Liquid biopsy: the experience of real life case studies

Liquid biopsy: the experience of real life case studies 10 th September 2018 Beatriz Bellosillo Servicio de Anatomía Patológica Hospital del Mar, Barcelona

Agenda Introduction Experience in colorectal cancer research samples Experience in lung cáncer research samples

Metastatic melanoma BRAF NRAS KIT Metastatic thyroid Papillary: BRAF Lung cancer Metastatic colon KRAS NRAS BRAF Metastatic serous low-grade ovarian BRAF BRCA1 BRCA2 EGFR KRAS ALK ROS RET BRAF H. Davies et al. Nature 2002 Detection Capability (mutant DNA/ total DNA) Cell-Free Tumor DNA 100% 10% Sanger Sequencing Circulating Tumor Cell 1% Pyrosequencing Cell-Free Normal DNA 0.1% Real-Time PCR 0.01% BEAMing/PCR digital

Hospital del Mar experience: NGS and liquid biopsy UMI based NGS DNApanels TNA based NGS panels 2012 2013 2015 2016 2011 2015 2016 2018 Junior 454 IonTorrent PGM IonChef IonTorrent S5 For Research Use Only. Not for diagnostic procedures.

Agenda Introduction Experience in colorectal cancer research samples Experience in lung cancer research samples

Oncomine TM Cell-free DNA Assays Oncomine cfdna Assays include library prep and analysis cfdna Isolation Library Prep Template Prep Sequencing Analysis Lab-created Report Applied Biosystems MagMAX cfdna Isolation Kit Oncomine cfdna Assays Ion Chef Instrument Ion GeneStudio S5 Systems Variant caller in Torrent Suite, Ion Reporter Software Oncomine Knowledgebase Reporter* Single blood tube Oncomine Colon cfdna Assay Refined variant data GENE LIST (14) AKT1 BRAF CTNNB1 EGFR ERBB2 FBXW7 GNAS KRAS KRAS MAP2K1 NRAS PIK3CAMAD4 TP53 APC KRAS/NRAS: G12/G13/Q61 BRAF: V600E PIK3CA: E545K, H1047R TP53: R175H R273H/C/L SMAD4: R361C/H Recurrent deleterious APC mutations (including p.r876*, p.r1114*, p.q1378*, and p.r1450*) Detect rare variants present down to 0.1% with Mean Sensitivity 90% Mean Specificity 98% For Research Use Only. Not for diagnostic procedures.

Heterogeneity and co-existence of mutations at progression of panitumumab based treatment - At least one mutation was detected in 94% of pts (15/16) - Median mutations per sample was 2.5 (range 1-13) Vidal et al, ESMO 2017

Heterogeneity and co-existence of mutations at progression of panitumumab based treatment Plasma Pre- Panitumumab Plasma Post-Panitumumab Pt APC TP53 CTNNB1 KRAS NRAS BRAF PIK3CA EGFR MAP2K1 ERBB2 AKT1 GNAS SMAD4 FBXW7 APC TP53 CTNNB1 KRAS NRAS BRAF PIK3CA EGFR MAP2K1 ERBB2 AKT1 GNAS SMAD4 FBXW7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NO PLASMA AVAILABLE 1 MUTATION IN THE SAME GENE 2 MUTATIONS IN THE SAME GENE 3 MUTATIONS IN THE SAME GENE - At least one mutation was detected in 80% of pts (8/10) - Median mutations per sample was 1.8 (range 1-4) - At least one mutation was detected in 94% of pts (15/16) - Median mutations per sample were 2.5 (range 1-13) Vidal et al, ESMO 2017

Agenda Introduction Experience in colorectal cancer research samples Experience in lung cancer research samples

% mutant alleles Retrospective case study #1 18 16 14 12 10 8 6 4 2 0 30.73% ND (< 0.02%) 1.068% 0.295% L858R EGFR L858R (T790M Not detected) 4.99% T790M 16.253% 0.948% 1.386% 27/08/2013 27/11/2013 12/11/2014 01/02/2015 04/03/2015 TKI Mutation % EGFR p.l858r 37% Mutation % EGFR p.l858r 21.% EGFR p.t790m Not detected EGFR p.t790m 1.9% TP53 p.r273c 17% TP53 p.r273c 9.5% Taus et al, CLC 2018

Oncomine TM Cell-free DNA Assays Oncomine Lung cfdna Assay GENE LIST (11) ALK BRAF EGFR ERBB2 KRAS MAP2K1 MET NRAS PIK3CA ROS1 TP53 169 hotspot mutations like EGFR: T790M, L858R, Exon19 del, C797S KRAS: G12X, G13X, Q61X ALK: 1151Tins, L1152R, C1156Y BRAF: V600E Detect rare variants present down to 0.1% with Mean Sensitivity 90% Mean Specificity 98% Mutational profile by NGS in NSCLC at the time of progression to TKI 3 1 4 2 3 N=13/22 cases T790M T790M+TP53 T790M+OTHER OTHER (TP53- RAS) UNKNOWN For Research Use Only. Not for diagnostic procedures.

Oncomine Lung Cell-Free Total Nucleic Acid Research Assay Single blood tube cftna extractio n Lung cftna Assay 0.1% LOD SNVs CNV Structural variation Sequencing of sample on Ion S5 System Analyze data in Ion Reporter Cloud software to generate test report Lung cftna Detects SNVs, indels + FUSIONS! Hotspot Genes ALK BRAF EGFR ERBB2 KRAS MAP2K1 Full- Length Genes Copy Number Genes Gene Fusions - MET ALK RET ROS1 Exon Skipping MET 2 2 4 N= 20 4 8 NO MUTATIONS FOUND EGFR MUTATIONS KRAS MUTATIONS FUSION DRIVER GENES OTHER MUTATIONS For Research Use Only. Not for diagnostic procedures.

Lung cancer case study #1 Lung cancer Research sample from 69 year woman Diagnosed with lung adenocarcinoma No positive biomarker for targeted therapy (EGFR-ALK- ROS1) PDL1 expression : 0% Standard chemotherapy

Lung cancer case study #1 Research sample from 69 year woman Diagnosed with lung adenocarcinoma Standard chemotherapy Clinical progression NGS screening of liquid biopsy research sample For Research Use Only. Not for diagnostic procedures.

Lung cancer case study #1 For Research Use Only. Not for diagnostic procedures.

Lung cancer case study #1 FISH probe Oncomine solid tumor CE-IVD assay For Research Use Only. Not for diagnostic procedures.

Lung cancer case study #1 Research sample from 69 year woman Diagnosed with lung adenocarcinoma Standard chemotherapy Clinical progression Potential targeted therapy was identified, and results may guide treatment decisions in the future (alectinib) For Research Use Only. Not for diagnostic procedures.

Lung cancer case study #2 Lung cancer Research sample from 48 year old woman Diagnosed of lung adenocarcinoma EGFR mutated (deletion in exon 19 and S768I in exon 20) Treatment with TKI (Afatinib) 5 month post-diagnosis: decrease of afatinib due to toxicity 7 months later slow progression Liquid biopsy : T790M not detected by ARMS real-time PCR, but positive for deletion in exon 19

Confirmation by digital PCR for T790M Osimertinib identified as potential treatment path in the future For Research Use Only. Not for diagnostic procedures.

Challenges of ctdna/tna analysis Dilution of tumoral/mutated DNA with wild type DNA Limited amount of cfdna/tna Small fragmented DNA Increase in the amount of information requested

Important considerations Optimize pre-analytical phase Blood extrac on Transport Plasma separa on DNA extrac on Adequate sample size to ensure minimum amount of cfdna/tna For some patients insufficient material

Important considerations Optimize pre-analytical phase Blood extrac on Transport Plasma separa on DNA extrac on Adequate sample size to ensure minimum amount of cfdna/tna For some patients insufficient material Ensure high quality cfdna Contaminating genomic DNA cfdna cfdna 22

Important considerations Optimize pre-analytical phase Blood extrac on Transport Plasma separa on DNA extrac on Adequate sample size to ensure minimum amount of cfdna/tna For some patients insufficient material Ensure high quality cfdna Participate in external quality controls and validation of technology

Current practice at Hospital del Mar for molecular testing Routine NGS 1º NGS Negative molecular marker - Re-biopsy - Repetition of liquid biopsy Pretreatment Rx Response Rx Progression Research

Molecular data should be discussed in multidisciplinary tumor boards

Take-home messages ctdna is a complementary strategy to tissue for molecular analysis that allows: Non-invasive analysis of tumor heterogeneity Dynamic biomarker monitoring NGS of ctdna provides a broad molecular analysis: SNVs, CNVs, fusions A comprehensive and dynamic monitoring of biomarkers is mandatory for real-time precision medicine Standardization and validation of the technology is necessary to ensure quality of the results Variant interpretation should be done in the potential clinical context in multidisciplinary teams

Disclaimer 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.

Servicio de Anatomía Patológica y Servicio de Oncología, Hospital del Mar Raquel Longarón Laura Camacho Conchi Fernández Lara Pijuan Alba Dalmases Gabriel Piquer Thermo-Fisher Chris Allen Francesco Acquadro Rosella Petraroli Edurne Arriola Clara Montagut Alvaro Taus Joana Vidal Joan Albanell