MET skipping mutation, EGFR

New NSCLC biomarkers in clinical research: detection of MET skipping mutation, EGFR T790M, and other important biomarkers Fernando López-Ríos Laboratorio de Dianas Terapéuticas Hospital Universitario HM Sanchinarro Madrid, Spain

Contents Background MET mutations causing exon 14 skipping Liquid biopsies Conclusions

Contents Background MET mutations causing exon 14 skipping Liquid biopsies Conclusions

NSCLC: emerging biomarkers in clinical research Emerging targeted agents for patients with genetic alterations Genetic alteration (i.e. driver event) High-level MET amplification or MET exon 14 skipping mutation RET rearrangements Available targeted agents with activity against driver event in lung cancer Crizotinib Cabozantinib Vandetanib HER2 mutations Trastuzumab (category 2B) Afatinib (category 2B) HER2, human epidermal growth factor receptor 2; MET, MET proto-oncogene, receptor tyrosine kinase; NCCN, National Comprehensive Cancer Network; NSCLC, non-small-cell lung cancer; RET, RET proto-oncogene. NCCN guidelines version 8.2017 NSCLC. Available from: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. Accessed: September 2017.

NSCLC: emerging biomarkers in clinical research ctdna H&E P40 TTF1 NSCLC- NOS x 1 x 1 x 1 H&E AC x 1 Lap B1 Diagnosis ALK translocation 720 ROS1 translocation PD-L1 expression EGFR mutation If EGFR/ALK/ROS1 negative Multiplexed genetic sequencing panels FISH IHC IHC or If Expert positive consensus opinion: in some clinical settings in which tissue is limited x and/or 10 (DNA) insufficient for molecular + testing, IHC FISH x 12 (RNA) physicians may in future consider T790M to testing use a cfdna assay for x 2 x 2 x 1 Progressed EGFR EGFR ctdna positive Multiplexed genetic If negative Expert sequencing consensus opinion: physicians may use cfdna PD-L1 expression panels methods to identify for EGFR T790M mutations in lung AC IHC patients with progression or acquired resistance to EGFR-TKIs Testing x10 (DNA) of the tumour sample will be in future recommended x 1 is recommended x12 (RNA) if the plasma result is negative Lap B2 Biomarkers 5% (real-time PCR) 30% (NGS) H&E x 1 AC, adenocarcinoma; ALK, anaplastic lymphoma kinase; cfdna, cell-free plasma DNA; ctdna, circulating tumour- DNA; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; H&E, haematoxylin and eosin; IHC, immunohistochemistry; NGS, next-generation sequencing; NOS, not otherwise specified; PCR, polymerase chain reaction; PD-L1, programmed death-ligand 1; ROS1, ROS1 proto-oncogene receptor tyrosine kinase. Adapted from Conde E, et al. Clin Transl Oncol. 2013;15:503-8, following the 2016 revised molecular testing guideline for selection of lung cancer patients (CAP, IASLC, AMP, draft statements 2016).

Contents Background MET mutations causing exon 14 skipping Liquid biopsies Conclusions

MET mutations causing exon 14 skipping Distribution of genotypes among 933 patients with non-squamous NSCLC NSCLC (2.6%, 2.7%, 3%, 5.3%) KRAS (34%) EGFR (19%) No oncogenic mutation identified (30%) ALK (3.9%) BRAF (3.8%) MET exon 14 (3.0%) PIK3CA (2.9%) ERBB2 (2.5%) NRAS (1%) RET (1%) ROS1 (1%) AKT (< 1%) HRAS (< 1%) MAP2K1 (< 1%) Sarcomatoid variant, with AC morphology (5%, 22%, 32%) Always with MET overexpression (IHC) MET amplification in ~ 15 21% Concurrent amplifications MDM2 (~ 35%) CDK4 (~ 20%) AKT, AKT serine/threonine kinase 1; BRAF, B-Raf proto-oncogene; CDK4, cyclin-dependent kinase 4 gene; ERBB2, erb-b2 receptor tyrosine kinase 2; HRAS, HRas proto-oncogene; KRAS, Kirsten rat sarcoma viral oncogene homologue; MAP2K1, mitogen-activated protein kinase kinase 1; MDM2, murine double minute gene; NRAS, NRAS proto-oncogene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. Awad MM, et al. J Clin Oncol. 2016;34:721-30. Mengoli MC, et al. Clin Cancer Res. 2016:22;3697-8. Saffroy R, et al. Oncotarget. 2017;8:42428-37. Liu X, et al. J Clin Oncol. 2016;34:794-802. Schrock AB, et al. J Thorac Oncol. 2016;11:1493-502. Tong JH, et al. Clin Cancer Res. 2016;22:3048-56.

MET mutations causing exon 14 skipping Example of MET alterations Deletion at the splice acceptor site (~ 41%) g.116411840 del49 g.116411857 del28 g.116411874 del26 g.116411880 del20 g.116411887 del28 g. 16411893 del30 g.116411897 del27 Deletion at the splice donor site (~ 11%) g.116412019 del25 g.116412026 del24 g.116412036 del12 g.116412040 del14 Mutation at the splice donor site (~ 48%) g.116412042 G>A g.116412042 G>C g.116412042 G>T g.116412043 G>A g.116412043 G>T g.116412043 G>C g.116412044 T>A g.116412044 T>C g.116411902 c.2888 TTAAGATCTGG Splice acceptor site 141 bases 47 AA Exon 14 g.1164122042 c.3028.cagaaggtata Splice donor site CBL, cobalamin. Amino acid sequence Regulatory sites: METex14 alterations, n (%) DLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPE 963 1009 PKC phospho site (S985) Caspase site (D1002) CBL docking site (Y1003) Base substitution splice donor 149 (49.1) Indel splice acceptor 100 (32.9) Indel splice donor 42 (13.8) Base substitution splice acceptor 4 (1.3) Base substitution noncoding adjacent spice acceptor 4 (1.3) Indel noncoding adjacent spice acceptor 3 (1.0) Whole exon 14 deletion 2 (0.7) Cortot AB, et al. J Natl Cancer Inst. 2017;109. Schrock AB, et al. J Thorac Oncol. 2016;11:1493-502.

MET mutations causing exon 14 skipping HGF, hepatocyte growth factor; PKC, protein kinase C. Cortot AB, et al. J Natl Cancer Inst. 2017;109.

MET mutations causing exon 14 skipping RNA panel Fusion drivers (n = 23) For Research Use Only. Not for use in diagnostic procedures Data courtesy of López-Ríos, F.

Case 1. MET mutations enriched in pan-negative lung adenocarcinomas Female, 72 years old Lung AC, stage IV Data courtesy of López-Ríos, F.

Case 1. MET mutations enriched in pan-negative lung adenocarcinomas Oncomine focus assay CNV: MET (x5,09 copies); confirmed with FISH (ratio 5:40) MET exon 14 deletion [ skipping exon 14 MET, MET(Ex13) MET(Ex15)]; confirmed with RT-PCR FISH MET+ IHC MET+ CNV, copy number variation; NF1, neurofibromin 1; RIT1, Ras-like in all tissues; RT-PCR, reverse transcriptase-polymerase chain reaction. Collisson EA, et al. Nature. 2014;511:543-50. Data courtesy of López-Ríos, F.

Case 2. MET mutations not always MET-amplified Oncomine focus assay No CNV; confirmed with FISH (MET) MET exon 14 deletion [ skipping exon 14 MET, MET(Ex13) MET(Ex15)]; confirmed with RT-PCR IHC MET+ FISH MET Data courtesy of López-Ríos, F.

Contents Background MET mutations causing exon 14 skipping Liquid biopsies Conclusions

Liquid biopsies: analytical options Available technologies for genotyping of plasma cfdna Characteristic Variants potentially detected Quantitation Speed and complexity Allele-specific PCR Known recurring mutations Semiquantitative (against standard curve) Rapid, relatively easy to interpret PCR assays Emulsion PCR Known recurring mutations Absolute or relative quantitation, wide dynamic range Rapid, relatively easy to interpret Amplicon-based targeted NGS Any exonic mutations, copy number gains Quantitation of relative AF, but vulnerable to PCR amplification bias Potentially rapid, less complex bioinformatics NGS assays Capture-based targeted NGS Exonic mutations intromic gene fusions, copy number gains Quantitation of relative AF Potentially slower more complex bioinformatics AF, allele frequency.

Liquid biopsies: sensitivity and specificity Performance of four different plasma assays, using a tissue a test result as a non-reference standard, for detection of EGFR mutations from ctdna in a set of 38 plasma samples from the AURA clinical trial Exon 19 deletion qpcr ARMS-PCR ddpcr TM BEAMingdPCR Sensitivity, % (n/n) 86 (24/28) 82 (23/28) b 93 (26/28) Specificity, % (n/n) 100 (10/10) 100 (10/10) b 100 (10/10) Concordance, % 89 87 b 95 L858R Sensitivity, % (n/n) 90 (9/10) 78 (7/9) 90 (9/10) 100 (10/10) Specificity, % (n/n) 100 (28/28) 100 (28/28) 100 (28/28) 93 (26/28) Concordance, % 97 95 97 95 T790M Sensitivity, % (n/n) 41 (7/17) 29 (5/17) 71 (12/17) 71 (12/17) Specificity, % (n/n) 100 (6/6) 100 (6/6) 83 (5/6) 67 (4/6) Concordance, % 57 48 74 70 a 24/38 samples were tested using local tissue assays and 14/38 samples were tested via central assays. b Comprehensive exon 19 deletion data not available. ARMS, amplification refractory mutation system; ddpcr Droplet Digital polymerase chain reaction; dpcr, digital polymerase chain reaction.

Liquid biopsies: sensitivity and specificity Concordance, sensitivity, specificity, PPV, and NPV of tissue and cfdna tests (n = 238) by EGFR mutation type Overall EGFR mutation-positive Exon 19 deletion L858R G719x L861Q Concordance, % 87.8 94.5 93.3 99.6 100.0 Sensitivity, % 75.0 82.5 62.2 50.0 100.0 Specificity, % 96.5 98.3 99.0 100.0 100.0 PPV, % 93.5 94.0 92.0 100.0 100.0 NPV, % 85.1 94.7 93.4 99.6 100.0 Tissue-positive patients, n 96 57 37 2 1 For Research Use Only. Not for use in diagnostic procedures NPV, negative predictive value; PPV, positive predictive value.

Promising technology for future use for liquid biopsies: NGS Assay Genes Selected SNV hotspots Oncomine lung cfdna assay ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 An example of our results Oncomine Lung cfdna Assay > 150 hotspots including EGFR: T790M, C797S, L848R, exon 19 del KRAS: G12X, G13X, Q61X BRAF: V600E ALK: exon 21-25 PIK3CA: E545K, H1047R, E542K Position Allele Frequency LOD chr7:55242470 EGFR p.l747_p753s 32.33% 0.05% chr7:55249071 EGFR p.t790m 15.87% 0.10% chr7:55249092 EGFR p.c797s 15.29% 0.10% LOD (20 ng) 0.1% LOD, log odds ratio; TP53, tumour protein 53. For Research Use Only. Not for use in diagnostic procedures Data courtesy of López-Ríos, F.

Case 3. Importance of broad profiling 2014 Lung AC Male, 70 years old IHC TTF1+/P40 EGFR exon 19 deletion (real-time PCR) No ALK fusion Treated with 3rd-generation TKIs 2016 Soft-tissue metastasis 52 genes NGS panel EGFR exon 19 deletion confirmation No ALK fusion T790M mutation C797S mutation Controls Position Control type Control status Flags Accepted by A01:A02:A03 Mutant control Valid B01:B02:B03 Specimens Position D01:D02:D03 Run name: 07-FEB-2014 15:16 EGFR P1 Negative control Sample ID Test result Mutation detected IHC TTF1+ For Research Use Only. Not for use in diagnostic procedures Valid Mutation result Flags Accepted by Exon 19 deletion Data courtesy of López-Ríos, F.

Case 3. Importance of broad profiling Case Tumour FFPE (NGS assay) Plasma Case Assay 1: NGS plasma panel Plasma Assay 2: PCR plasma T790M T790M T790M EGFR exon 19 deletion EGFR exon 19 deletion EGFR exon 19 deletion C797S C797S Not included in panel For Research Use Only. Not for use in diagnostic procedures Data courtesy of López-Ríos, F.

Promising technology for future use for liquid biopsies: NGS total NA Oncomine Lung cftna Assay ALK BRAF EGFR ERBB2 KRAS MAP2K1 MET NRAS PIK3CA RET ROS1 TP53 Enhanced content Amplicons: 58 Total genes: 12 Key hotspot mutations in 11 genes Increase in hotspot SNVs and indels Fusions (49) ALK, RET, ROS1 1% LOD CNV MET MET exon 14 skipping single library (DNA and RNA) SNV LOD down to 0.1% with 20 ng input SNV, single-nucleotide variants; TNA, total nucleic acid. For Research Use Only. Not for use in diagnostic procedures

Oncomine Lung cftna Assay: early experience Analysis of plasma samples from patients diagnosed with NSCLC and previously characterized by NGS (Oncomine TM Focus Assay) or FISH analysis (FFPE samples). Oncomine Lung cftna Assay Controls (n=4) run in parallel according to manufacturer s instructions. 1) Run performance OK: the assay works! For Research Use Only. Not for use in diagnostic procedures

Oncomine Lung cftna Assay: early experience 2) Analysis: A real sample FFPE tumor sample (FISH analysis) Plasma sample (Oncomine Lung cftna Assay) ALK positive For Research Use Only. Not for use in diagnostic procedures

Contents Background MET mutations causing exon 14 skipping Liquid biopsies Conclusions

Conclusions Broad profiling is recommended in NSCLC MET mutations causing exon 14 skipping is an emerging biomarker that can be detected with targeted NGS Using NGS in liquid biopsies allows for the detection of mutations, CNV, and rearrangements

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