EGFR. Pathway and biomarkers. Alex Soltermann

Transcription

1 EGFR Pathway and biomarkers Alex Soltermann

2 EGFR = HER1 signaling pathway EGFR Cheng Mod Pathol 2012 Chromosome 7p11.2, spans 200kb, 28 exons, 464 aa, 170 kda protein 2

3 Signal transduction pathways controlled by the activation of EGFR Ciardello N Engl J Med 2008 Oncogenic addiction EGFR/HER1 HER2 HER3 HER4 3

4 Mechanisms of acquired resistance to EGFR TKIs Mitsudomi Cancer Sci 2007 MET (Hepatocyte growth factor receptor) amplification leads to gefitinib resistance by HER3-dependent PI3K activation (Engelman, Science 2007) MET reduces susceptibility to irreversible EGFR inhibitor in EGFR-T790M mutant lung cancer (Yamada, Clin Cancer Res 2010) Oncogenic shift 4

5 Global, in-vivo, site-specific phosphorylation dynamics in signaling networks Olsen Cell

6 Drug sensivity and resistance mutations Cheng Mod Pathol

7 Frequency of EGFR mutation 1. REASON, German register for epidemiology and evaluation of the EGFR status in newly diagnosed patients with NSCLC stage 3b/4, P. Schirmacher, Pathology Heidelberg, DGP meeting: EGFR Mut PT 9%, LN-Mets 10.4%, Distant Organ-Mets 17% 2 Exone 19 und 21 7%, 3-4 Exone 11.1% 2. C. Zhou, Department of Oncology, Shanghai Pulmonary Hospital, USZ meeting: EGFR mut 28%, KRAS mut 5.2% 3. D. Zimmermann, Molecular Pathology USZ 73/345 (21%) mutated Pao Nature Med

8 EGFR on TBB How much DNA needed per exon: 2, 5, 10, 50 or ng? F. Herth, Pneumology Heidelberg: 21g needle 2 500, 19g needle 3 600, cell block 4 000, 14g needle 6 000, TBB , Cryobiopsy cells? Representative tumor cells? 8

9 EGFR-Mutation: > 50% tumor cells/surface >50% Tu on core surface >50% Tu cell block surface Cell block: 100 µl sediment + plasma and thrombin p.l858r exon 21 p.g719d exon 18 PCR + Sanger sequencing: 25% mutated allele. Pyro: 10% Goal: Parallel <10% mut allele 9

10 Cutting record lung biopsy, LCM on cytology micrometer micrometer 1. step, 7 series (HE, EVG, AB-PAS, 4 LS) 2. step, 7 series (HE, EVG, AB-PAS, 4 LS) 3. step, 7 series (HE, EVG, AB-PAS, 4 LS) 4 micrometer cut for ALK-FISH punch core for Mol Path 21 surfaces EGFR mutation/fish on Papanicolaoustained cytologic smears by laser microdissection with pressure catapulting system (Savic Br J Cancer 2007)

11 Diagnostic workup Switzerland 2012 Biopsy/Cytology Squamous Cell Ca Adenocarcinoma NSCLC-NOS Stage 3/4 No further examination Reflex Testing: If EGFR negative, automatically ALK Goal 2013 EGFR / KRAS (mutation analysis) negative ALK (FISH) Adeno phenotype TTF1+, p63- negative Squamous phenotype p63+, TTF1- No further examination EGFR, KRAS, BRAF, HER2 mut in parallel ALK IHC following FISH BRAF/HER2-neu (mutation analysis) (not established)

12 Histosubtypes of lung adenocarcinoma 1. Lepidic 2. Acinar 3. Papillary 4. Micropapillary 5. Solid Travis J Thorac Oncol 2011 IASLC/ATS/ERS consensus 12

13 EGFR mutations among adeno-ca subtype Travis J Thorac Oncol 2011 IASLC/ATS/ERS consensus 13

14 TRU, terminal respiratory unit Yatabe Pathology International 2007 TRU is composed of alveolar cells, Clara cells, and non-ciliated bronchioloalveolar epithelium, expressing TTF-1 = NKX2 and surfactant 14

15 Intratumoral heterogeneity of EGFR mut Sun J Thorac Oncol 2012, Korea EGFR mut: Mixed acinar/lepidic non-mucinous 68%, mixed papillary/acinar 65%, mixed solid/ acinar 38%, micropapillary/acinar 30%, acinar/ lepidic mucinous 13%, esp in TTF-1+. No intratumoral heterogeneity among 2-3 areas Yatabe, USCAP 2011, Japan No heterogeneity among 100 areas Chen Oncologist 2012 Primary lesions detected at different times, PT with matched metastatic lymph nodes, multiple pulmonary nodules, PT with matched distant metastases. Multiple pulmonary nodules group has the highest discordance rate of 24% Petrausch MEMO 2012 Vincenten J Thorac Oncol 2012 TTF-1 has a high negative predictive value of >95% for EGFR mut (surrogate for chemo) 15

16 Key candidate genes lung squamous cell carcinoma KRAS mut 1%, HRAS und NRAS mut ev. <10% BRAF mut 4% FGFRs 20% amp, 12% mut (in Zürich FGFR1 amp 20%) DDR2 mut 4% PI3K-CA amp 20%, mut 6% SOX2 amp 20% MDM2 amp 10% MET amp 6% PDGFRA amp 8% p53 mut 65% NRF2/KEAP mut 12% PTEN mut 10%, protein loss 70% EPHA2 mut 7% HER2 mut 3%, HER2 amp <5% LKB1 mut 5% AKT mut 5% EGFRvIII mut: in-frame deletion of exon 2 to 7 Corr with increased CN No EGF binding Insensitive to erlotinib/gefitinib but sensitive to neratinib EGFR amp 7%, EGFR mut like L858R rare, EGFRvIII mut 5%, EGFR protein 84% Hammerman, AACR 2012; Heist, J Thorac Oncol

17 Therapy strategy TP53, RB1 (all) CREBBP and PTEN mut FGFR1 amp No EGFR mut Peifer, Nat Genet 2012 FGFR1 Pelosi Lung Cancer

18 Sarcomatoid lung carcinoma Pelosi Lung Cancer

19 EGFR and other alterations 1. Double alterations 68 frozen Zürich NSCLC samples sent for WGS: 8 EGFR mutations, 5 together with p53 mut, 1 together with PI3K-CA mut Concurrent EGFR exon 19 deletion plus ALK rearrangement (5 loss) responding to erlotinib (Popat J Thorac Oncol 2012). ALK IHC neg. 19

20 EGFR and other alterations 2. Correlation with ERCC1 from NER (nucleotide excision repair) and with TS (thymidylate synthase) EGFR mut corr with low ERCC1 and low TS, ALK alt corr with low TS (Zhou, pers. commun) ERCC1 low in high pt, larger size (Arbogast Appl Immunohistochem Mol Morphol 2010) 3. Correlation with epithelial-mesenchymal transition EMT inversely corr with EGFR TKI response: epithelial 59% versus mesenchymal 35.7% (Zhou, pers. comm, Fuchs Cancer Res 2008) High cytosolic vimentin and periostin corr with high grade and high pt, larger size, respectively (Soltermann Clin Cancer Res 2008) 20

21 EGFR FISH and SISH Silver-enhanced in situ hybridization (Wulf Am J Surg Pathol 2012) Identical results between FISH and SISH in 114/116 pats in 2 cohorts Cappuzzo/Colorado 4 ( 4 signals in 10% to 40% cells) versus 5 critical Preferential amplification of the mutated gene due to mutant allele-specific imbalances (MASI) High EGFR CN on FISH corr with Response to gefitinib (Cappuzzo, JCO 2007, Hirsch JCO 2005) and to cetuximab, panitumumab in ColonCa (Sartore-Bianchi, JCO 2007) 21

22 EGFR total protein and cetuximab H-Score Intensity 0,1,2 or 3 multiplied by frequency of stained cells (0, 10 for 1-10%, 50 for 11-50% and 100 for % positive cells Range 0 to 300, 180 used as cut-off No correlation with survival on Zurich TMA 22

23 Antibodies against EGFR mut and phospho-egfr L858R? Tyr1173 Ab against most common EGFR mutations: E746_A750 del in exon 19 and L858R in exon 21 Ab against phospho-egfr: Tyr992, Tyr1045, Tyr1068, Tyr1148, Tyr

24 Conclusions 1. EGFR mutation rate in Europe around 15%, in Asia higher. Intratumoral heterogeneity may not be a major issue 2. Multiplex package of EGFR, KRAS, BRAF, HER2 mutation plus ALK IHC and FISH most suitable strategy for Amount and preservation of tumor tissue, copy number of mutant alleles, imbalanced PCR amplification and relative number of contaminating wild-type alleles influence the sensitivity of detection 24