Lung Cancer Biomarkers: A Practical Update

Transcription

1 Lung Cancer Biomarkers: A Practical Update Lynette M. Sholl, M.D. Associate Pathologist, Brigham and Women s Hospital Associate Professor, Harvard Medical School Boston, MA

2 Disclosures Consultant for Foghorn Therapeutics, AstraZeneca, LOXO Oncology

3 Objectives Identify clinically relevant genetic alterations in lung carcinomas Appreciate appropriate approaches to biomarker testing in lung cancers Recognize biomarkers of immunotherapy response in lung cancer

4 Case 60 year old woman, never smoker, presents with difficulty breathing CT scan showed markedly enlarged chest and neck lymph nodes as well as a large left-sided lung mass A biopsy was performed: TTF-1 +, p40 metastatic lung adenocarcinoma

5 Oncogenic drivers in non-squamous non-small cell lung carcinoma No/unknown driver KRAS NTRK MAP2K1 AKT1 RET ROS1 NRAS ERBB2 PIK3CA MET BRAF ALK EGFR

6 Targeted therapy improves outcomes. (Lung Cancer Mutation Consortium I and II) Driver = EGFR, ALK, MET Driver = EGFR, ALK, ROS1, BRAF, MET, ERBB2, RET No targeted therapy Targeted therapy No driver Kris et al. JAMA Aisner et al. Clin Cancer Res 2018.

7 Driver oncogene mutual exclusivity principle : Immuno-oncology benefit Aisner et al. Clin Cancer Res 2018.

8 How should we manage our patient, a symptomatic female non smoker with lung adenocarcinoma? 1) Treat now with EGFR TKIs for EGFR mutation, ask questions later 2) Treat now with crizotinib for ALK/ROS1 rearrangement, ask questions later 3) Start chemotherapy 4) Order NGS testing for all possible targets and wait until it is done (2-3 weeks) to start therapy 5) Start immunotherapy

9 Joint CAP/IASLC/AMP Guidelines (January 2018) J Thorac Oncol. doi: /j.jtho Arch Pathol Lab Med. doi: /arpa CP J Mol Diagn. doi.org/ /j.jmoldx

10 2018 CAP-IASLC-AMP Lung Cancer Guidelines: What to test? Must test EGFR ALK ROS1 Enriched in light and never smokers Test for clinical trials BRAF** ERBB2 MET RET KRAS Who to test? All advanced-stage lung adenocarcinoma patients, irrespective of clinical characteristics Patients with tumor histologies other than adenocarcinoma when clinical features suggest probability of an oncogenic driver Light/never smoker Age<50 *ASCO and NCCN guidelines recommend up front testing for BRAF V600E for all advanced lung adenocarcinoma patients.

11 EGFR mutation is key biomarker

12 Testing for EGFR Use molecular diagnostics approaches for hotspot mutations in EGFR exons 18 to 21 with at least 1% prevalence The following methods are NOT recommended for selection of patients for EGFR TKI therapy: FISH for EGFR amplification total EGFR expression by immunohistochemistry EGFR mutant-specific immunohistochemistry EGFR mutation hotspots Codons 709 and 719 Exon 19 deletion Codon 768 Exon 20 insertions Codon 790 Codons 858 and 861

13 ALK translocations in NSCLC: 3-5% of lung adenocarcinomas, responsive to TKIs

14 ALK Fluorescence in situ hybridization (FISH) The gold standard Abbott/Vysis breakapart kit is the FDA-approved companion diagnostic for treatment with crizotinib Challenges: Costly Time consuming Technically demanding ~10% fail rate Improper fixation Poorly preserved cells Insufficient tumor cellularity Images from Lindeman et al. Molecular Testing Guidelines, JTO 2013 ALK FISH positive (split 3-5 signals) ALK FISH negative ALK FISH positive (single 3 ALK)

15 Unexpected/discordant case example: 55 year old female light smoker with lung mass Lung biopsy confirms adenocarcinoma PET scan detects metastases Genomic testing: ALK rearrangement by FISH in 74% of cells, per report EGFR mutation negative Crizotinib started progressive disease Genomic testing repeated

16 Unexpected/discordant case example: 55 year old female light smoker with lung mass ALK (5A4) IHC Lung biopsy confirms adenocarcinoma PET scan detects metastases Genomic testing: ALK rearrangement by FISH in 74% of cells, per report EGFR mutation negative Crizotinib started progressive disease Genomic testing repeated FISH Panel Next Gen Sequencing: KRAS c.34g>t (p.g12c) TP53 c.473g>t (p.r158l)

17 Guideline: ROS1 molecular or cytogenetic testing should be performed on all lung adenocarcinoma patients, irrespective of clinical characteristics. ROS1-fusion+ Lung tumors show deep, sustained responses to ROS1 inhibitors (crizotinib) Shaw et al: 72% ORR, 19.2 month PFS Mazieres: 80% ORR, 9.1 month PFS Operational and testing challenges: ROS1 fusion events are rare (1-2% of LUAD) Highly enriched in never/light smokers, but not absolutely Cytogenetic/molecular screening methods may be costly. Large number of partner genes No companion diagnostic for crizotinib in ROS1+ LUAD Shaw AT et al. N Engl J Med 2014;371:

18 Testing for ROS1: Use FISH, RT-PCR or NGS. ROS1 IHC should be used as a screening test, with molecular/fish confirmation of positive results. ROS1

19 82 year old former smoker with lung mass and supraclavicular lymphadenopathy. Lymph node excision: METASTATIC SQUAMOUS CELL CARCINOMA, poorly differentiated. The findings are not specific to a primary site. Clinical and radiographic correlation is needed.

20 Targeted testing performed at large reference lab: Mutation analysis shows: EGFR wild type BRAF wild type FISH analysis shows: No ALK translocation detected ROS1 translocation detected

21 Molecular results Sequencing results: Targeted fusions (not detected): CCDC6-ROS1.C6R34 CD74-ROS1.C4R33.NGS CD74-ROS1.C6R32.COSF1202 CD74-ROS1.C6R34.COSF1200 CEP85L-ROS1.C8R36 EZR-ROS1.E10R34.COSF1267 GOPC-ROS1.G4R36.COSF1188 GOPC-ROS1.G8R35.COSF1139 KDELR2-ROS1.K4R34 LRIG3-ROS1.L16R35.COSF1269 SDC4-ROS1.S2R32.COSF1265 SDC4-ROS1.S2R34 SDC4-ROS1.S4R32.COSF1278 SDC4-ROS1.S4R34.COSF1280 SLC34A2-ROS1.S13R32.COSF1259 SLC34A2-ROS1.S13R34.COSF1261 SLC34A2-ROS1.S13R36 SLC34A2-ROS1.S4R32.COSF1197 SLC34A2-ROS1.S4R34.COSF1198 TPM3-ROS1.T7R35.COSF1273

22 ROS1 IHC results:

23 Final ( Integrated ) pathology report: A. LEFT AXILLARY MASS, EXCISION: METASTATIC SQUAMOUS CELL CARCINOMA, poorly differentiated, involving lymphoid tissue. Immunohistochemistry performed at BWH demonstrates the following staining profile in lesional cells: Negative - ROS1 (see NOTE) Per report, immunohistochemistry performed at the outside institution shows the following staining profile: Positive - AE1/AE3, p63 Negative - MART1, CK7, CK20, PSA, TTF-1 Per report, molecular testing performed at Large Reference Laboratory showed the following: EGFR: mutation negative ALK: FISH negative ROS1: FISH positive PD-L1: Positive (10%) (Clone 22C3) Microsatellite: Stable RNA fusions showed the following: CDKN2A R58* PIK3CA M1043V NOTCH1 Q1547* NOTCH1 Q1282* TP53 splice site mutation TP53 R248Q BRCA2 P3150L NF2 Q298* MSH2 E786K CD44 >=10 copy number amplification NOTE: The morphologic, immunohistochemical, and molecular phenotype of this tumor supports the diagnosis of squamous cell carcinoma. The negative ROS1 immunohistochemical result, together with the absence of ROS1 fusion in the reported RNA assay, indicates that a functional activating ROS1 fusion is NOT present in the tested tumor. The positive ROS1 FISH result likely reflects nonspecific underlying genomic instability, including at or near the ROS1 locus. This patient is unlikely to benefit from ROS1-directed therapy (e.g. crizotinib).

24 BRAF V600E Should be tested in the front line setting for all patients with advanced non-squamous NSCLC (ASCO and NCCN guidelines)

25 Targeting BRAF V600E in lung cancer: Dabrafenib+trametinib in the 1 st line 64% response rate 10.4 month duration of response Planchard et al. Lancet Oncol. October 2017

26 BRAF mutational patterns in Lung D594E D594G D594N D594Y G464V G466L G466R G466V G466V,N581Y G469A V600E 40% G469E G469R G469S G469V G596R K601E K601N K601N,A598D L597Q L597R N581I N581S N581Y V600_W604insR V600E W531C,G596R

27 Target RET fusion ERBB2 mut KRAS MET ex14 or amp 2018 Biomarker Guideline Updates- in need of updating Strength of evidence Inadequate Inadequate Adequate Study types Single arm phase 2 clinical trial Single arm phase 2 clinical trial Single arm phase 2 clinical trial Correlative studies Clinical trial references Outcomes Comments Drilon et al. Lancet Oncol 2016; 17(12): Yoh K et al. Lancet Respir Med. 2017;5(1): Kris et al. Ann Oncol 2015 n/a Cabozantinib 28% DCR Vandetanib 53% objective response Dacominitib 12% DCR n/a Inadequate Case series n/a n/a DCR = disease control rate FISH or capture-based NGS; Insufficient evidence for use of IHC No response in patients with ERBB2 amp. May be used in sequential testing Requires carefully designed NGS and/or RNA studies Neither FISH nor IHC have established criteria for positivity.

28 NTRK and RET as evolving biomarkers

29 NTRK fusion: A new tumor-type agnostic biomarker for response to the TKI larotrectinib FDA approved larotrecitinib for all solid tumors with NTRK gene fusions by NGS or FISH. Overall response rate of 75% >12 month duration of response for 39% of patients These fusions are very rare in lung cancers (Feng et al. ESMO 2018: 0 cases of 1366 screened), more common in salivary tumors, pediatric tumors and sarcomas PAN TRK IHC screening? A Drilon et al. N Engl J Med 2018;378:

30 Slide 2 Presented By Alexander Drilon at 2018 ASCO Annual Meeting

31 Slide 10 Presented By Alexander Drilon at 2018 ASCO Annual Meeting

32 RET fusion updates September 2018: A selective RET inhibitor has been granted breakthrough therapy designation by the FDA for NSCLC after progression on chemo and immunotherapy Update on phase 1 and 2 trials expected in 2019

33 So many targets, so little tissue...

34 Sample adequacy challenges Quantity Quality Tissue size = DNA content Tumor content = mutant fraction Adverse factors: Delayed fixation Inadequate fixation Excessive fixation Acid or heavy-metal fixatives (decalcification)

35 Up-front histology slide protocol for small biopsies 1 2x 10x 3x 20 H&E Diagnostic IHC (TTF-1 + p40) ALK, ROS1, PD-L1 IHC Dissection for molecular testing Back-up slides (IHC, FISH) H&E

36 Sample Selection Fix biopsy specimens immediately, resection specimens within 30 minutes Use 10% buffered formalin Avoid decalcification Many standard decal solutions are HCl-based and will destroy nucleic acids EDTA-based solutions are gentler and compatible with molecular testing ANY cytology sample with adequate cellularity is ok for testing, including smear preps: Sequencing quality metrics to FFPE samples Hwang et al. Cancer Cytopath 2017 Roy-Chowdhuri et al. Mod Pathol 2017 Image courtesy of D. Hwang

37 Biomarkers for Immunotherapies

38 In lung cancers, smoking high TMB neoantigen formation adaptive immunity In lung adenocarcinoma, high neoantigen burden associated with: Longer overall survival Upregulated immune related genes including PD-L1 and IL-6 High PD-1 expression in neoantigen-reactive T cells PD-L1 CD8 McGranahan et al. Science 2017.

39 PD-1 inhibitor Pembrolizumab outperforms platinum chemo in the first line treatment of NSCLC* (with 50% PD-L1 expression) Progression free survival: Overall survival: *With improved quality of life as compared to chemo Brahmer et al. Lancet Oncol Reck et al. NEJM 2016

40 PD-1 inhibitor Nivolumab offers no benefit over platinum chemo in the first line treatment of NSCLC (with 5% PD-L1 expression) Carbone DP et al. N Engl J Med 2017;376:

41 Pembrolizumab significantly improves progression free and overall survival for patients with non-squamous NSCLC receiving 1 st line platinum chemotherapy. Immune-mediated adverse events in ~23% of pembro+chemo patients L Gandhi et al. N Engl J Med DOI: /NEJMoa

42 Impact of mutational status on IO outcomes Meta analysis of >2200 patients receiving pembro, nivo or atezolizumab monotherapy vs. docetaxel EGFR mutation: IO offers no benefit, possibly detrimental increased risk of pneumonitis KRAS mutation: IO provides 35% reduction in risk of death, but KRAS status not sufficient to predict benefit Smoking status: Not predictive of benefit with IO Lee CK JAMA Oncology 2017

43 Where are we with TMB? Tumor mutation burden

44 TMB predicts superior PFS with Ipilimumab-Nivolumab in the 1 st line irrespective of tumor PD-L1 expression Overall survival data (Oct 2018) shows significant benefit of Ipi+Nivo over chemo irrespective of TMB status (HR = 0.77 for TMB high vs 0.78 for TMB low groups). MD Hellmann et al. N Engl J Med 2018;378:

45 How to define high tumor mutation burden? Whole exome sequencing Carbone et al. NEJM Hellmann et al. AACR annual meeting 2018.

46 In silico downsampling studies to mimic commerciallyavailable 523 (1.9Mb) and 170 (0.53Mb) gene NGS panels Confidence intervals tighter around larger panel Bucchalter et al. Int J Cancer Sep 21. [Epub ahead of print]

47 WES vs panel sequencing TMB is comparable But the rules for how you calculate TMB matter and panels overestimate TMB relative to WES. Ahmet Zehir and Shweta Chavan, Memorial Sloan Kettering Cancer Center Higher depth of sequencing permits detection of low-level variants Specific selection of genes known to be mutated in cancers Optimization for uniform coverage Differential variant calling algorithms Tumor only sequencing introduces germline variants, variants due to clonal hematopoiesis

48 TMB standardization efforts Friends of Cancer Research Qualitätssicherungs-Initiative Pathologie GmbH (QuIP) collaboration QuIP: Compare TMB estimates derived from WES-derived reference standards (NSCLC and others) with commercial NGS panels and labdeveloped tests at academic institutions.

49 NSCLC Testing and Treatment Algorithm EGFR, BRAF mutation Targeted tx Adenocarcinoma ALK/ROS1/?NTRK rearrangement ALK/ROS1 TKI Stage IIIB-IV NSCLC PD-L1 expression TBD: May % TPS 1-49% TPS N/A or 0-100% TPS 1st line pembro.* 2 nd line pembro.* 2 nd line nivo, atezo. TMB? >10Mut/Mb??Ipilimumab/Nivolumab Squamous cell carcinoma PD-L1 expression *Or 1 st line pembrolizumab+chemo irrespective of PD-L1 score 50% TPS 1-49% TPS N/A or 0-100% TPS 1st line pembro. 2 nd line pembro. 2 nd line nivo, atezo.

50 Back to our patient: Rapid IHC results PD-L1 ALK ROS1

51 Now, how should we manage our patient, a symptomatic female non smoker with lung adenocarcinoma? 1) Treat now with frontline pembrolizumab monotherapy 2) Treat now with frontline combined pembrolizumab + platinum chemotherapy 3) Treat with crizotinib for ROS1 overexpression 4) Confirm the presence of a ROS1 fusion by FISH or molecular methods 5) Start with immunotherapy; if no response then switch to crizotinib

52 Our patient ROS1 IHC positive FISH confirmation requested The test failed. Sequencing for confirmation DNA (Hybrid capture) Requires intronic coverage Often shallow and of lower quality Analytic sensitivity may be low RNA (RNAseq, Anchored multiplex PCR) RNA degrades readily specimen failure Use of both DNA and RNA based assays increases testing complexity DNA-based NGS confirmed the presence of an SLC34A2-ROS1 fusion event.

53 Our patient: Crizotinib therapy led to symptomatic improvement within days. No evidence of cancer progression at 1 year At diagnosis 8 months post Crizotinib

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