Advances in Pathology and molecular biology of lung cancer. Lukas Bubendorf Pathologie
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1 Advances in Pathology and molecular biology of lung cancer Lukas Bubendorf Pathologie
2 Agenda The revolution of predictive markers Liquid biopsies PD-L1
3 Molecular subtypes (non-squamous NSCLC) Tsao AS et al., JTO 2016
4 AC / AC phenotype Stage IIIB/IV Mutation analysis EGFR KRAS HER2 BRAF NGS (Panel) Immunohistochemistry ALK ROS1 Negative Positive Positive 5 days 1 day ALK FISH Others: Resistance mutations PD-L1 IHC EGFR IHC MET amplification METex14 Negative Negative ROS FISH RET FISH Swiss Lung Pathology Group Recommendations 2014
5 2004 Bronchus (Biopsy): Infiltrates of an adenocarcinoma
6 2017 Bronchus (Biopsy): Infiltrates of a TTF1 positive adenocarcinoma Result of NGS mutation analysis using Oncomine Solid Tumor Panel (tumor cell proportion 90%): Predictive results: EGFR: no mutation KRAS: p.g12v (74%) BRAF: no mutation HER2: no mutation Other results: TP53: p.e294fs (69%), significance: unknown Result of predictive immunohistochemistry: ALK (5A4, Leica): negative ROS1 (D4D6, Cell Signaling): negative PD-L1 (22C3, Dako): Immune cells: <1% Tumor cells: 60%
7 2017 Lung (transthoracic biopsy): TTF-1 positive NSCC, consistent with AC Result of NGS mutation analysis using Oncomine Solid Tumor Panel (tumor cell proportion 90%): Predictive results : EGFR: no mutation KRAS: no mutation BRAF: no mutation HER2: no mutation Other results: TP53: p.q192_h193del (20%), significance: unknown Result of gene sequencing: MET (Exon 14 skipping Mutation): no mutation Result of Fluorescence in-situ Hybridization ALK: no rearrangement ROS1: no rearrangement Result of predictive IHC: ALK (5A4, Leica): negative ROS1 (D4D6, Cell Signaling): negative PD-L1 (22C3, Dako): Immune cells: <1% Tumor cells: 30% MET (SP44, Ventana): positive (moderate in 80%)
8 Managing tumor material for biomarker testing
9 ALK Immunohistochemistry Very high concordance with ALK FISH (90-100%) Screening for subsequent FISH analysis May replace FISH
10 Benchtop Sequencer for next-generation sequencing (NGS) MiSeq Personal Sequencer (Illumnia) Ion Proton Diagnostic biopsies (formalin-paraffin) 10ng DNA tumor cells 20% tumor cell content IonTorrent (ThermoFischer)
11 DNA cells DNA Extraction PCR Amplification Data analysis by trained people NGS Dr Luca Quagliata PD Dr. phil. Michel Bihl
12 Next generation sequencing - Why it concerns pathologists- Review material for suitability Estimating the percentage of tumor cells Understanding the principles Sign out of the report in conjunction with histology (integrated report) Collaboration with the molecular lab
13 Acquired resistance in NSCLC ALK TKI EGFR TKI T790M Doebele et al, Clin Cancer Res 2012 Cortot & Jänne, Eur Respir Rev 2014
14 Months: p53 p.g245v EGFR Exon 19 p.e746-t751delinsf KRAS p.g13d EGFR p.t790m EGFR p.c797s Therapy TKI-1 Erlotinib TKI-2 Afatinib Pem/ Cis Nivolumab Osimertinib Bronchoalveoläre Bronchoalveolar lavage Lavage Gewebe Biopsie Biopsy Pleural Pleuraerguss Liquid Biopsy (Plasma) Gewebe effusion Liquid biopsy (plasma) Biopsie Biopsy
15 Liquid biopsies 1. Circulating cell-free tumor-dna (ctdna) 2. Circulating tumor cells (CTC) Source: Sysmex
16 Clinical applications of ctdna analysis Prognosis Presence / amount of ctdna Monitoring during treatment Early detection of residual disease or progression Predictive marker testing Instead of biopsy in frail, untreated patients At the time of resistance ( resistance mutation)
17 New Engl J Med 2015 n=138 n=62 Osimertinib Partial of full remission in approx. 60% of the patients Approved for T790M mutated NSCLC
18 Special aspects of ctdna analysis Associated with tumor stage / tumor volume Sensitivity around 70-80% ( consider rebiopsy in case of negative result) Testing prior to treatment Pre-analytical factors (type of tubes etc) NGS panels optimized for cfdna
19 Cancer Immunotherapy Immune check-point inhibitors
20 The dilemma of predictive PD-L1 IHC 5 drugs, each with its own PD-L1 IHC assay Drug Antibody Platform TC IC Nivolumab 28-8 clone Dako + Pembrolizumab 22C3 clone Dako + Atezolizumab SP142 clone Ventana + + Durvalumab SP263 clone Ventana + Avelumab - Dako + 4 antibodies 2 immunostainer platforms Different cut-offs (<1%, 5%, 10%, 25%, 50%) Different scoring: Tumor cells +/- Immune cells
21 Pembrolizumab (Keynote-001/-010) PD-L1 IHC DAKO 22C3 Assay: Tumor Proportion Score TC <1% TC 1-49%: TC 50% x5 x40 Garon EB et al, NEJM 2015; Herbst RS et al, Lancet, 2015
22 Keynote-024 First-line Pembrolizumab for advanced NSCLC and a PD-L1 TPS 50% (Dako 22C3 assay) OR R Pembrolizumab Group (N=154) Chemotherapy Group (N=151) 44.8% 27.8% PD-L1 testing is entering diagnostic routine Reck M et al, NEJM 2016
23 Harmonization Comparison between different PD-L1 IHC Assays Blueprint project German Harmonization study Cross-validation between platforms (using Assay as the gold standard)
24 Blueprint Projekt - comparing 4 PD-L1 Assays - 22C SP263 SP142 % positive % Tumor tumor Staining cells Hirsch F et al, J Thorac Oncol 2016
25 Swiss Cross-Validation Studies To evaluate if 22C3 is applicable to other immunostainer platforms using the 22C3 on DAKO AS48 as gold standard 22C3 «gold standard» DAKO Link 48 (not widely available in CH) Ventana Benchmark XT Leica BondMax
26 PD-L1 22C3 IHC: TPS > 50% ASL-48 Benchmark Ultra Bondmax
27
28 Conclusions These are exciting times for pathologists Rapid technology development NGS is becoming a standard Liquid biopsies are following IHC remains a strong value Morphology will survive remember: garbage in garbage out
29
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