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 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 PHYSICIAN PATHOLOGIST general treatment visit Result Pathological studies sample PATHOLOGIST Lab

Future paradigm PATIENT PHYSICIAN PHARMA LAB Personalised treatment visit PHYSICIAN PHARMA Result Molecular testing sample LAB Pathologist

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. Lung cancer

Treatment of Lung Cancer Small Cell Lung Cancer (SCLC) chemotherapy Radiation Non-Small Cell Lung Cancer (NSCLC) surgery radiation 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 Receptor antibodies

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 %) Herceptin

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)

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 Young age Vogelstein et al, Science Aug 22, 2013 NEJM May 30, 2015

Somatic mutations in cancer P Breast NSCLC Colon Prostate TP53 23 34 48 16 KRAS < 10 15-25 35 5 PIK3CA 26 4 22 2 EGFR 10-30 MLL3 7 10 12 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 % PTEN : 25 % (loss) P

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 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) P

EGFR mutations

EGFR Resistance : T790M mutation Inhibitors of EGFR with the T790M mutation : AZD9291 CO-1831

EGFR resistance : KRAS and BRAF mutations TREATMENT RELAPSE EGFR KRAS WILD

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 activation is caused by ROS1 fusion to different partners ROS1 mutations are sensitive to Crizotinib (Xalkori) P

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 TISSUE BIOPSY EGFR TREATMENT RELAPSE EGFR KRAS WILD

Liquid biopsy LIQUID BIOPSY TREATMENT EGFR KRAS BRAF WILD

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 ? Refer to our consultation : Email ctDNA@GENDIA.net to ask for an appointment Send blood : Email ctDNA@GENDIA.net to ask for tubes

www.circulatingtumorDNA.net