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

SINGLE NUCLEOTIDE VARIANT

COPY NUMBER VARIANT (CNV)

FUSION GENES

ALK

EML4

MICROLESIONS MACROLESIONS

FISH

Sanger

qPCR

CGH

Microtome

Nucleic Acid Extraction

Fusion Genes

CNV Copy Number Variation

SNV Single Nucleotide Variation

Conventional approach

Copy Number variant

FISH

Sanger

qPCR

CGH Nucleic Acid Extraction

Fusion Genes

Single Nucleotide Variant

NG

S

NGS approach

Oncomine Focus Assay

52 unique genes 269 amplicons in DNA panel, 271 amplicons in RNA panel

DNA Panel

RNA Panel

Hotspot genes, n=35 Fusion drivers, n=23 ALK RET ROS1 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 MET BRAF RAF1 ERG ETV1 ETV4 ETV5 ABL1 AKT3 AXL EGFR ERBB2 PDGFRA PPARG

Copy Number Variants, n=19 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

ALK AR BRAF CCND1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2

FGFR3 FGFR4 KIT KRAS MET MYC MYCN PDGFRA PIK3CA

Thermo Fisher All Rights Reserved For Research Use Only. Not for use in diagnostic procedures.

COPY NUMBER VARIATION DETECTION

Depth ratio: tumoral DNA/Bioinformatic baseline (48 XY patients)

Normalisation with reference genes Gender information+++ Tumoral cellularity+++

Detection threshold : At least 4 copies At least 10 amplicons involved

Ref. genes Chromosome

APC 5

BIRC2 11

BRCA1 17

DCUN1D1 3

MED12 X

NF1 17

ALK AR BRAF CCND1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2

FGFR3 FGFR4 KIT KRAS MET MYC MYCN PDGFRA PIK3CA

19 genes

*For Research Use Only. Not for use in diagnostic procedures.

Gene A Gene D primer 5’a

primer 3’d

Gene A Gene B

Amplicon AB primer 5’a

primer 3’b

1- DIRECT DETECTION OF 269 KNOWN TARGETED FUSION BREACKPOINTS

FUSION TRANSCRIPTS DETECTION: PRINCIPLES (AMPLISEQ STRATEGY)

Possibility to identify new fusion transcripts between genes included in the RNA panel

ALK RET ROS1 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 MET BRAF RAF1 ERG ETV1 ETV4 ETV5 ABL1 AKT3 AXL EGFR ERBB2 PDGFRA PPARG

2- IMBALANCE ASSAY PRINCIPLE (ALK, ROS1, RET and NTRK1)

Fusion partner

Driver oncogene

012345

Expression 5' Expression 3'

Oncogène driver

Partenaire defusion

5’ 3’

Fusion breakpoint

Oncogenic Fusion Transcript

012345

5' expression 3' expression

Driver oncogene

Fusion partnerReciprocal Fusion Transcript

Imbalance expression between the 5' assay and the 3' assay of the driver gene

5’ 3’

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

0.0

5.0

10.0

15.0

20.0

25.0 ALKRETROS1NTRK1NTRK2NTRK3FGFR1FGFR2FGFR3METBRAFRAF1ERGETV1ETV4ETV5ABL1AKT3AXLERBB2PDGFRAPPARG

*For Research Use Only. Not for use in diagnostic procedures.

Relative histotype representation in Oncomine Focus Fusion panel

0

5

10

15

20

25

30

35

40

45

nb FT in histotype N = 269 FT

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.

ION REPORTER CNV detection RNA alteration detection

OVAT

Selection of predefined pathogenic variants (Thermofisher database)

Oncomine Knowledge Base Reporter

Edition of clinical trials available

Tumoral sample information entry

TORRENT SUITE SERVER

Annotated vcf Bam files

Raw data

SNV detection

Option 1 : Automatic interpretation of predefined variants with OVAT

ION REPORTER CNV detection RNA alteration detection

OVAT

Selection of predefined pathogenic variants (Thermofisher database)

Oncomine Knowledge Base Reporter

Edition of clinical trials available

Tumoral sample information entry

TORRENT SUITE SERVER

Annotated vcf Bam files

Raw data

SNV detection

Option 2 : Manual interpretation of pathogenicity by the biologist

Biological interpretation and validation of variants Evaluation of pathogenicity

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.

OKR Example of Met alteration

Administrations approvals Trials available

Thermo Fisher All Rights Reserved For Research Use Only. Not for use in diagnostic procedures.

Clinical trial description

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) TRIAL TYPE PROMOTOR TARGETS DRUG NAMES

MOST Umbrella Centre Léon Bérard

ABL1, KIT, PDGFRA/B, DDR1/2, CSF1R

Nilotinib

PIK3CA, PIK3R1, AKT12, mTOR, TSC1/2,

PTEN Everolimus

VEGFR1-3, PDGFRB, FLT3, BRAF (non

V600), CRAF, KRAS, RET

Sorafenib

ERBB2 Lapatinib

VEGFR1-3, PDGFRA/B, KIT Pazopanib

AcSé Crizotinib Basket

Unicancer

ALK1, ROS1, MET Crizotinib

AcSé Vemurafénib

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

• 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

02468

1012141618

Repartition of histological types in our series(%)

4 tumor types representing 52% of the samples

DESCRIPTIVE ANALYSIS of ALTERATIONS AND GLOBAL YIELD IN BIP PROTOCOL

0.02.04.06.08.0

10.012.014.016.018.0

73% 23% 4%

Mutations

Amplifications

RNA alterations

Patie

nt (

%)

45.3 33 37.6

0

50

Oncomine focus assay global yield

All Somatic alterations Novel somatic alterations Actionable alterations

Alteration types distribution %

Tumor recruitment versus Histotype fusion panel representation

05

1015202530354045

• Panel under-exploited for thyroid and CNS fusion detection in our series

0

5

10

15

20

NSCLC CERVIX COLORECTAL ENDOMETRIAL GASTRIC OVARIAN PANCREAS SARCOMA BREAST UROTHELIAL

Yield by tumoral type (all somatic alterations)

Histotype recruitment (%) Histotype contribution to global yield (%)

COMPARATIVE YIELD OF RELEVANT ALTERATIONS: BY TUMORAL TYPE

COMPARATIVE YIELD OF RELEVANT ALTERATIONS: PRIMARY VERSUS METASTATIC SITES

0

50

100

150

200

Primary site Metastatic site

Number of samplesscreened

Number of samples withat least one actionablealteration

300

82

4 0

50

100

150

200

250

300

350

All tumor sequenced Complementary CGH Actionable Targets detected by CGH

Is CGH-array has additional value to Oncomine Focus assay to screen tumors?

4/82 cases : - PTEN homoz del - AKT2/VEGFA amp - AR amp - BRCA2 homoz del

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

Dr Emmanuel Khalifa Dr Benjamin Bonhomme Félix Lefort Magali Philips Isabelle Hostein Gaelle Pérot Jennifer Chiron Mathilde Beunardeau Céline Auzanneau

Molecular Pathology Lab

Larry Blanchard Mélanie Muller Laetitia Nègre-Mayeur Agnès Ribeiro Marie-Cécile Blard Marlène Boucheix Nathalie Mérillon-Tortevoie Charlotte Primois Léa Ries Ghislaine Sierankowski

Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.