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DNA diagnosis of lung cancer Patrick Willems GENDIA •Antwerp, Belgium Treatment of Lung Cancer – Small Cell Lung Cancer (SCLC) • chemotherapy • radiation – Non-Small Cell Lung Cancer (NSCLC) • surgery • radiation • chemotherapy • targeted treatment • immunotherapy Personalized cancer treatment • Immunotherapy to stimulate immune response to cancer PD-1 inhibitors PD-L1 inhibitors CTLA-4 inhibitors • Targeted therapy with designer drugs that target the genetic cause of the tumor mAB: Herceptin TKI: Gleevec Problems in personalized cancer treatment • Immunotherapy Extremely expensive (100-300.000 Euro/year) Few biomarkers (companion diagnostics) • Targeted therapy with designer drugs Very expensive (50-100.000 Euro/year) Biomarkers (companion diagnostics) Problems in personalized cancer treatment The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary Cancer biomarkers tumor material (biopsy) blood (liquid biopsy) Market for tumor biomarkers in Liquid biopsies TARGETS DRUGS SEQUENCING Liquid biopsy market for tumor biomarkers: 40 Billion USD per year (Illumina estimate) Current paradigm PATIENT general visit treatment PHYSICIAN Result sample Pathological studies PATHOLOGIST Lab Future paradigm PATIENT Personalised visit treatment PHYSICIAN PHARMA Result sample Molecular testing Pathologist LAB Mortality UK, 2009-2011 Cancer Morbidity and Mortality Canada, 2007 New cancers per year in Belgium • Lung : 7.100 • Colon : 6.500 • Prostate : 8.800 • Breast : 9.700 TOTAAL : 65.000 Lung cancer • 14 % of all cancer • 80 % is non–small cell lung cancer (NSCLC) • Belgium : 7.100 new cases per year • Worldwide : 10 million new cases per year • Worldwide : 8 million fatalities per year • The main cause (20-30%) of cancer-related death in both men and women : • More women die of lung ca than breast, cervical and uterine ca combined. • More men die of lung ca than prostate and colorectal ca combined. Treatment of Lung Cancer – Small Cell Lung Cancer (SCLC) • chemotherapy • Radiation – Non-Small Cell Lung Cancer (NSCLC) • surgery • radiation • chemotherapy • immunotherapy • personalised targeted treatment Immunotherapy for NSCLC • CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) : ipilimumab • PD-1 (programmed death-1) : nivolumab, pembrolizumab • PD-L1 (programmed death-1 ligand) BMS-935559, MPDL3280A Inhibition immune checkpoints Biomarkers for immunotherapy for Lung Ca Few biomarkers for immunotherapy First real biomarker : Tumor load (amount of mutations-driver and passenger) Response to pembrolizumab (PD-1 inhibitor) better if high mutation load Science, April 3, 2015 (Rizvi et al) MSI as Biomarker for immunotherapy MMR deficiency Genomic instability Large mutation load in tumor (driver and passenger) Many mutant proteins - neoantgens Immune response Immunotherapy for NSCLC – Extremely expensive (100-300.000 Euro/year) – No biomarkers to select patients Targeted therapy with designer drugs • Receptor antibodies (---- ab) • • • • • HER2 : Trastuzumab (Herceptin) EGFR : Cetuximab, Pertuzumab MET : AMG102 VEGF : Befacizumab Tyrosine Kinase Inhibitors : TKI (---- ib) • • • • • • BRC-ABL : Imatinib (Gleevec) KRAS : Tipifarnib BRAF : Sorafenib MEK ERK mTOR : Everolimus Receptor antibodies inhibit receptor kinases by interfering with ligand-receptor binding Preventing intracellular signaling Herceptin Herceptin (Trastuzumab) Inhibits HER2 dimerisation / activation and the downstream signaling pathways MAPK and AKT/mTOR Active when there is HER2 overexpression • Breast ca (25 %) • Gastric ca (20 %) Tyrosine kinase inhibitors (TKI) TKI inhibits a Tyrosine kinase by binding to its kinase domain Preventing phosphorylation (activation) of target Gleevec Gleevec (Imatinib) inhibits Tyrosine kinases by binding to its kinase domain Thereby preventing phosphorylation (activation) of targets : • BCR-ABL (CML) • cKIT (GIST, Mastocytosis) • PDGFR (GIST) Targeted treatment – Non-Small Cell Lung Cancer (NSCLC) • surgery • radiation • chemotherapy • personalised targeted treatment • immunotherapy – Small Cell Lung Cancer (SCLC) • chemotherapy • radiation Targeted treatment of NSCLC Expensive, but many biomarkers to select patient Personalised targeted treatment targets specific somatic mutations that cause NSCLC These mutations are patient-specific These mutations can be detected by molecular studies of : tumor (biopsy) blood (liquid biopsy) Progress in lung ca treatment Problems in targeted cancer treatment The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary The mutations leading to lung ca are the biomarkers to guide targeted therapy Inheritance of cancer • Majority of cancers are caused by genetic anomalies in the tumor (somatic mutations) • Minority of cancers is inherited (germline mutations) : • Breast Cancer : 10 % • Colon cancer : 5-10 % • Prostate cancer : low • Lung cancer : very low Inheritance of lung cancer • NO germline mutations • MANY somatic mutations Driver and passenger gene mutations TUMOR MUTATIONS EXPLANATION HNPCC 1782 Genomic instability Lung 150 Mutagen (smoke) Melanoma 80 Mutagen (sun) Colon with MSS 73 Breast 60 Prostate 40 Leukemia 10 Fast (acute) tumor Pediatric tumors 10 Young age Vogelstein et al, Science Aug 22, 2013 NEJM May 30, 2015 Somatic mutations in cancer Breast NSCLC Colon Prostate TP53 23 34 48 16 KRAS < 10 15-25 35 5 26 4 22 2 EGFR < 10 10-30 < 10 4 MLL3 7 10 12 5 < 10 < 10 < 10 4 PIK3CA CTNNB1 Somatic mutations in adeno ca NSCLC TP53 : 34 % EGFR : 10-30 % KRAS : 15-25 % MLL3 : 10 % STK11 : 9% CDKN2A : 8% ALK fusions : 5 % HER 2 : 2% BRAF : 1-2 % Ros fusions : 2 % Somatic mutations in adeno ca NSCLC Cell growth and survival pathway Genetic testing for lung cancer – EGFR: deletions in exon 19 L858R mutation in exon 21 T790M mutation in exon 20 – KRAS: mutations of codons 12 and 13 – BRAF: V600E, G469A and D594G mutations – ALK-EML4 fusion EGFR Mutations in lung cancer EGFR mutations : 10 % (Europe) 30 % (Asia) women, non-smokers, adenocarcinoma (NSCLC) 90% of EGFR mutations : L858R in exon 21 (Sensitivity to TKIs) Small deletions in exon 19 (Sensitivity to TKIs) T790M in exon 20 (Resistance to TKIs) First-generation EGFR tyrosine-kinase inhibitors : Erlotinib (Tarceva) Gefitinib (Iressa) Second-generation EGFR tyrosine-kinase inhibitors : Dacomitinib Afatinib (Gilotrif) EGFR mutations EGFR Resistance : T790M mutation Inhibitors of EGFR with the T790M mutation : AZD9291 CO-1831 EGFR resistance : KRAS and BRAF mutations TREATMENT E G F R K R A S W I L D RELAPSE KRAS Mutations in lung cancer KRAS mutations : 15-25 % in NSCLC smokers 90% of KRAS mutations : codon 12 (90 %) codon 13 (5-10 %) KRAS Mutations are contraindications for EGFR TKI BRAF Mutations in lung cancer BRAF mutations : 1-4 % in NSCLC 55 % of KRAS mutations : V600E BRAF Mutations are contraindications for EGFR TKI ALK Mutations in lung cancer • ALK mutations : 5 % in NSCLC • ALK activation is caused by EML4-ALK fusion generated by inv(2)(p21p23) • ALK mutations are sensitive to ALK inhibitors : Crizotinib (Xalkori) Ceritinib (Zykadia) ROS1 Mutations in lung cancer ROS1 mutations : 2 % ROS1 activation is caused by ROS1 fusion to different partners ROS1 mutations are sensitive to Crizotinib (Xalkori) Why perform genetic studies on tumor DNA ? • Initial diagnosis and prognosis Initial therapy • Monitoring recurrence – metastasis Secundary therapy Prognosis according to EGFR mutations Prognosis according to BRAF mutations Why perform genetic studies on tumor DNA ? • Initial diagnosis and prognosis Initial therapy • Monitoring recurrence – metastasis Secundary therapy Monitoring recurrence - metastasis • Clinical : imaging • Tumor markers : CEA • Circulating tumor cells (CTC) • Circulating tumor DNA (ctDNA) Diagnostic tests on tumor DNA • Solid tumor biopsy FFPE Frozen Fresh • Liquid biopsy : Circulating tumor DNA (ctDNA) Blood plasma/serum Advantages liquid biopsies • No tissue biopsy needed • No FFPE fixation • Profiling the overall genotype of cancer • primary cancer • circulating cells • metastases • Better evaluation of : • reaction to therapy • development of resistance Why liquid biopsies for Lung cancer ? • The main cause (20-30%) of cancer-related death • High percentage of driver oncogenic mutations • Druggable targets • Lung biopsy difficult – liquid biopsy easy • Relatively cheap test • Association with expert group of Rafael Rosell Circulating tumor DNA (ctDNA) ctDNA ctDNA from tumor tissue is released through secretion, necrosis and apoptosis, but mainly through apoptosis Ct DNA cell-free DNA (cfDNA) is released from healthy, inflamed or cancerous tissue undergoing apoptosis or necrosis a small fraction of cfDNA is circulating tumor (ctDNA) very sensitive technology is needed to detect mutations in ct DNA cell-free DNA (cfDNA) • Cell-free DNA (cfDNA) in plasma of healthy individuals : Mandel and Métais (1948) • A proportion of cfDNA in pregnant women is fetus-derived (cffDNA) : Lo et al. (1997) • Non-Invasive Prenatal testing (NIPT) for Down syndrome: 2012 : start 2015 : > 1 million tests Market : 4 billion USD • Increased concentrations of cfDNA in the circulation of cancer patients : Leon et al. (1977) • A proportion of cfDNA is tumor-derived : Stroun et al. (1987) • Circulating tumor DNA (ctDNA) testing (liquid biopsy) : 2015 : start Market : 40 billion USD Advantages of liquid biopsies vs FFPE • No biopsy needed • Better representation of : • • • • Total mutation load Mutations in metastatic cells Reaction to therapy Development of resistance Tissue biopsy • • E G F R K R A S W I L D TISSUE BIOPSY EGFR TREATMENT • RELAPSE Liquid biopsy • • E G F R K R A S B R A F W I L D LIQUID BIOPSY TREATMENT Companies focusing on ctDNA • Pangaea Biotech • • • • • • • • • • • • • • Cynvenio BGI Agena Bioscience Boreal Genomics Chronix Biomedical Genomic Health Guardant Health Inivata Molecular MD Myriad Genetics Natera Personal Genome Diagnostics Sysmex Inostics Trovagene Liquid biopsy market for tumor biomarkers: 40 Billion USD per year Companies focusing on ctDNA Most companies have an expensive test (5000 USD) based upon NGS (Next Generation sequencing) detecting many mutations in many cancer genes of which the majority are currently “nondruggable” Sensitivity to detect mutant sequence • Sanger sequencing : 10 % • Next gen sequencing (NGS) : 1 % • NGS + specific technology : 0.1 % Pangaea biotech • Simple, relatively cheap test for lung cancer • Spin off company from IOR (Instituto oncologico Rosell) • CEO : Rafael Rosell • Focusing on Lung cancer • Technology : Mutant Allele - specific PCR using PNAs Mutant Allele - specific PCR PCR amplification of wild type allele is blocked by PNA probe, while mutant is allele is PCR-amplified ctDNA testing for lung ca 1. DESCRIPTION : ct DNA testing on liquid biopsies : • EGFR: deletions in exon 19, L858R mutation in exon 2, T790M mutation in exon 20 • KRAS: codon 12 and 13 mutations • BRAF: V600E, G469A and D594G mutations 2. SAMPLE : blood in specific test kits with Streck tubes provided by GENDIA 3. TURNAROUND TIME : 3 weeks 4. PRICE : < 1000 Euro How offer ctDNA testing to your patients ? 1. Refer to our consultation : Email [email protected] to ask for an appointment 2. Send blood : Email [email protected] to ask for tubes www.circulatingtumorDNA.net