EGFR mutation testing: what is the best choice?

EGFR? EGFR mutation testing: what is the best choice? Dekairelle Anne-France, PhD Center for Applied Molecular Technologies Prof GALA Jean-Luc, MD, PhD In response to ligand binding, EGFR is activated via dimerization and subsequent autophosphorylation of the tyrosine kinase domain Phosphorylation activates downstream pathways cell growth and proliferation UCL Dec 2010 EGFR-targeted therapies : TKI Advanced NSCLC : one disease, one treatment? Doublet chemotherapy has been a gold standard for years Panitumumab (Vectibix) Cetuximab (Erbitux) Trastuzumab (Herceptin) Zalutumumab Nimotuzumab Matuzumab. Iressa (Gefinitib) Tarceva (Erlotinib) Imatinib (Glivec) Lapatinib «One size fits all» therapy is not effective enough Cell growth Angiogenesis Invasion Metastasis Schiller JH, et al. NEJM 2002;346:92-98 EGFR in NSCLC? Response rate to EGFR TKIs Clinical benefits of EGFR-TKIs demonstrated in NSCLC BUT : Drug improvement in patients with lung cancer is not universal among patients. Clinical observation It is more effective in lung cancer patients with the following characteristics: - Asian patients - Females - Never smoked - Adenocarcinoma histology + Asian patients 1

Why study EGFR in NSCLC? Use of molecular genetics for EGFR: Rationale! Clinical benefits of EGFR-TKIs demonstrated in NSCLC BUT : Drug improvement in patients with lung cancer is not universal among patients. Clinical observation: It is more effective in lung cancer patients with the following characteristics: - Asian patients - females - never smoked - adenocarcinoma histology EGFR mutations have been discovered to be associated with response to EGFR inhibition Activating mutation in the EGFR underlying responsiveness of NSCL cancer to gefitinib (Lynch et al, NEJMed, 05/2004) EGFR mutations in lung cancer : correlation with clinical response to gefitinib therapy (Paez et al, Sciences, 06/2004) EGFR gene mutations are common in lung cancer from «never smokers» and are associated with sensitivity to gefitinib (Pao et al, PNAS, 09/2004) Maemondo M et al. N Engl J Med 2010; 362: 2380-8. EGFR mutation & treatment with an EGFR-TKI in non-asian patients Recurrent Mutations in NSCLC Reck M Expert Rev. Anticancer Ther. 10(6), 955 965 (2010) 2

Basic Principle of Individualized Medicine Pneumologists/Oncologists, Pathologists, Labs Lung Cancer Anapath The case of (mutated) EGFR Epithelial Growth Factor Receptor Lab Global cohort of treated patients Non responders Responders: 12-15% Integrated chain From the patient to the result Patient selection Patient selection Pneumologist / oncologist Pneumologist / oncologist Sampling Sampling DNA Extraction DNA Extraction Analysis Analysis What type of sample? Issues? Integrated chain From the patient to the result Pathologist Identification of the tumor zone Main issue Small tumor & small sample!!! A wide range of samples Surgical Tumor (rare if early stage...) Paraffin-Embedded-Formalin-Fixed Biopsy Cytology Unfixed Fixed in alcohol (cytospins) Embedded in paraffin Frozen specimens Bronchiolo Alveolar Lavage: Very few cells! However sometimes effective!!!! Slides are properly labelled (internal codification number) 3

Integrated chain From the patient to the result Patient selection Pneumologist / oncologist 1 Colored slide + Biopsies & FFPE 10 to 12 white slides (5µ) Surgical sample 6 White slides (5µ) Sampling DNA Extraction Molecular Analysis Pathologist Geneticist / Mol Biologist EGFR : Gene organization Mutated EGFR gene: impact on the EGFR protein! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Kinase domain Much stronger inhibition of phosphorylation in presence of TKI 26 27 28 TK domain www.egfr-info.com EGFR mutations in the kinase domain (exons 18 to 21)!! Acquired RESISTANCE mechanism!! T790M : change in the catalytic cleft of the EGFR TK domain Methionine substitution introduces a bulkier side chain, causing steric hindrance that is thought to interfere with binding of erlotinib * Mainly present in relapsed tumors after an initial response and secundary to EGFR-TKIs therapy * Accounts for ~50% acquired resistance to EGFR-TKIs L747S / D761Y / insertion exon 20 : Modest resistance Huang S et al. Clin Cancer Res 2004;10:8195-8203 2004 by American Association for Cancer Research Zhang and Chang 2008. Int J Med Sci 4

Molecular methods? What are the molecular methods used to identify EGFR mutations in NSCLC? Sequencing Gold-Standard, complete BUT tedious, time consuming, not very sensitive PCR-RFLP RFLP + GeneScan Applied only on well identified mutations, tedious, costly Real-Time PCR Rapid and sensitive but only on well identified mutations SNP assay (pyrosequencing?, microarrays, ) Not yet published.but also on well identified mutations TheraScreen Assay - Commercialized...but only on well identified mutations - not flexible for techno improvement - not attractive in academic R&D laboratories What is our current EGFR workflow? What is our current EGFR workflow? Pre-screening 5

Protocol Scraping Visual slide quality assessment Scraping : + overnight proteinase K DNA extraction DNA quantification HRM on the LightCycler 480 Real-Time PCR System Purification (positive or doubtful HRM) Sequencing Manually DNA extraction Automated DNA quantification Use of the NucleoSpin Tissue Kits (Macherey-Nagel) In progress : comparative assays for an automated extraction via the EZ1 Biorobot (Qiagen) Nanodrop ND-1000 In the daily practice : fast screening! Primers Exon 18 DNA High throughput Screening phase (High Resolution Melting or HRM) WT: ~90% Mutated: ~10% Direct sequencing Do et al., BMC Cancer 2008, 8:142-155 Fragment Size : 199 pb 163921 agcccatgcc gtggctgctg gtccccctgc tgggccatgt ctggcactgc tttccagcat ggtgagggct gaggtgaccc ttgtctctgt gttcttgtcc cccccagctt gtggagcctc ttacacccag tggagaagct cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat caaagtgctg ggctccggtg cgttcggcac ggtgtataag gtaaggtccc tggcacaggc ctctgggctg ggccgcaggg cctctcatgg tctggtgggg agcccagagt Mutation Identification 6

Primers Exon 19 Heideman et al., Cellular Oncology 2009, 31 : 329-333. Primers Exon 20p Heideman et al., Cellular Oncology 2009, 31 : 329-333. Fragment Size : 146 pb. 164701 cggctccaca gccccagtgt ccctcacctt cggggtgcat cgctggtaac atccacccag atcactgggc agcatgtggc accatctcac aattgccagt taacgtcttc cttctctctc tgtcataggg actctggatc ccagaaggtg agaaagttaa aattcccgtc gctatcaagg aattaagaga agcaacatct ccgaaagcca acaaggaaat cctcgatgtg agtttctgct ttgctgtgtg ggggtccatg gctctgaacc tcaggcccac cttttctcat gtctggcagc tgctctgctc tagaccctgc tcatctccac atcctaaatg ttcactttct atgtctttcc Fragment Size : 122 pb. 171361 catgcgaagc cacactgacg tgcctctccc tccctccagg aagcctacgt gatggccagc gtggacaacc cccacgtgtg ccgcctgctg ggcatctgcc tcacctccac cgtgcaactc atcacgcagc tcatgccctt cggctgcctc ctggactatg tccgggaaca caaagacaat attggctccc agtacctgct caactggtgt gtgcagatcg caaaggtaat cagggaaggg agatacgggg aggggagata aggagccagg atcctcacat gcggtctgcg ctcctgggat agcaagagtt tgccatgggg atatgtgtgt gcgtgcatgc agcacacaca cattccttta Primers Exon 20d Heideman et al., Cellular Oncology 2009, 31 : 329-333. Primers Exon 21 Adapted from Heideman et al., Cellular Oncology 2009, 31 : 329-333. Fragment Size : 97 pb. 171361 catgcgaagc cacactgacg tgcctctccc tccctccagg aagcctacgt gatggccagc gtggacaacc cccacgtgtg ccgcctgctg ggcatctgcc tcacctccac cgtgcagctc atcacgcagc tcatgccctt cggctgcctc ctggactatg tccgggaaca caaagacaat attggctccc agtacctgct caactggtgt gtgcagatcg caaaggtaat cagggaaggg agatacgggg aggggagata aggagccagg atcctcacat gcggtctgcg ctcctgggat agcaagagtt tgccatgggg atatgtgtgt gcgtgcatgc agcacacaca cattccttta Fragment Size : 199 pb 181681 tggatcagta gtcactaacg ttcgccagcc ataagtcctc gacgtggaga ggctcagagc ctggcatgaa catgaccctg aattcggatg cagagcttct tcccatgatg atctgtccct cacagcaggg tcttctctgt ttcagggcat gaactacttg gaggaccgtc gcttggtgca ccgcgacctg gcagccagga acgtactggt gaaaacaccg cagcatgtca agatcacaga ttttgggctg gccaaactgc tgggtgcgga agagaaagaa taccatgcag aaggaggcaa agtaaggagg tggctttagg tcagccagca ttttcctgac accagggacc aggctgcctt cccactagct gtattgttta acacatgcag gggaggatgc tctccagaca ttctgggtga HRM protocole Primers : 0,15 µm - MgCl2 : 2,5 mm DNA : 10 to 15 ng Activation : 95 C 10 Amplification (50 cycles) : 95 C 10 65 C 10 : Touchdown 1 /cycle the first 10 cycles - 55 C for the last 40 cycles 72 C 30 : recording of the data HRM : 97 C 1 40 C 1 70 C 1 95 C : Continuous Aquisitions : 25. Thus 25 acquisition/ C from 70 to 95 C Cooling : 40 C 30 Results (05.12.2010) Comparison HRM-sequence analysis (validation carried out on at least 40 samples) 58 % Insufficent DNA quality or quantity: no HRM nor sequence analysis! 4 DNA amount insufficient for sequencing but sufficient for HRM 8 Results comparison HRM and sequence analysis of exons 18 to 21 46 Informative rate = 79.3 Discrepant results after HRM and sequence analysis 0 Concordance = 100.0 Total number of samples received since 5/02/2010 243 Insuffisant DNA quality or quantity for HRM 26 HRM analysis (followed by selective sequence analysis of the "positive samples") 217 Informative rate = 89.3 a. HRM-negative samples 189 b. HRM-positive samples 28 % HRM-positive = 12.9 * Mutated samples % Mutated samples 8.8 19 = exon 18 E709K; G719C 1 exon 19 del E746-A750 6 exon 19 del L747-S752; P753S 1 exon 19 del L747-E749; A750P 1 exon 19 del L747-S755 InsTRD 1 exon 20 R776G et exon 21 L861Q (double mutation) 1 exon 21 V851I 1 exon 21 L858R 6 exon 21 L861Q 1 Samples with HRM-detectable polymorphism * 9 % Irrelevant SNP = 4.1 7

Identification of EGFR Factors impacting on the success rate? Patients selection influence /Geographical influence: Comparison (05.12.2010) Patient selection? Quality of the pre-analytical step? (quality/quantity sample DNA) Analytic step (methodology) North South Lux TOTAL Nb samples (n=243) 55 120 68 Unsatisfactory (test failure) 7 9 10 HRM analysis Informative Rate 48 87,27 111 92,50 58 85,29 a. HRM-negative samples 39 101 49 Ana path Lab b. HRM-positive samples 9 10 9 * Mutated samples % Mutated Samples 7 14,58 7 6,31 5 8,62 * Polymorphism % Irrelevant SNP 2 4,17 3 2,70 4 6,90 If St Luc only (n=33): Informative Rate: 97% - mutation rate 12% Identification of EGFR Factors impacting on the success rate? Influence of the analytical method Therascreen versus HRM (Preliminary data 05.12.2010) Patient selection? Quality of the pre-analytical step? (quality/quantity sample DNA) Analytic step (methodology) Ana path Lab Slides from the tumor Same DNA sample Sample HRM-Seq Therascreen 1 WT WT 2 WT WT 3 pos, L858R No Sample 4 WT WT 5 WT WT 6 WT WT 7 WT WT 8 pos, del 747-752, mut P753S exon 19 pos, del exon 19 9 WT WT 10 WT WT 11 onvoldoende DNA onvoldoende DNA 12 WT WT 13 WT WT 14 onvoldoende DNA WT 15 pos, del 746-750 exon 19 pos, del exon 19 16 WT WT 17 WT WT 18 WT WT 19 HRM pos Exon 21 (seq in progress) pos, L858R, exon 21 20 WT WT 21 WT WT 22 WT WT 23 WT WT 24 WT WT 25 HRM pos Exon 20p (seq in progress) pos, Ins exon 20 26 WT WT 27 WT WT 28 WT WT 29 WT WT 30 WT WT 31 HRM pos Exon 21 (seq in progress) WT 32 WT WT 33 WT WT Data from : Jessa Hospital Clinical Laboratory for Molecular Diagnostics Hasselt TheraScreen Allow to detect the more prevalent mutations (described until now) : 28 activating mutations 1 resistance mutation (T790M). List: 19 Del exon 19 T790M L858R L861Q G719X S768I 3 Ins exon 20 Based on a real-time PCR with the ARMS (Amplification refractory mutation system) and Scorpions technologies Sensitivity: 1% BUT limited to already well identified mutations!! Costs HRM (1/3) based on a cohort of 200 patients/year LC480 HMR Master (5*1mL) HTVA 480 euros Per well 10 µl Per well HTVA Analysis in triplicat Exons 18 to 21 (2 for 20) 6 controls (3- / 3+) 4 patients / series 0.96 euros HTVA 3 wells/amplicon & patients 5 amplicons per patients 90 wells/experiment 90*0.96 = 86.4 euros For one patient 86.4 / 4 = 21.6 HTVA Updated from data of Dr El Housni (Erasme) 8

Costs HRM (2/3) based on a cohort of 200 patients/year Plates (50/box) HTVA One plate For one patient 370 euros 7.4 euros Primers HTVA / synthesis ~26 euros Life Time : 3 years For one primer & 1 patient One analysis 7.4 / 4 = 1.85 HTVA 600 patients 0.043 euros HTVA 2 primers / 5 amplicons Costs HRM (3/3) based on a cohort of 200 patients/year Sequencing Expected cases 10% Patients a year 200 Sequences 200*2/10 = 40 Patients in triplicate Price for 1 seq 40*3 = 120 seq 5 euros HTVA Price for seq in 1 year 600 euros HTVA For one patient (distributed on all patients) 3 HTVA For one patient 0.43 HTVA TOTAL HRM For one patient 28.9 TVA HRM + Sequencing included For a mutated sample 32 TVA Updated from data of Dr El Housni (Erasme) Updated from data of Dr El Housni (Erasme) Costs Therascreen Turn around time HRM analysis Limited component = Mix STD One kit : 500 µl - 5 µl for one well - 40 µl for one plate MAX 12.5 plates for one kit Number of patients / plate : 10 Price of 1 kit : 12.960 euros HTVA 15.681 TVA Price for 1 plate : 1206,3 TVA 10 patients / plate : 120,6 TVA / pt 5 patients / plate : 241,3 TVA 3 patients / plate : 402,1 TVA 2 patients / plate : 603,1 TVA 1 patient / plate : 1206,3 TVA Tu p.m.: We a.m.: We p.m.: Th a/p.m.: Fr a.m.: Scraping of the tumour zone DNA extraction (first part) DNA extraction (final steps) DNA quantification (Nanodrop) HRM analysis STOP for wild-type samples (~75%): 1.5 day DNA purification Cycle sequencing analysis Sequence analysis Report for suspected mutated samples: 3 days Start WT? Mut Updated from data of Dr El Housni (Erasme) Mo Tu We Th Fr Sa So Mo Tu We Th Fr Sa So Mo Tu Thanks for your attention Bonnes fêtes!! Prettige Feesten!! 9