CANCER BIOMARKER DETECTION STRATEGIES TO DIRECT PRECISION MEDICINE GLORIA RIBAS, PhD Days on Personalised Medecine, Wien, Austria 24 25 February, 2017
PERSONALIZED MEDICINE One size fits all treatments have become obsolete
SCHEDULING OF TUMOR BIOPSIES AND THE OPORTUNITIS FOR GENOMIC ANALYSIS Von Hoff D, et al. J Clin Oncol 2010 Dienstmann R, Rodón J, Tabernero J. J Clin Oncol 2013
SIGNALLING PATHWAYS ALTERED IN CANCER
RESISTANCE TO HER2 BREAST CANCER TREATMENT MUTATIONS IN ONGOGENE PI3KCA BREAST CANCER ER/PGR + HER2+ NEO ADYUVANCY PIK3CA MUTATIONS GOOD RESPONSES LESS COMPLETE PATHOLOGICAL RESPONSES IN SEARCH OF NEW THERAPIES
COLORECTAL CANCER Metastasic Cancer CRC anti EGFR therapies Cetuximab, panitumumab + QMT respondants MUTATIONS KRAS 40 50% NON respondants IMPROVED SURVIVAL We are participating in a H2020 Motricolor proyect with three clinical trials based on molecular profiling. Patients will have dedicated therapies according to their genomic profile.
LUNG CANCER NSCLC 80% MUTATIONS (L858R) EGFR Asian ethnicity, female sex, adenocarcinoma histology, light/never smokers MUTATION K RAS /N RAS (20 25%)/(1%) TRASLOCATION EML4 ALK (1 7%) ALK TK inh THERAPIES crizotinib anti EGFR THERAPIES erlotinib o gefitinib NO respondedores respondedores respondants ios ensayos clínicos novedosos Amplification MET (20%) MUTATION T790M EGFR (50%) EGFR mutations and K ras mutations are almost always mutually exclusive. the lack of response to an EGFR TKI is likely more closely linked and better predicted by the lack of an EGFR mutation than the existence of a K ras mutation.
MELANOMA LATE STAGE MELANOMA Aprox 50% melanomas MUTATION V600 (*) B RAF Non respondants vemurafenib y dabrafenib B-RAF respondants MEK inhibitors, trametinib (Mekinist) y cobimetinib (Cotellic), show reduction of some melanomas with BRAF mutations
SETUP OF A PRESCREENING PROGRAM IN CANCER: SAMPLE TIMELINES SOURCE STORAGE COST PLATFORM SELECTION SCOPE SUCCES IN MATCHING PATIENTS TO THERAPY ROBUSTNESS SENSITIVITY
SAMPLE TYPES OF BIOLOGICAL TISSUES ADVANTAGES DISADVANTAGES BIOLOGICAL MATERIAL IN GOOD SHAPE FOR ÓMIC STUDIES BEST QUALITY AND QUANTITY NOT ALWAYS STORED APPROPRIATELY, NOT ALWAYS ACCESIBLE SINGLE POINT IN TIME FRESH FROZEN FFPE TMA MOST COMMONLY CONSERVED IN CLINICAL SETTINGS, (ALLOWS RETROSPECTIVE AND ARCHIVED MATERIAL). CONTAMINATED WITH NORMAL CELLS (MIN % SHOULD BE ESTABLISHED) SUBOPTIMAL EXTRACTION OF BIOLOGICAL MATERIAL (RNA, DNA, PROTEINAS) DEGRADED STATIC SAMPLE LIQUID BIOPSIES EASY TO OBTAIN IN RUTINE CLNICAL ANALYSIS (WILL FACILITATE MONITORING DISEASE, MINIMUN INVASIVE CONTAINS (PLATELET DERIVED CELLS, INMUNE CELLS, AND IN THE SERUM (EXOSOMES, ctdna, CTCs). LOW ABUNDANCE AND FRAGMENTED BIOLOGICAL MATERIAL BUT PRESENT REQUIRED ULTRASENSITIVE DETECTION METHODS
SAMPLE CLONAL HETEROGENEITY TUMORS ARE CLONALLY HETEROGENEOUS DIFFERENT SECTIONS CAN HAVE DIFFERENTS ALTERATIONS METASTASIS CAN DIFFER FROM PRIMARY TUMOR TUMOR CAN EVOLVE AT PROGRESSION Allison K., et al., Oncology Journal 2014 Gerlinger, N Engl J Med 2012
TIMELINES In a clinical scenario, timelines are very short. In Phase I trials, 2-3 weeks could be considered acceptable as total tournaround time Sanger sequencing (several genes) can take up to 2 weeks Assays developed to test 1 mutation in 1 oncogene can be done in < 1 week Multiplex Assays can be done whithin 1 week NGS for large list of candidate genes, of sequencing exomes o whole genome may take 6-8 weeks. The bottle neck is in the analysis. Should not last for more than two weeks.
ROUTINE SCREENING PROGRAM AT OUR HOSPITAL CLINICIAN requests For somatic mutation profiling MOLECULAR PATHOLOGY LAB selects FFPE blocks Evaluates tumor content and cuts slices Min 3-4 days (max 1 week) CANCER FX GENOMICS LAB Extracts DNA Quantifies and QCs Performs mutation detection Results are manually curated And are sent back to the clinician 1 week x 8 patients Total prescreening Program Turnaround time 2 weeks
PLATFORM SELECTION SANGER SEQUENCING NGS JUNIOR ROCHE MASSARRAY SEQUENOM <2010...... 2013...... 2014... NGS MISEQ, ILLUMINA DIGITAL PCR, SYSMEX INOSTICS... 2016...... 2017...
SANGER SEQUENCING - Quality - long reads - Costs - Analog trace peaks MIN ALLELE Detection 25% Gene A Gene B gdna Long Fragments 150-800bp One trace, from all fragments labelled
TYPE OF FUNCIONAL GENES IMPORTANCETO DESIGN ASSAYS ONCOGENES: Hostspots AKT1, BRAF, KRAS, PIK3CA, NRAS, HRAS... SUPPRESORES DE TUMORES: great diversity of alterations TP53, PTEN, P53, NF1, VHL... MUT indels
NGS FIRST GENERATION Nº GENE CHR 1 AKT1 14 2 BRAF 7 3 KRAS 12 4 KRAS 13 5 PIK3CA 3 6 PIK3CA 3 7 PIK3CA 3 8 NRAS 1 9 EGFR 7 10 EGFR 7 11 EGFR 7 EXON LONG MUT E3 125 p.e17k E15 119 p.v600f E2 122 p.g12+/p.g13+ E3 179 p.q61+ E2* 428 p.e110k E10* 125 p.e542+/p.e545+ E21* 6000 p.t1025+/p.h1047+ E2 128 p.g12+/p.g13+ E19 99 p.e747del E20 186 p.t790m E21 156 p.l858r NEXT GENERATION SEQUENCING: NGS JUNIOR (ROCHE) GENOTING Y GENETIC DIAGNOSTIC (UCIM, UV INCLIVA) u 6 GENES (AKT1, BRAF, KRAS, PIK3CA, NRAS and EGFR) 15 HOTSPOTS
MUTATION PRE SCREENING BY SEQUENOM OncoCarta Panelv1: 19 Oncogenes/238 hostspots Clia Vall D Hebron Panel: 5 additional oncogenes, 8 genes /86 hotspots Incliva Panel: ERBB2,ERBB3,ERBB4 Fleitas et al. Cancer Treat Reviews, 2016
TYPES OF TUMORS ANALYZED BY MASSARRAY TECHNOLOGY Widely used technology Mass spectrometry technology remains a good validation technology and is optimal when only hotspots are pursued Fleitas et al. Cancer Treat Reviews, 2016
D770_N771>AGG P772_H773insV L597S V600E MUTATED GENES AND SPECIFIC MUTATIONS FOUND TUMORES GASTROINTESTINAL TUMORS R970C G12S Q61R 35,5% A146V G12D G12S Q61K C634W C634Y D52N 22,6% G1049R C420R E542K KRAS PIK3CA KIT RET BRAF EGFR MET NRAS TUMORES BREAST/UTERUS/ENDOMETRY R201H P772_H773insV E839K G13D D1071N 35% C420R E542K H1047R R24C 17% E17K G12D, G12D Y253H Q61K PIK3CA KRAS ABL1 AKT1 CDK1 EGFR GNAS KIT NRAS PDGFRA
MUTATIONS FOUND ACROSS TUMOR SAMPLES 197 samples analysed (49% HAD 1 MUTATION IN 16 DIFFERENT GENES % CRC, % Breast) Mutations in KRAS and PIK3CA were detected in 40/97 (41.2%) and 30/97 (30.9%) patients respectively. Thirty one patients (32.0%) had mutations in two genes, 20 of them (64.5%) initially diagnosed with colorectal cancer co occurrence of mutation involved mainly KRAS, PIK3CA, KIT and RET Ibarrola Villava et al., Oncotarget 2015
OPTIONS OF TARGETED THERAPIES 101 PATIENTS WERE CANDIDATES TO BENEFIT FROM TARGETED THERAPIES 75 had actionable mutations 26 were KRASwt and could have possible be treatedwithanti EGFR agents 5 anti EGFR 15 OTHER THERAPIES 8 were selected for clinical trials 5 got PI3K/AKT inhibitors 1 anti IGF1, 2 anti ERBB3 TARGETED THERAPIES 28% ACTUALLY BENEFITED FROM TARGETED THERAPIES 34% HAD STANDARD THERAPY (CO MORBIDITIES, POOR PERFORMANCE STATUS, CONCURRENT 2º `NEOPLASM, OR LOSS OF FOLLOW UP 26% DIS NOT PROGRESS WITH CURRENT THERAPY Ibarrola Villava et al., Oncotarget 2015
THEY ARE MORE GENES MUTATED OUT THERE? Cancer genes identified from a data set of 4,742 tumours. Down-sampling analysis shows that gene discovery is continuing as samples and tumour types are added. DISCOVERY AND SATURATION ANALYSIS OF CANCER GENES ACROSS 21 TUMOUR TYPES MS Lawrence et al. Nature 000, 1-7 (2014)
GENOMIC PLATFORMS TO SCREEN A PANEL OF CANDIDATE GENES MALDI TOFF NGS masa complete genome non extended primer Complete exome customized panels Method to select wanted sections
IILLUMINA SEQUENCING: MISEQ APPLICATIONS IN CANCER GENOMICS Resequencing candidate gene-exons approach NimbleGen Seq Capture HaloPlex (Agilent) Hybridization Bead capture
MUTATION DETECTION EGFR L747P MUTATION
DISCOVERY AND SATURATION ANALYSIS OF CANCER GENES ACROSS 21 TUMOUR TYPES Cancer genes in selected tumour types. Lawrence et al. Nature 1 7 (2014) doi:10.1038/nature12912
MORE COMPLEX ANALYSIS
The plasma/serum contain several components CTCs, exosomes, cfdna, ctdna To have high levels of cfdna could be due to several reasons:: Inflammation Wounds Malignant lesions Sport LIQUID BIOPSY Healthy individuals: 3,000 5,000 Genomic Equivalents/ml Oncologic patients: av 10,000 Genomic Equivalents/ml Diaz LA & Bardelli A, JCO 2014:32:579 586
BEAMING DPCR Pre amplification Emulsion PCR Hybridization Wild-type signal Flow Cytometry Flow cytometry analysis Wild-type DNA Mutant & Wild-type DNA Mutant DNA Detection Capability (mutant DNA/ total DNA) 100% 10% 1% 0.1% Sanger Sequencing Pyrosequencing Real-Time PCR Mutant signal 0.01% BEAMing
MIN RESIDUAL DISEASE/EARLY MOLECULAR RECURRENCE Before Surgery Day of Surgery After Surgery Day 3 After Surgery Day 244 Normal signal Residual Mutant cfdna Mutant signal MUTATIONAL LOAD MINIMAL RESIDUAL DISEASE EARLY MOLECULAR RELAPSES Diehl et at. Nat Med. 2008
CENTRAL LAB FOR LIQUID BIOPSIES VALENCIA SYSMEX BASED ON FLOW CITOMETRY NRAS c13 NRAS c12 NEG 3mutand beads/0.08% POS 211 mutand beads/0.46% NATIONAL NETWORK OF CENTRES USING SYSMEX PLATFORM IN MAIN HOSPITALS. OncoBEAM" RAS CRC Kit We have data of already 30 patients. We detected 69% mutations (KRAS, NRAS) KRAS c13 POS 52029 mutand beads/15.8% POS KRAS c13 10829 mutand beads/12.3%
COMPARATIVE NGS MassArray Digital PCR Base calling/ Variant calling Fit for purpose validated pipeline defined performance for variant calling and frequency detection Automatic sofware generate variant calling and frequency detection Automatic score for quality control Automatic generation of results Mutation annotation Querying knowledge base stringent criteria quality control locked down pipeline Manual inspection data Locked system Clinical interpretation Unambigous, clinically relevant interpretation for end user Clinical report generation Free to decide What has to be included (ethical considerations) Genetic counseling considerations Reporting variants of unknown significance Report inclusión criteria Possible utilization in individual ls healthcare Fit for purpose clinical report Decide data to be shown Automatic generation Of clinical report Medical record generation Report data inclusion criteria
COST EFFECTIVENESS OF A TECHNIQUE IN A SREENING PROGRAM The higher the multiplexing is, higher the odds that a therapy may be matched The number of therapies (for now) is finite, so are the number of relevant targets to be asessed KRAS MEK inhibitor PIK3CA PI3K inhibitor BRAF BRAF inhibitor FGFR3 FGFR3 inhibitor EGFR EGFR inhibitor
GENOMIC LANDSCAPE BEFORE AND AFTER ANTI-EGFR THERAPY IN ADVANCED CRC ANTI-EGFR IN ADVANCED CRC The population of patients with no MAPK-pathway genomic alterations before treatment ( all wild-type ) is more likely to respond to epidermal growth factor receptor (EGFR) monoclonal antibodies (mabs). The high overlap in primary (left) and acquired (right) resistance mechanisms reinforces that clonal selection is a major determinant of the clinical outcome. Only EGFR mutations have not been identified in pretreatment lesions. In a substantial proportion of the samples, resistance is polyclonal, with co-occurring RAS mutations and EGFR or PI3K catalytic subunit-α (PIK3CA) mutations. ampl., amplification; mut., mutation. BREAST CANCER /PRIMARY AND METASTASIS : PIK3CA GENE MUTATIONS Dienstmann et al., Nature Reviews Cancer 17, 79 92 (2017) Cejalvo et al,, Breast Can rev Treat, 2015
EMERGING POSITIVE PREDICTIVE BIOMARKERS FOR TREATMENT SELECTION IN ADVANCED CRC Dienstmann et al., Nature Reviews Cancer 17, 79 92 (2017)
EVOLUTION OF PRECISION MEDICINE PARADIGMS IN COLORECTAL CANCER. The shift from a clonal perspective for targeted therapies ( one-gene, one-drug and multi-gene, multi-drug ) to a clonal stromal immune perspective ( multimolecular, multi-drug ) reflects increased understanding of the biology of the disease and advances in biomarker drug co-development. EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; inh., inhibitor; mab, monoclonal antibody; MSI, microsatellite instability; PD1, programmed cell death protein 1; PIK3CA, PI3K catalytic subunit-α; POLE, DNA polymerase-ε; TGFR, transforming growth factor-β receptor; TKI, tyrosine kinase inhibitor.
PROSPECTS FOR CLINICAL TRANSLATION OF MOLECULAR TESTS IN CRC Dienstmann et al., Nature Reviews Cancer 17, 79 92 (2017)
CLINICAL TRIAL ONCOLOGY DEPARTMENT INCLIVA Open clinical trials 2016 Active clinical trials 18 16 14 12 10 8 6 4 2 0 Fase I Fase II Fase III Fase IIIb We are participating in a H2020 Motricolor project with three clinical trials based on molecular profiling. Patients will have dedicated therapies according to their genomic profile.
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Cejalvo et al., Breast Cancer Res Treat, 2016