Oncomine Focus assay panel and Oncomine Knowledgebase Reporter.

Oncomine Focus assay panel and Oncomine Knowledgebase Reporter. How it can help to identify relevant alteration and early phase trials. Dr Isabelle SOUBEYRAN Dr Emmanuel KHALIFA Molecular Pathology Unit Institut Bergonié France ESMO 2017- THERMO FISHER SCIENTIFIC SYMPOSIUM

NGS panel for testing mutations, copy number variations and RNA alterations OKR to process NGS data and link it to trials Our experience

MAIN TYPES OF MOLECULAR ALTERATION TO BE SCREENED MICROLESIONS SINGLE NUCLEOTIDE VARIANT COPY NUMBER VARIANT (CNV) MACROLESIONS FUSION GENES EML4 ALK

Conventional approach Microtome FISH Fusion Genes qpcr Nucleic Acid Extraction CGH CNV Copy Number Variation Sanger SNV Single Nucleotide Variation

NGS approach FISH Fusion Genes Nucleic Acid Extraction qpcr NGS CGH Copy Number variant Sanger Single Nucleotide Variant

52 unique genes 269 amplicons in DNA panel, 271 amplicons in RNA panel Oncomine Focus Assay Hotspot genes, n=35 Copy Number Variants, n=19 Fusion drivers, n=23 AKT1 ALK AR BRAF CDK4 CTNNB1 DDR2 EGFR ERBB2 ERBB3 ERBB4 ESR1 FGFR2 FGFR3 GNA11 GNAQ HRAS IDH1 IDH2 JAK1 JAK2 JAK3 KIT KRAS MAP2K1 MAP2K2 MET MTOR NRAS PDGFRA PIK3CA RAF1 RET ROS1 SMO DNA Panel ALK AR BRAF CCND1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2 FGFR3 FGFR4 KIT KRAS MET MYC MYCN PDGFRA PIK3CA ALK RET ROS1 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 MET BRAF RAF1 ERG ETV1 ETV4 ETV5 ABL1 AKT3 AXL EGFR ERBB2 PDGFRA PPARG Thermo Fisher All Rights Reserved For Research Use Only. Not for use in diagnostic procedures. RNA Panel

COPY NUMBER VARIATION DETECTION 19 genes Detection threshold : At least 4 copies At least 10 amplicons involved ALK AR BRAF CCND1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2 FGFR3 FGFR4 KIT KRAS MET MYC MYCN PDGFRA PIK3CA Ref. genes Chromosome APC 5 BIRC2 11 BRCA1 17 DCUN1D1 3 MED12 X NF1 17 *For Research Use Only. Not for use in diagnostic procedures. Depth ratio: tumoral DNA/Bioinformatic baseline (48 XY patients) Normalisation with reference genes Gender information+++ Tumoral cellularity+++

ALK RET ROS1 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 MET BRAF RAF1 ERG ETV1 ETV4 ETV5 ABL1 AKT3 AXL EGFR ERBB2 PDGFRA PPARG Gene A FUSION TRANSCRIPTS DETECTION: PRINCIPLES (AMPLISEQ STRATEGY) 1- DIRECT DETECTION OF 269 KNOWN TARGETED FUSION BREACKPOINTS Gene B primer 5 a Amplicon AB primer 3 b Driver oncogene 2- IMBALANCE ASSAY PRINCIPLE Fusion partner (ALK, ROS1, RET and NTRK1) Fusion breakpoint 5 3 5 4 3 2 1 0 Expression 5' Expression 3' Oncogène driver Partenaire de fusion Imbalance expression between the 5' assay and the 3' assay of the driver gene Possibility to identify new fusion transcripts between genes included in the RNA panel primer 5 a Gene A Gene D primer 3 d 5 3 Oncogenic Fusion Reciprocal Fusion Transcript Transcript 5 4 3 2 1 0 5' expression 3' expression Driver oncogene Fusion partner Confirmation for the fusion calls Improve sensitivity by providing indirect evidence of a fusion other than those targeted by the panel *For Research Use Only. Not for use in diagnostic procedures.

Relative representation of the 23 driver oncogenes in Oncomine Focus Fusion panel 25.0 ALK RET ROS1 NTRK1 20.0 NTRK2 NTRK3 FGFR1 FGFR2 15.0 FGFR3 MET BRAF RAF1 10.0 ERG ETV1 ETV4 ETV5 5.0 ABL1 AKT3 AXL ERBB2 0.0 PDGFRA PPARG *For Research Use Only. Not for use in diagnostic procedures.

Relative histotype representation in Oncomine Focus Fusion panel N = nb FT in histotype 269 FT 45 40 35 30 25 20 15 10 5 0 Oncomine Fusion panel optimized for NSCLC, CNS and thyroid tumor testing *For Research Use Only. Not for use in diagnostic procedures.

Oncomine Knowledge Base Reporter Efficient and intuitive tool for screening clinical trials Updated several times a year Allows to filter according to type of cancer, gene and trials *For Research Use Only. Not for use in diagnostic procedures.

Option 1 : Automatic interpretation of predefined variants with OVAT Tumoral sample information entry TORRENT SUITE SERVER Raw data Annotated vcf SNV detection Bam files ION REPORTER CNV detection RNA alteration detection OVAT Selection of predefined pathogenic variants (Thermofisher database) Oncomine Knowledge Base Reporter Edition of clinical trials available

Option 2 : Manual interpretation of pathogenicity by the biologist Tumoral sample information entry TORRENT SUITE SERVER Raw data Annotated vcf SNV detection Bam files ION REPORTER CNV detection RNA alteration detection Biological interpretation OVAT and validation of variants Evaluation of pathogenicity Selection of predefined pathogenic variants (Thermofisher database) Oncomine Knowledge Base Reporter Edition of clinical trials available

Oncomine Knowledge Base Reporter Functions and settings Allows to upload variant file filtered by OVAT (automatic interpretation) Allows to interrogate relevance of gene alteration (manual interpretation) Option tools to filter trials according to country/center availability Thermo Fisher All Rights Reserved For Research Use Only. Not for use in diagnostic procedures.

GLOBAL ANALYSIS IN THE CONTEXT OF BIP PROTOCOL AT BERGONIE INSTITUTE (about the 300 first samples screened by Oncomine Focus assay)

BERGONIE INSTITUTE PROFILING (BIP) Goal : Identify relevant molecular alterations in cancer research High throughput analysis : NGS Oncomine Focus assay Inclusion criteria : Solid malignant tumor or hematological malignancy Advanced disease Performance status < 2 Measurable disease according to RECIST 1.1 TRIAL TYPE PROMOTOR TARGETS DRUG NAMES MOST Umbrella Centre Léon Bérard AcSé Crizotinib AcSé Vemurafénib Basket ABL1, KIT, PDGFRA/B, DDR1/2, CSF1R PIK3CA, PIK3R1, AKT12, mtor, TSC1/2, PTEN VEGFR1-3, PDGFRB, FLT3, BRAF (non V600), CRAF, KRAS, RET ERBB2 VEGFR1-3, PDGFRA/B, KIT ALK1, ROS1, MET Nilotinib Everolimus Sorafenib Lapatinib Pazopanib Crizotinib Unicancer Basket BRAF Vemurafénib STRTRK-2 Basket IGNYTA Inc NTRK1-3, ALK1, ROS1 Entrectinib LOXO-101 Basket LOXO Oncology NTRK1-3 Larotrectinib KIDES Basket Orion FGFR1-4, VEGFR1-3 ODM203 JNJ-42756493 Basket Janssen RD FGFR1-4 Erdafitinib BET-1155121 Basket GSK Ampli N-MYC GSK525762 MEDIOLA Basket AstraZeneca BRCA / PDL1 MEDI4736 + Olaparib GSK-2118436 Basket GlaxoSmithKline BRAF Dabrafenib

Repartition of histological types in our series(%) 18 16 14 12 10 8 6 4 2 0 4 tumor types representing 52% of the samples

DESCRIPTIVE ANALYSIS of ALTERATIONS AND GLOBAL YIELD IN BIP PROTOCOL 50 0 % Oncomine focus assay global yield 45.3 33 37.6 All Somatic alterations Novel somatic alterations Actionable alterations Alteration types distribution 23% 4% Mutations 73% Amplifications RNA alterations Patient (%) 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0

Tumor recruitment versus Histotype fusion panel representation 45 40 35 30 25 20 15 10 5 0 Panel under-exploited for thyroid and CNS fusion detection in our series

COMPARATIVE YIELD OF RELEVANT ALTERATIONS: BY TUMORAL TYPE 20 15 10 5 0 Yield by tumoral type (all somatic alterations) NSCLC CERVIX COLORECTAL ENDOMETRIAL GASTRIC OVARIAN PANCREAS SARCOMA BREAST UROTHELIAL Histotype recruitment (%) Histotype contribution to global yield (%)

COMPARATIVE YIELD OF RELEVANT ALTERATIONS: PRIMARY VERSUS METASTATIC SITES 200 150 100 50 Number of samples screened Number of samples with at least one actionable alteration 0 Primary site Metastatic site

Is CGH-array has additional value to Oncomine Focus assay to screen tumors? 350 300 300 250 200 150 100 82 4/82 cases : - PTEN homoz del - AKT2/VEGFA amp - AR amp - BRCA2 homoz del 50 0 4 All tumor sequenced Complementary CGH Actionable Targets detected by CGH Conclusion : In our series, CGH-array yields up only few additional relevant alteration when no new targetable alteration is detected by Oncomine Focus panel *For Research Use Only. Not for use in diagnostic procedures.

COMPARATIVE YIELD OF ACTIONABLE ALTERATIONS: ONCOMINEFOCUS ASSAY VERSUS 426 GENES PANEL 426 genes panel (568 tumors) Oncomine focus assay (52 genes) on 300 tumors Tumors screened: n=300 Tumors with at least one relevant alteration: n=136 (37,6%) *For Research Use Only. Not for use in diagnostic procedures.

Take home messages Globally Oncomine focus panel yield allows to identify > 37% of tumors with at least 1 actionable alteration The RNA panel is optimized for non epidermoid NSCLC theranostic screening Under-exploitment of panel in our series Screening yield in a trial depends on panel volume as number of potential drugs available Oncomine Knowledge base Reporter is an efficient and intuitive tool for screening clinical trials

THANKS Molecular Pathology Lab Dr Emmanuel Khalifa Dr Benjamin Bonhomme Félix Lefort Magali Philips Isabelle Hostein Gaelle Pérot Jennifer Chiron Mathilde Beunardeau Céline Auzanneau Larry Blanchard Mélanie Muller Laetitia Nègre-Mayeur Agnès Ribeiro Marie-Cécile Blard Marlène Boucheix Nathalie Mérillon-Tortevoie Charlotte Primois Léa Ries Ghislaine Sierankowski

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