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Biomarker Assessment for Selection of Therapy in NSCLC
Marileila Varella Garcia, PhD UC SOM, Depts. Medicine and Pathology Livorno, Italy April 13, 2012
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Disclosures Research Grants/Contracts: Boehringer Ingelheim Genentech
Imclone Consultant: Abbott Molecular
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Outline NSCLC: Molecular Heterogeneity and Targeted Therapy
Testing Platforms: Strengths and Limitations Clinical Assays: ALK Novel Targets: ROS1 and RET Novel Multiplex FISH Reagents: 4-target Enumeration and 4-target Break-Apart
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Molecular Heterogeneity in NSCLC
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Testing Platforms DNA-based RNA-based Protein-based
mutation analyses, FISH, BRISH, SNPs RNA-based RT-PCR, quantitative PCR, gene expression arrays Protein-based IHC
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Selecting Clinical Assays
Defined reagent, standardized scoring system, validated cut-off TEST A ALK Break Apart FISH TEST B complete concordance False negatives or accurate detection of primary refractory population? e.g. RT-PCR False positives or missed true positives? e.g. IHC TEST C Partial concordance ? TEST D Partial concordance ? ? TEST E Partial concordance Accuracy, Precision, Cost, Feasibility Precision, Cost, Feasibility STANDARDIZATION
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ALK Dual Color, Break Apart FISH assay
Abbott Molecular 2p23 normal 3’ 5’ Rearrangement positive - split Rearrangement positive - single 3’ ALK inversion with EML4-ALK fusion
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Bright-Field In Situ Hybridization
CISH ALK+ by split ALK+ by single 3’ALK Kim et al., JTO, 6(8): , 2011 Few publications but already include DIFFERENT: a) criteria for classification of split signal b) cut-offs for classification as ALK+
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ALK Protein Expression by IHC: Several Reagents
Novocastra clone 5A4 DAKO clone ALK1 Cell Signaling clone D5F3 Paik et al, JTO 2011 Yi et al, JTO 2011 Mino-Kenudson et al., CCR 2010 Antibody Authors Journal, Year Group ALK1 Boland HP 2009 Mayo DAKO Yi JTO 2011 Yang JTO 2012 Popat LC 2011 London, Royal Marsden Hospital Rodig CCR 2009 MGH/BWH/DFCC Shaw JCO 2009 Mino-Kenudson CCR 2011 Takeuchi MP 2009 Japan, The Cancer Institute D5F3, Cell Signaling Technology Salido Barcelona/Colorado 5A4, AbCam Novocastra Paik Seoul National University Koh McLeen-Florin Grenoble, France
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ALK IHC: Heterogeneity in Scoring Systems
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Standardization is urgently needed!!!
But likely lack of standardization is not the only reason for discrepancies…
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Discrepancies Are Likely Also Biology-Based
Comparison FISH, PCR and IHC Pt. ID % cells FISH positive (BA) EML4-ALK FISH EML4-ALK Variant-specific RT-PCR Insight Genetics qPCR ALK IHC (D5F3) ALK IHC (D9E4) 1 24% POS E13;A20* NEG 2 33% E13;A20 3 37% E6;A20 4 43% 5 47% 6 53% 7 59% E18;A20 8 77% 9 10 80% 11 12 93% Expression-based Genomic-based *Positive after 2 rounds of PCR Varella-Garcia et al., unpublished data
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Acquired Resistance to Crizotinib in ALK FISH+
Multiple mutations in ALK tyrosine kinase domain New oncogenic driver: EGFR and KRAS Loss of the ALK activation by fusion Gain in copy number of the ALK rearrangement Implications in selection of further therapies: 2nd generation ALK inhibitor Other options Doebele et al., Clin Cancer Res 2011
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Chromosomal Rearrangement
Novel Target in NSCLC: ROS1 Fusion Chromosomal Rearrangement Tumor type 1st ROS1 exon Partner Exon Number of Cases Reference CD74-ROS1 t(5;6)(q32;q22) NSCLC 35 6 1 Rikova et al., 2007 Lung adenocarcinoma 34 2 Li et al., 2011 NR 5 Bergethon et al., 2012 32-34 3 Takeuchi et al., 2012 Doebele 2012 EZR-ROS1 inv(6)(q22q25.3) 10 GOPC(FIG)-ROS2 240 kb interstitial del(6)(q21q21) Glioblastoma 36 7 1-cell line U118MG Charest et al., 2003 likely deletion Cholangiocarcinoma Gu et al., 2011 PLOS 242.5 kb interstitial deletion Low Malignant Potential Serous Ovarian Carcinoma Birsch et al., 2011 LRIG3-ROS1 t(6;12)(q22;q14.1) 16 SLC34A2-ROS1 t(4;6)(p15.2;q22) 33-35 4 1-cell line HCC78 12 SDC4-ROS1 t(6;20)(q22;q12) 32 TPM3-ROS1 t(1;6)(q21.2;q22) 8 Unknown 39 lung cancer clinical cases + HCC78 cell line 7 distinct fusions (in 26) + unknown (in 13)
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ROS1 FISH Positive = Split 3’-5’ or Single 3’
ROS1 FISH probe for NSCLC TMA ROS1 FISH+ are likely to benefit from Crizotinib Bergethon et al, 2012; Doebele et al., under review
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Novel Target in NSCLC: RET
Reference KIF5B-RET Fusion Number of Patients Frequency in Tested Cohorts Ju et al, 2012 K16R12 1 proband K15R12 1 /5 ADC (20%) K23R12 1 /15 ADC (6.7%) Kohno et al, 2012 3 6/319 ADC (1.9%) K24R8 Takeuchi et al, 2012 8 12/1121 ADC (1.0%) K22R12 K23R22 K24R11 Lipson et al, 2012 8* 12/585 ADC (2.0%) K15R11 1* Total Patients 33 Activated RET is a target for Vandetanib Ponatinib Sorafinib
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KIF5B-RET fusion detected by FISH Negative Positive
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FGFR1/FGFR2 4-target Enumeration
Novel FISH Reagents FGFR1/FGFR2 4-target Enumeration FGFR1/CEP8 FGFR2/CEP10
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FGFR1/FGFR2 Enumeration
Novel FISH Reagents FGFR1/FGFR2 Enumeration FGFR1/FGFR2 FGFR1/CEP8 FGFR2/CEP10
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Multiplex FGFR1/FGFR2 Enumeration
Novel FISH Reagents Multiplex FGFR1/FGFR2 Enumeration FGFR1/CEP8 FGFR2/CEP10 FGFR1/CEP8 FGFR2/CEP10 FGFR1/CEP8 FGFR2/CEP10
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Multiplex ALK-ROS1 Break-Apart (~10% ADC)
Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart (~10% ADC) 3’ALK/5’ALK 3’ROS1/5’ROS1 Positive for ALK Negative for ROS1
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Positive for ALK rearrangement Negative for ROS1 rearrangement
Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart (~10% ADC) normal Single 3’ 3’ALK/5’ALK 3’ROS1/5’ROS1 Positive for ALK rearrangement Negative for ROS1 rearrangement
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Negative for ALK rearrangement Positive for ROS1 rearrangement
Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart normal Single 3’ Single 3’ 3’ALK/5’ALK 3’ALK/5’ALK 3’ROS1/5’ROS1 3’ROS1/5’ROS1 Negative for ALK rearrangement Negative for ALK Positive for ROS1 rearrangement Positive for ROS1 Time is over, for updates follow us on and
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CONCLUSIONS Discovery of molecular markers for sensitivity and resistance to targeted therapy agents has brought new excitement to the NSCLC field Greater understanding of molecular pathways will make biomarker design more efficient It is unlikely that any single technical platform will reach 100% diagnostic specificity and sensitivity for any given marker For each marker, a panel of assays should be optimized, standardized and validated Multiplexing is highly recommended due to scarcity of tumor material in advanced NSCLC
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THANKS Margaret Skokan Wilbur Franklin, MD Mariana Theodoro
Adelita Mendoza Yong Gon Cho Antonella Flacco Severine Kako Tara O’Brien Liang-Guo Xu Nathan Schulte Wilbur Franklin, MD Dara Aisner, MD, PhD Robert Doebele, MD, PhD Ross Camidge, MD, PhD Paul Bunn Jr, MD Anna Baron, PhD Abbott Molecular, for novel probe sets
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