Molecular classification of lung cancer for clinical practice

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2 Centre Jean Perrin Centre de Lutte contre le Cancer d'auvergne Clermont-Ferrand - France - Molecular classification of lung cancer for clinical practice Frédérique Penault-Llorca

3 In the beginning.. There was lung cancer And it was treated with surgery or radiotherapy Then came small cell lung cancer The. Adenocarcinoma Squamous cell carcinoma Neuroendocrine tumors Large cell carcinoma Adenosquamous carcinoma Sarcomatoid carcinoma Other carcinomas (LEL, NUT) Salivary gland-type tumors Papillomas Adenomas Mesenchymal tumors Lymphoproliferative disorders Tumors of ectopic origin Metastases to the lung 2

4 In 2008, a Therapeutic Reason to Subclassify NSCLC Came Along Adeno vs Squamous Differential responses to pemetrexed (better in non SCC) Contra-indication for bevacizumab for SCC Pathological subtyping became important NSCLC-NOS o lo ge a epta le!! Immunohistochemical refinement of diagnosis TTF1, p63, p40, CK5/6 Scagliotti GV et al. J Clin Oncol. 2008;26. 3

5 Then Came EGFR Mutation Testing Known since 2004 Licensing of EGFR TKIs for EGFR mutant lung cancer EGFR mutation testing became mandatory Reflected in guidelines A feature of adenocarcinoma biology Mok TS et al. N Eng J Med. 2009;361. 4

6 Guidelines for EGFR Testing Test adenocarcinomas Caveats in SCC never smokers Range of mutations to be tested Independent of methodology Lindeman NI et al. CAP/IASLC/AMP J Thorac Oncol Jul;8(7): Kerr KM et al. ESMO Annals of Oncology. 2014;25:

7 Then Came ALK Rearrangement Testing Fusion gene identified in 2007 Crizotinib licensed in 2010 FDA FISH testing FISH-IHC correlation IHC Multiplex PCR for fusion mrnas Fusion gene by Next Generation methods A feature of adenocarcinoma biology Shaw AT et al. NEJM Soda M, et al. Nature. 2007;448:

8 Guidelines for ALK Testing Test adenocarcinomas Caveats in never smokers Screening by IHC Confirm by FISH IHC as primary test? Lindeman NI et al. CAP/IASLC/AMP J Thorac Oncol Jul;8(7): Kerr KM et al. ESMO Annals of Oncology. 2014;25:

9 Guidelines for ROS1 Testing Test adenocarcinomas Caveats in never smokers Screening by IHC Confirm by FISH IHC as primary test? Lindeman NI et al. CAP/IASLC/AMP J Thorac Oncol Jul;8(7): Kerr KM et al. ESMO Annals of Oncology. 2014;25:

10 2018

11 Lindeman NI et al J Thorac Oncol Mar;13(3): Update of the 2013 guidelines in 2018 The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity (20% instead of 50%) Recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for laboratories with NGS clinical trials IHC as an alternative to FISH for ALK and/or ROS1 testing use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors use of cell-free DNA to ule i ta geta le utatio s when tissue is limited or hard to obtain.

12 Algorithm without NGS Non-small cell lung cancer Squamous cell carcinoma EGFR & ALK testing ONLY if never or long time exsmoker Chemotherapy If negative Non-squamous cell carcinoma (de facto ADC) EGFR mutation ALK ROS1fusion gene testing EGFR or ALK/ROS1 TKI if positive Chemotherapy If negative Tissue is an issue Tests can be performed on biopsies, surgical specimens, cytology (EBUS) 11

13 21/05/ Driver oncogenes in ADC NKTR1Fusion (neurotrophic tyrosine kinase1) and response to entrectinib lartrectinib 0,1% ADK MET exon 14 splicing and response to crizotinib and other MET inhibitors Driver oncogenes in SCC TCGA, Nature 2014

14 Kerr KM. J Clin Pathol 2013;66: ROS1 fusion genes % Adenocarcinomas Fusion correlates with protein (IHC) Sensitive to crizotinib HER2 mutations 1-2% Adenocarcinomas NTRK fusion Mutually exclusive of EGFR, KRAS MPRIP-NTRK1 and CD74-NTRK1 Traztuzumab? Neratinib? 3.3% of onco- egative ade o a i o as Trk inhibitors Vaishnavi A et al. Nat Med 2013; 19, Drilon A, et al N Engl J Med. 2018;378: CD74-NRG1 fusion Sea h i onco- egative ade o a i o as ERBB3 and PI3K-AKT pathway activation Mucinous adenocarcinomas Potential therapeutic target Fernandez-Cuesta L et al. Can Disc 2014; jan30 epub BRAF mutations 2.5-4% Adenocarcinomas Incl V600E other V600 Smoking vs non-smoking Vemurafenib KRAS mutations 25-35% Adenocarcinomas MEK inhibition? If STK11 mut are associated aggressive phenotype RET fusion genes ~1% Adenocarcinomas Vandetanib & others MET upregulation 4% amplification, ~50% overexpression Biomarker issues Failed trials

15 Clin Can Res 2012;18, FGFR1 amplification Biomarker issues Definition of amplification 20% may be overestimate? Ponatinib FGFR1 inhibitor Wynes MW et al. Clin Cancer Res 2014;20: Discoid Domain Receptor 2 mutation Prevalence 3.8% Good in vitro target mirna & Dasatinib Limited clinical evidence Hammerman PS et al. Cancer Discov 2011 PI3Kinase ~30% amplification ~6% mutations addictive?? Inhibitors exist MET Inhibitors exist So far no success EGFR TKI vs MoAb Mutations rarity (viii 8%) Targeting the receptor IGFR1 Figitumumab Some effect in squamous Toxicity STK11 Identifies a KRAS aggressive subtype Especially when exon 1-2 mut

16 Novel Therapies for SCLC Fall Into 5 Categories That Target Key Aspects of Cancer Biology 1 1. Sabari et al. Nat Rev Clin Oncol. 2017;14:

17 It is worthwhile finding an Actionable genetic alteration in Lung cancer Driver detected Targeted Rx Kris MG et al. JAMA 2014; 311,

18 IMMUNOTHERAPY

19 21/05/ Classification of NSCLC Clinical Histological Molecular Host factors Immune response Prognostic and predictive value Tumor heterogeneity Evolution during the disease Optimization of the use of tumor specimens REVOLUTION OF NGS

20 Nivolumab SP263 Ventana 22C3 Dako 28-8 Dako Extracellular IHC Tests PD-L1 Drug Clone PD-L1Epitope Ab Dilution (kit) Automate Detection Provider Pembrolizumab SP263 Ventana Extracellular 3 µg/ml 2 µg/ml Dako Link 48 Dako Link 48 EnVision FLEX EnVision FLEX Atezolizumab SP142 Ventana Intracellular 7 µg/ml Ventana Benchmark Ultra OptiView + amplification Durvalumab Avelumab SP263 Ventana Dako Intracellular Intracellular 1.25 µg/ml N/A Ventana Benchmark Ultra Dako OptiView EnVision FLEX

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25 Incorporating PD-L1 Testing Into Practice Se o d o g eate li e o fi st li e the apy Will reflex testing be possible? Yes Will reflex testing be required? Yes for first line Will testing require rebiopsy? No At least five drugs, at least five biomarkers Will the pathologist know which one(s) to do? Prospect for a single biomarker test? PD-L1 IHC is a reasonable biomarker; expectations also have to be reasonable 25

26 LEVEL OF PD-L1 POSITIVE CELLS MATTERS!

27 Biomarkers for Immunotherapy? PD-L1 Negative Less response PD-L1 Positive (predictive of response) More response? 1% 5% 10% 50% cell positive Intensity of staining? Immune cell staining? Several therapeutics Several companion diagnostics Gandhi L, et al. AACR Abstract CT105.

28 KEYNOTE-024 Study Design (NCT ) Key Eligibility Criteria Untreated stage IV NSCLC Pembrolizumab 200 mg IV Q3W (2 years) PD-L1 TPS 50% ECOG PS 0-1 No activating EGFR mutation or ALK translocation R (1:1) N = 305 No untreated brain metastases No active autoimmune disease requiring systemic therapy Platinum-Doublet Chemotherapy (4-6 cycles) PD a Pembrolizumab 200 mg Q3W for 2 years Key End Points Primary: PFS (RECIST v1.1 per blinded, independent central review) Secondary: OS, ORR, safety Exploratory: DOR a To be eligible for crossover, progressive disease (PD) had to be confirmed by blinded, independent central radiology review i fo atio s ie tifi ue o validée pa l auto isatio de Mise su le Ma hé des su sta es a tives itées and all safety criteria had to be met.

29 KN-024 Results Progression-Free Survival Overall Survival i fo atio s ie tifi ue o validée pa l auto isatio de Mise su le Ma hé des su sta es a tives itées Reck et al, ESMO 2016

30 Putting data in context NSCLC patients in daily practice PDL1 Or NSCLC patients with PDL 5 % PS, BM AI, steroids The pool of patients who can benefit from immunotherapy in the front line setting has to be enlarged Pembrolizumab PS 0/1, no untreated BM, no AI, no steroids JC. Soria ESMO 2016

31 Incorporating PD-L1 Testing Into Practice On going and still there for few years! Alternative Biomarker Strategies for Immunomodulatory Therapy? Smoking History Smoking signature (genetic) Mutational burden Mismatch repair Microsatellite instability Polymerase E mutations Immune gene signatures Immune cell infiltrates 31

32 OTHER BIOMARKERS FOR IO

33 Mutational load and response to anti-pd1/pdl-1 Yarchoan M et al. N Eng J Med 2017

34 Parameters and potential biomarkers of anti-tumor immune response Technical and clinical validations are mandatory Modified from Peters, AACR 2017; Topalian, Nat Rev Cancer 2016

35 Checkpoints modulators: next steps Define biomarkers of response Scoring combining checkpoint expression, immune infiltration characteristics, tumor antigen load and host factors like microbiome. Large prospective studies needed Consider variations of those characteristics in space and time, with modifications induced by conventional and targeted therapies Cancer immunogram Blank et al. Science 2016 PD-1/PD-L1 inhibition alone

36 CLINICO-PATHOLOGICAL CORRELATIONS (OR HOW TO SELECT PATIENTS WHEN MOLECULAR BIOLOGY ACCESS IS LIMITED)

37 Molecular alteration Histologi c type Frequency Targeted therapy Correlation morphology IHC test KRAS mut ADC 20-30% indirect YES NO YES Smoking EGFR mut ADC 10-15% YES YES YES* NO ALK transl ADC 3-5% YES YES YES NO BRAF mut ADC 1-3% YES NO YES* YES ROS1 trans ADC 1-2% YES YES YES NO RET trans ADC 1-2% YES YES NO NO HER2 mut ADC 1-2% YES NO NO? 21/05/2018 FGFR1 amp PIK3CA mut SCC 20% YES NO NO YES SCC 15% YES NO NO YES DDR2 mut SCC 2% YES NO NO YES 37

38 Screening for ALK and ROS1 by IHC Nuclear Pan-Trk IHC Expression NTRK1 fusion IHC ALK clone D5F3 IHC BRAF V600E: covers 50% of mutations in NSCLC FISH ALK, ROS1, RET for confirmation Alternative approaches to molecular biology

39 21/05/ Typical associations with histologic subtypes Non mucinous ADC Mucinous ADC In situ lepidic non muc Papillary Acinar Solid Micropapillary Solid/cribriform/signet ring aspects

40 Typical associations Non smoker Female Asian Papillary ADC TTF1+ EGFR Mutations 21/05/2018 Smoker Mucinous ADC TTF1- KRAS mutation Non smoker M=F Solid, signet ring, cribriform ADC TTF1+ (and CK5+) ALK, ROS or RET rearrangements Patients less than 40 years old with lung adenocarcinoma have up to 50% chances to have either EGFR or ALK/RET /ROS alterations 40

41 MET Exon 14 mutations 3% in lung adenocarcinoma 22% in sarcomatoid tumors CRIZOTINIB

42 CONCLUSION

43 Take-home messages Reflex test PD-L1 for all NSCLC Reflex IHC ALK, ROS1 for adenocarcinoma If young patients FISH ALK ROS1 even if IHC is negative Correlation with morphology can help to do some triage when the access to molecular test is limited (for instance mucinous ADK, smokers KRAS mut)

44 Algorithm in advanced NSCLC Advanced NSCLC Adenocarcinoma NSCLC, nos NSCLC subtyping Morphology ± IHC+PD-L1 NGS lung panel Squamous cell carcinoma NGS lung panel EGFR, RAS, BRAF, HER2. + ALK and ROS1 (IHC/FISH) Screening for other targets

45 Alternative to NGS Directly from FFPE RT-PCR Results in 150mn Hands on machine: 2mn EGFR, RAS, BRAF >10% Tum cells 51 mutations in exons Average se sitivity of to 5%

46 Future Emerging targets (NTRK fusions) Mutations, fusions and amplifications Stand alone tests rapidly become non-viable Tissue issues Costs Time issues Tissue still an issue Amount Processing Multiplex testing => NGS Blood testing as a surrogate? 46

47 Personal algorithm in advanced NSCLC Advanced NSCLC Adenocarcinoma NSCLC, nos NSCLC subtyping Morphology ± IHC + reflex PD-L1 Squamous cell carcinoma ALK and ROS1 (IHC/FISH) (reflex testing) NGS lung panel + ARCHER panel for fusion (ALK, ROS1, RET, NTRK ) TMB, MSI, POLE

48 Liquid biopsy in LUNG oncology T790M PMC full text: Front Oncol. 2015; 5: 209. Published online 2015 Sep 30. doi: /fonc