Disclosures Genomic testing in lung cancer No disclosures Objectives Understand how FISH and NGS provide complementary data for the evaluation of lung cancer Recognize the challenges of performing testing on very small specimens Describe how genomic testing is used to choose lung cancer therapy UMMC lung cancer panel ALK and ROS1 by FISH NGS BRAF EGFR ERBB2 HRAS JAK2 KRAS MET NRAS PIK3CA Sample type Can be performed on: FFPE specimens Cytologic specimens (i.e., FFPE cell blocks) Must have minimum 20% tumor involvement What does laboratory receive? One H & E (can be shared between MDL and Cytogenetics) Ten 10-micron thick unstained recuts Four 3-5 micron thick unstained recuts on positively charged slides Positively charged is critical otherwise will lose tissue during processing for FISH Multiple cuts: Increases quantity of DNA obtained from tissue Permits reset of probes if initial hybridization suboptimal 1
What happens to those slides? H&E examined in MDL Tumor percentage estimated Tumor marked on H&E if macrodissection is needed; otherwise entire specimen scraped off for DNA extraction H&E examined in Cytogenetics Tumor marked Slides processed for FISH; technologists find corresponding area on FISH slide 1950s - Histologic subtypes Embedded Tissue Biopsy Microtome Small cell carcinoma (~10-15%) Non-small cell carcinoma (NSCLC) (~85-90%) Adenocarcinoma Squamous cell carcinoma Large cell carcinoma FISH Signal Pattern on Thin Section 2
Major histologic classifications (2004 WHO and 2012 IASLC/ATS/ERS) Distinct morphologic patterns Papillary Acinar Solid Lepidic Micropapillary Morphologic classification 3 major subgroups by gene expression profiling Nonmucinous bronchioloalveloar ca Mixture of patterns incl lepidic Purely invasive with no lepidic Association of morphologic subtypes and mutation status is imperfect both are needed for full classification Why is histologic subtyping important? 1990-2000s: All pts with NSCLC received essentially the same treatment Overall survival 33% at 1 year, 11% at 2 years New therapy regimens like Elvis sightings they frequently created excitement but usually did not last long Relationship between histology and therapeutic response 2004: NSCLC with adenocarcinoma component Pts with EGFR mutations may respond to gefitinib 2008: SCC: Cisplatin plus gemcitabine Adenocarcinoma: Cisplatin plus pemetrexed Ding L et al, 2008, Nature 455:1069 1013 nonsynonymous mutations (point mutations, insertions/deletions) 26 high-frequency mutation genes 3p deleted early in carcinogenesis Weir BA et al, 2007, Nature 450:893 26 large-scale gains/losses CDKN2A often deleted 3
Weir BA et al, 2007, Nature 450:893 31 small-scale gains/losses NKX2-1 (TTF1) transcription factor involved in lung development Most common genomic abnormalities in lung adenocarcinoma Aberration Mutations Fusions Copy number alterations TP53, KRAS, EGFR, BRAF, MET ALK, ROS1, RET Gains: NKX2-1, TERT, EGFR, MET, KRAS, ERBB2, MDM2 Losses: LRP1B, PTPRD, CDKN2A Pathway alterations RTK-RAS-RAF (~75%) MAPK and PI3K signaling JAK-STAT TP53 (60-65%) Cell cycle regulation (~65%) Devarakonda et al, 2015, Lancet Oncol 16:e342 Swanton C and Govindan R, 2016, N Engl J Med 374:19 Drug classes EGFR tyrosine kinase inhibitors Erlotinib Gefitinib Afatinib ALK inhibitors Crizotinib Ceritinib Median PFS 9-14 mos (5-7 mos for other therapies) EGFR (7p11.2) Encodes HER1 (ERBB1) member of epidermal growth factor receptor family Transmembrane tyrosine kinase receptor After it binds ligand, it dimerizes with other members of ERBB family (just like HER2) Upregulates signaling pathways: RAS/MAPK pathway PI3K/AKT pathway Cellular proliferation, decreased apoptosis Extracellular (ligand-binding) Transmembrane Intracellular (tyrosine kinase) (exons 18-25) EGFR EGFR abnormalities Mutation, amplification Both may be present heterogeneously within a tumor Clinical associations: Female, East Asian, never-smokers Well-differentiated adenoca Lepidic, papillary or acinar histology Brambilla E and Gazdar A, 2009, Eur Respir J 33:1485 4
EGFR mutations 3 major categories In-frame deletions of exon 19 (85-90% of cases) Insertions in exon 20 Associated with primary or acquired resistance to TKIs Missense mutations in exons 18-21 Most frequent: Exon 21 L858R Exon 21 c.2573t>g (Leu858Arg) EGFR targeted therapies Tyrosine kinase inhibitors Monoclonal Abs against extracellular ligand-binding domain E.g., cetuximab, panitumumab Small molecular tyrosine kinase inhibitors E.g., gefitinib, erlotinib Assoc. with increased response rate: Female, never smoker, Asian ethnicity, adenocarcinoma EGFR mutation-positive Better response to & longer progression-free survival (PFS) with gefitinib or erlotinib vs conventional chemo EGFR-mutation-NEGATIVE Better response to & longer PFS with conventional chemo Testing for mutation (rather than amplification) more predictive of TKI response 5
Resistance Most pts with EGFR mutations relapse in 10-14 mos due to drug resistance Repeat biopsies show acquisition of other additional mutations in EGFR (e.g., T790M) or other pathways (HER2, MET, PIK3CA) Median survival after development of resistance: 10-12 mos ALK (2p23) Rearranged in ~3-5% of tumors Clinical associations Younger, light or never smokers Advanced clinical stage ALK rearrangement Oncogenic EML4-ALK fusion results from inv(2)(p21p23) Variable fusion sites within EML4 and ALK Other translocation partners KIF5B TFG Subtle rearrangement requires breakapart probe FFDA-approved assay companion diagnostic Adjacent red and green signals <2 signal diameters apart or overlapping are considered ONE fused signal Diffuse signals adjacent or connected by fiber are considered ONE fused signal 6
POSITIVE POSITIVE 2 ALK signals, both rearranged 2 ALK signals, 1 rearranged 1 intact ALK signal, 1 rearranged signal with missing G (isolated 3 pattern) 1 intact ALK signal, 2 rearranged signals with 1 missing G NEGATIVE 1 intact signal, 1 diffuse signal 1 intact signal, 1 just slightly separated CLASSIFICATION Classification by FISH NUMBER OF CELLS Positive Negative Equivocal >25/50 (50%) with positive signal pattern <5/50 (10%) with positive signal pattern 5-25/50 (10-50%) with positive signal pattern 1 intact signal, 1 rearranged signal with missing R (isolated 5 pattern) EML4-ALK EML4-ALK 2-7% of NSCLC In adenoca Younger never-smokers with higher-stage dz Assoc with solid growth, frequent signet-ring cells with abundant intracellular mucin Mutually exclusive with EGFR and KRAS mutations in NSCLC EML4-ALK Constitutive activation of ALK tyrosine kinase activity Inhibited by ALK TKI: Crizotinib Partial response and median progressionfree survival ~10 mos Superior to standard chemo 7
Crizotinib resistance > 70% of ALK-rearranged NSCLC rx d with crizotinib relapse within first year Mechanisms Alterations within the specific gene Esp. L1196M and G1269A Activation of alternate oncogenic pathways Inadequate drug exposure (drug interactions, pharmacokinetic differences) Crizotinib resistance Bypass pathways KIT amplification Activating mutations in EGFR and KRAS IGF1 receptor pathway activation Resistance also with newer generation drugs ROS1 (6q22) rearrangements ~1-2% of NSCLC 9 different fusion partners thus far In adenocarcinoma Younger light or never-smokers of Asian ethnicity Often high-grade dz Mutually exclusive with ALK rearr Katayama R et al, 2015, Clin Cancer Res 21:2227 ROS1 fusion partners 3 5 Davies KD and Doebele RC, 2013, Clin Cancer Res 19:4040 8
ROS1 fusion partners ROS1 (6q22) rearrangements Upregulate MAPK pathway Sensitive to crizotinib or foretinib Acquired resistance has been reported (G2032R) Davies KD and Doebele RC, 2013, Clin Cancer Res 19:4040 BRAF BRAF D549N ~3-5% of lung adenocarcinoma V600E (~50% of mutations); also G469A and D594G Strongly associated with smoking history Micropapillary component Poor prognosis Clinical trials for BRAF inhibitors KRAS mutations Negative predictor of response to EGFR TKIs ~30% of lung adenoca in Western (non-asian) pts, Clinical associations Older, usually smokers (~2% nonsmokers) Male prevalence More aggressive tumors Associated with papillary or micropapillary histology; also high frequency in lepidic-pattern mucinous adenoca RET 10q11.2 Rearrangements in ~1-2% of NSCLC (esp. poorly-differentiated adenocarcinoma) Several different fusion partners Often: never- or light smokers; younger Cabozantinib 9
HER2 (ERBB2) Mutations in ~2% NSCLC In-frame insertion in Exon 20 (not amplification as in breast, GI ca) Assoc with: Adenocarcinoma Younger age Female East Asian ethnicity Never smokers Thomas A et al, 2015, Nat Rev Clin Oncol doi: 10.1038/nrclinonc.2015.90 10