown and confirmed by Sanger's sequencing. In particular, the left panel shows Sanger electropherograms for Rs homo- (C;C), hetero- (C;G) and homo-zygote (G;G) patients. In the middle panel the same patients are represented by sequence BAM files uploaded in IGV software: each horizontal gray line represents single sequence independently evaluated.

26 Exons are labeled in blue and amplified regions in red. In left bottom part of the figure are shown representative genotypes identified by NGS and confirmed by Sanger's sequencing., The left panel shows the high correlation between NGS data and Sanger sequencing for allelic frequencies > 15%. In the right part of the figure Kaplan- Meier curves show association between non synonymous variations in the PIK3CA.4 amplicon or specific PIK3CA p.i391m mutation and early disease progression

27 Small molecules that inhibit PI3K signaling, and/or the mtor pathway, have activity in preclinical MPM models Mikami et al, Oncol Rep, 2010 Altomare et al, Oncogene, 2005 Hoda et al, J. Thoracic Oncology, 2011 López-Lago et al, Mol. Cell. Biol, 2009

28 Modified RECIST measurements per independent radiographic review * * * * * * * PTEN loss * * Data cut-off 18 May 2012 * Biomarker analyses performed * PIK3CA R88Q Kindler, IMIG 2012

29 Significant association between accumulation of validated non synonymous variations identified by NGS and disease progression (Log-rank p value=0.02) A trend was identified between accumulation of validated non synonymous variations and overall survival (logrank p value=0.055).

30 CDKN2A, NF2, BAP1 genes are frequently altered in MPM. Germline BAP1 mutations associated to mesothelioma, uveal melanoma, renal cell carcinoma and cholangiocarcinoma Role of Merlin as TSG proven in preclinical models and Merlin null MPMs are more sensitive to FAK inhibitors. A phase II study is ongoing. Our study investigated NGS showed that most frequently mutated genes are TP53, KDR, KIT, PIK3CA with variations that affect protein structure Synonymous/intronic variations in HRAS, SMARCB, STK11, PDGFRA were also observed. BAP1 non synonymous mutations were associated with the lack of nuclear expression of BAP1 protein. Accumulation of mutations in several key pathways progressively increase the risk of progression. CSF-1R may be a potential, relevant target in MPM. Presented by: G. V. Scagliotti

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32 COLLECTION OF BIOLOGICAL SAMPLES (Clinical units) Plasma/serum (see SOP) Pleural liquid (see SOP) Tissue (see SOP) Paraffin embedded tissues Patients annotation SAMPLES STORAGE & MANIPULATION (Biological units) RNA/DNA Exome sequencing & RNAseq Exosomes Cell cultures from tissues Cell cultures from pleural liquid 3D cultures PDX RESPONSES TO BIOLOGICAL AND CLINICAL QUESTIONS (Biological & Clinical units) Patients stratifications Role of Metabolism Role of inflammation and host response Response to therapeutic regimens Reverse engineering experiments Circuit analysis MESOLINE s strategy includes 3 sequential and partially interconnected steps respectively committed to the collection of the samples (biobanking), the preparation of suitable tools (data sets, cellular and animal models) to be differently interrogated to fill clinical gaps.

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