Molecular Pathology and Lung Cancer A. John Iafrate MD-PhD Department of Pathology Massachusetts General Hospital Boston, MA aiafrate@partners.org
Disclosures Preliminary patent application NGS AMP Fusion (Iafrate, Zheng, Le) ArcherDx: Equity holder, Consultant, Royalties SAB/Consultant: Roche, Chugai, Constellation, DebioPharm, Pfizer
Genetic Alterations in Adenocarcinoma Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing, Cell 2012
Mutational Profiling in Lung Adenocarcinoma ROS1 1.5% BRAF 2% HER2 2% CTNNB1 2% ALK 3% PIK3CA 4% TP53 5% AKT 1% NRAS 1% RET 1% IDH1 <1% No Mutation ~40% EGFR 15% KRAS 23%
Mutation Frequencies in Common Cancers Cancer Genome Group, Broad Institute
FDA approved Companion Diagnostics in NSCLC Diagnosis Mutation/Target Drug Diagnotic NSCLC EGFR Erlotinib Gefitinib Afatinib Roche Cobas Qiagen Therascreen Qiagen Therascreen NSCLC EGFR T790M Osimertinib Roche Cobas V2 NSCLC ALK Crizotinib Alectinib Ceritinib NSCLC ROS1 Crizotinib MGH FISH* Abbott FISH; Ventana IHC D5F3 FDA-Approved* FDA-Approved* NSCLC PD1 Pembrolizumab PDL1 IHC (DAKO) Source: FDA website
NGS Based Snapshot Panel Anchored Multiplex PCRbased ~190 target amplicons across 40 genes High-quality sequence: - Staggered start sites - 1000X median coverage Molecular indexing - Bi-template coverage ~2% analytical sensitivity Fast turn-around (<2 weeks) Cost-effective (<$200 reagents) Small tissue amounts (5-10
NGS based Profiling in Lung Adenocarcinoma >85% of samples with detectable driver mutation
EGFR Transmembrane receptor tyrosine kinase involved in epithelial development and function Kinase domain mutations in lung ACA linked with non-smoking and response to EGFR TKIs (2004) Resistance to approved TKIs current focus of R&D Zhou and Yao, JTO 2016
EGFR kinase domains inhibitors: clinical response in 10% of patients Pre-treatment Gefitinib (Iressa) Erlotinib (Tarceva) 3 months post-iressa Nakagawa K et al., Ann Oncol. 2003 Jun;14(6):922-30.
High Response Rate to First Line Anti-EGFR Therapy in Advanced EGFR-mutant Lung Adenocarcinoma Sequist et al., J Clin Oncol. 2008 May 20;26(15):2442-9
EGFR and Iressa Pan-Asia Study (IPASS) Mok et al., NEJM 2009
EGFR and Iressa Pan-Asia Study (IPASS) Mok et al., NEJM 2009
KRAS Sequencing: What Drugs Not to Use 21-kDa GTPase involved in signal transduction in MAPK pathway EGFR Mutations are common in human cancer including in: 90% pancreatic ca, 50% colon ca, 30% lung NSCLC Mutations in KRAS are downstream of EGFR; such tumors would not respond to anti-egfr therapy
Mechanisms of acquired drug resistance NSCLC 37 patients w/ EGFR-mutant tumors responded to EGFR inhibitors and later relapsed Sequist et al., Science Translational Medicine, 2011
EGFR T790M mutation is associated with EGFR TKI resistanc The T790M mutation causes steric hindrance that prevents TKI binding Mutations are common cause of acquired TKI resistance Following mutations in re-biopsy or cfdna can help guide therapeutic decisions
T790M Specific Inhibitors Osimertinib (in development Rociletinib) Prior EGFR TKI FDA cleared assay, T790M positive
MET amplification mediates EGFR TKI resistance even in presence of EGFR sensitizing mutations
NSCLC Case 1 Therapy erlotinib resistance to therapy 1/30/08 3/31/08 (RUL 6.8x4.5 cm) (RUL 2.6x1.8 cm) response to therapy tumor mass reduced by >50% 2/25/09 (RML 2 cm) new nodules developed consistent with recurrence
NSCLC Case 1 Molecular Testing Pre-Rx 08 Resistant 09 Therapy Rx on clinical trial FISH analysis: MET gene amplification MET+EGFR inhibitor Engelman et al., Science, 2007 therapy Bean et al., PNAS, 2007 response to therapy tumor mass reduced by ~30%
MET-amplified NSCLC (<1% prevalence): Crizotinib response Day 7 Day 25 Dr. Ignatius Ou
Crizotinib : Benefits of Real-time Screening Crizotinib: Potent & selective ATP competitive oral inhibitor of MET, ALK, and ROS1 kinases and their oncogenic variants Telomere 2p23 region Centromere 3 5 t(2;5) ALK gene breakpoint region ~250 kb ~300 kb
ALK FISH in NSCLC Case 1 Telomere 2p23 region Centromere ALK 29.3 EML4 42.3 3 5 t(2;5) ALK gene breakpoint region ~250 kb ~300 kb WT (non-split) signal Split signal
ALK IHC Comparison in NSCLC Mino-Kenudson et al., CCR 2010
ALK rearrangement Histology MGH experience Signet Ring Cells N % None detected 38 32% < 10% 43 36% > 10% 39 32% Nishino M, Mark EG, Iafrate AJ, Mino-Kenudson M
Tumor Responses to Crizotinib by Patient Decrease or increase from baseline (%) 60 50 40 20 20 10 0 20 40 40 70 60 100 80 100 30% reduction PD SD PR CR Best Percent Change in Tumor Size (n=105 evaluable patients) Camidge R et al. Poster 366 presented at the 35 th ESMO, 2010
Timeline for PF2341066 and ALK in NSCLC PF2341066 Inhibits ALK activity PF2341066 FIP May PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH- McDermott) Phase III study of Crizotinib in ALK positive NSCLC starts 2005 2006 2007 2008 2009 2010 2011 Identification of PF2341066 PF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house) Discovery of EML4- ALK fusions in NSCLC (CREST) Japan Science & Technology Agency) Objective responses demonstrated in ALK fusion positive NSCLC and IMT FDA Approval of Crizotinib in ALK positive NSCLC
ALK inhibitor resistance
2 nd Generation ALK Inhibitors: Alectinib
Surprising Re sensitization to Crizotinib
ROS1 rearrangement in NSCLC
ROS1 rearrangement in NSCLC
Clinical Characteristics of ROS1 rearranged NSCLC Age, median Smoking All Patients ROS1 (n=18) ALK (n=31) ROS1 NEG (n=1055) P value No. % No. % No. % No. % ROS1 vs. NEG 62 49.8 51.6 62.3 <.001 Never 239 22% 14 78% 13 42% 225 21% <.001 Light 62 6% 1 6% 1 3% 61 6% Smoker 695 65% 2 11% 3 10% 693 66% NA 77 7% 1 6% 14 45% 76 7%
ROS1-translocated NSCLC (1.5% prevalence): Crizotinib response
Summary of Tumor Responses in Evaluable Patients with Advanced ROS1+ + NSCLC* Best change from baseline (%) 100 80 60 40 20 0 20 40 60 7 43+ 24 9 25+ 8+ 15+ 28+ 10+ 36+ Best overall response 12 PD SD PR CR Overall response rate = 50% n=20 evaluable patients; 1 CR and 9 PRs Disease control rate = 70% at 8 weeks 45+ 18 44+ 16+ 80 7+ 100 44+ 54+ 80+
Actionable Rearrangements in LUNG Tumors Gene Pathway Prevalence NTRK1,2,3 MAPK <1% FGFR1,2,3 MAPK <1% BRAF MAPK <1% ALK MAPK 3 5% ROS1 MAPK 1.5% RET MAPK 1.5% PRKACA camp <1% MET MAPK 3 5% PRKCA camp <1% ERBB4 MAPK <1% PRKCB camp <1% NRG MAPK <1%
Detecting Rearrangements Immunohistochemistry Antigen expression in tumor but not in normal Ab availability Ab specificity Qualitative analysis FISH Technically challenging Expert analysis Poor scalability and multiplexing ability RT PCR Requires knowledge of both partners Limited scalablity and multiplexing ability
Anchored Multiplex PCR (AMP) Zheng et al. Nat Med 2014
Assay Design for ROS1 Translocation
Solid Tumor Fusion Assay (50 genes) ADCK4 AKT3 ALK AXL B2M BRAF BRD4 CCDC6 CD74 CHTOP CTBP1 EGFR ERBB2/4 EWSR1 FGFR1 FGFR2 FGFR3 GAPDH INSR INSRR JAK1 JAK2 MAST1 MAST2 MET MUSK NFIB NOTCH1 NOTCH2 NRG1 NTRK1 NTRK2 NTRK3 NUMBL PDGFB PDGFRA PPARG PRKACA RAF1 RET RHOA ROS1 TMPRSS2
Solid Fusion Assay ALK 12 ANK3 1 BRAF 2 EGFR 7 EWSR1 1 FGFR2 12 FGFR3 2 FGR 2 JAK2 1 MAML2 4 MAST1 2 MET 18 NFIB 2 NOTCH1 1 NRG1 1 NTRK1 2 NTRK3 2 PRKACA 2 PRKCB 1 RET 21 RNF26 1 ROS1 12 ZFPM2 1 Positive 110 Negative 1118
Met exon 14 skipping: RNA or DNA?
Case Genomic DNA alteration Deletion in 3 splice site of intron 1 13 (32 bp deletion) Point mutation in 5 splice site of 2 intron 14 (G>A) SNaPShot Age Gender Stage Smoking Status wt 79 wt 65 Female IB Female IV Never Never Histology ADC ADC 3 Not available Point mutation in 5 splice site of 4 intron 14 (G>A) Deletion in 3 splice site of intron 5 13 ( (12 bp deletion) Deletion in 3 splice site of intron 6 13 (14 bp deletion) Deletion in 3 splice site of intron 7 13 (27 bp deletion) Deletion in 3 splice site of intron 8 13 (24 bp deletion) Deletion in 3 splice site of intron 9 13 (10 bp deletion) Point mutation in 5 splice site of 10 intron 14 (G>A) Deletion in 3 splice site of intron 11 13 (32 bp deletion) wt 73 wt 84 PI3K CTNNB 48 wt 44 wt 78 wt 75 wt 68 wt 69 wt 81 Female IIB Never SQC Female IA Never ADC Female IIA Never ADC Male IV Never SQC Female IB Never SARC Female IA Never ADC Female IV Former ADC Male IIB Never ADC Female IV Never ADC
Met Exon 14 Skipping: Crizotinib Response 74 yo Male non-smoker Stage IV adenocarcinoma 3/18/2015 4/19/2015 Failed multiple lines of chemotherapy AMP detected MET exon 14 skipping Crizotinib Heist et al., in press.
45 yo Male 30py smoker Stage IV adenocarcinoma Failed multiple lines of chemotherapy AMP detected NTRK1 fusion Clinical trial of entrectinib NTRK Fusions in NSCLC
CD74 NRG1 in Mucinous ADC Fernandez-Cuesta L et al, Cancer Discovery 2014
Mucinous Adenocarcinoma & KRAS J Mol Diagn 2007;9:320-6.
Mucinous Adenocarcinoma & KRAS 90% KRAS All smokers J Mol Diagn 2007;9:320-6.
KRAS mutation profiling Clin Cancer Res 2014;20:3921-3
TP53 mutation 535C>G H179D Reference: 939 Variant: 168 CD74-NRG1 fusion + TP53m Of 50 cases (30 NGS and 20 SNaPShot), only one case had TP53 mutation. In TCGA, of 230 cases, 105 (46%) cases have TP53 mutation.
IMA in TCGA Mucinous (n=12) Non mucinous (n=218) TP53 mutant 1 104 TP53 wild 11 114 P=0.008
Palmetto MAC MolDx Program NSCLC comprehensive genomic profiling policy (81445, 81455) Patient has been diagnosed with advanced (Stage IIIB or IV) NSCLC Patient is a lifetime non-smoker or former light smoker with <15 pack year history of smoking Patient previously tested negative for EGFR, ALK, and ROS1 through non-cgp methods Testing is performed by a lab that satisfies Palmetto GBA s published AV criteria.
NGS MAC NGS Program NSCLC panel policy proposal for 81445 made with input from 5 academic medical centers Newly diagnosed NSCLC patients with advanced (stage IIIB or IV) NSCLC, who are not treatable by resection or radiation with curative intent, and who are suitable candidates for therapy at the time of testing. No smoking related rules No comment on prior testing No test quality standards
Summary Targeted therapies are a part of routine lung cancer management. Managing resistance is the next frontier. No test is perfect FISH, IHC and NGS all have challenges. The best test choice is a mix of performance, TAT, cost and comprehensiveness. NGS panel testing is replacing other molecular assays in mutation/fusion detection in the clinical lab.
Characteristics of Lung Cancer Small cell carcinoma Squamous carcinoma >98% smokers Adenocarcinoma >75% smokers Nat Rev Cancer 2007;7:778-90. Nat Genet 2012;44:1074-5
Genetic Alterations in Squamous Cell Carcinoma Comprehensive genomic characterization of squamous cell lung cancers, Nature 2012
Genetic Alterations in Squamous Cell Carcinoma Copy Number Alterations Comprehensive genomic characterization of squamous cell lung cancers, Nature 2012
Genetic Alterations in Small Cell Carcinoma Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer, Nature Gen. 2012
MiSeq Rearrangement Assay Validation
TTF1 (aka Nkx2.1) 38-kDa transcription factor normally expressed in adult thyroid and lung tissue and is essential in lung development Positive by IHC in >75% of nonmucinous lung adenocarcinomas Most common focal amplification in genome-wide analysis TTF1 may have a role in pathogenesis