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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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Presentation on theme: "Biomarker Assessment for Selection of Therapy in NSCLC Marileila Varella Garcia, PhD UC SOM, Depts. Medicine and Pathology Livorno, Italy April 13, 2012."— Presentation transcript:

1 Biomarker Assessment for Selection of Therapy in NSCLC Marileila Varella Garcia, PhD UC SOM, Depts. Medicine and Pathology Livorno, Italy April 13, 2012

2 Disclosures Research Grants/Contracts: Boehringer Ingelheim Genentech ImcloneConsultant: Abbott Molecular

3 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

4 Molecular Heterogeneity in NSCLC

5 Testing Platforms   DNA-based   mutation analyses, FISH, BRISH, SNPs   RNA-based   RT-PCR, quantitative PCR, gene expression arrays   Protein-based   IHC

6 Selecting Clinical Assays TEST A TEST B complete concordance 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 C Partial concordance ? ? TEST D Partial concordance TEST D Partial concordance ? ? ? ? TEST E Partial concordance TEST E Partial concordance ? ? Accuracy, Precision, Cost, Feasibility Precision, Cost, Feasibility Defined reagent, standardized scoring system, validated cut-off STANDARDIZATION ALK Break Apart FISH

7 2p23 ALK Dual Color, Break Apart FISH assay Abbott Molecular normal 3’ 5’ Rearrangement positive - split Rearrangement positive - single 3’ ALK inversion with EML4-ALK fusion

8 Kim et al., JTO, 6(8): , 2011 ALK+ by split Bright-Field In Situ Hybridization CISH ALK+ by single 3’ALK Few publications but already include DIFFERENT: a) criteria for classification of split signal b) cut-offs for classification as ALK+

9 Paik et al, JTO 2011 Novocastra clone 5A4 ALK Protein Expression by IHC: Several Reagents Yi et al, JTO 2011 DAKO clone ALK1 Mino-Kenudson et al., CCR 2010 Cell Signaling clone D5F3 AntibodyAuthorsJournal, YearGroup ALK1BolandHP 2009Mayo DAKOYiJTO 2011Mayo YangJTO 2012Mayo PopatLC 2011London, Royal Marsden Hospital RodigCCR 2009MGH/BWH/DFCC ShawJCO 2009MGH/BWH/DFCC Mino-KenudsonCCR 2011MGH/BWH/DFCC TakeuchiMP 2009Japan, The Cancer Institute D5F3, CellMino-KenudsonCCR 2011MGH/BWH/DFCC Signaling TechnologySalidoJTO 2011Barcelona/Colorado 5A4, AbCamTakeuchiCCR 2009Japan, The Cancer Institute NovocastraPaikJTO 2011Seoul National University KohJTO 2011Seoul National University McLeen-FlorinJTO 2012Grenoble, France

10 ALK IHC: Heterogeneity in Scoring Systems

11 Standardization is urgently needed!!! But likely lack of standardization is not the only reason for discrepancies…

12 Pt. ID % cells FISH positive (BA) EML4-ALK FISH EML4-ALK Variant-specific RT-PCR Insight Genetics qPCR ALK IHC (D5F3) ALK IHC (D9E4) 124%POS E13;A20*NEG 233%POS E13;A20NEG POS 337%POS E6;A20POS 443%POS E13;A20POS 547%POS E6;A20POS 653%POS E13;A20POS 759%POS E18;A20POS 877%POS E6;A20POS 977%POS E13;A20*NEG 1080%POS E6;A20NEG 1180%POS E13;A20POS 1293%POS E13;A20POS Discrepancies Are Likely Also Biology-Based Comparison FISH, PCR and IHC *Positive after 2 rounds of PCR Expression-based Genomic-based Varella-Garcia et al., unpublished data

13 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 Doebele et al., Clin Cancer Res 2011 Implications in selection of further therapies:  2 nd generation ALK inhibitor  Other options

14 Fusion Chromosomal Rearrangement Tumor type 1st ROS1 exon Partner Exon Number of Cases Reference CD74-ROS1t(5;6)(q32;q22)NSCLC 3561 Rikova et al., 2007 Lung adenocarcinoma3462 Li et al., 2011 Lung adenocarcinomaNR 5 Bergethon et al., 2012 Lung adenocarcinoma Takeuchi et al., 2012 NSCLC 3462 Doebele 2012 EZR-ROS1inv(6)(q22q25.3) Lung adenocarcinoma34102 Takeuchi et al., 2012 GOPC(FIG)-ROS2 240 kb interstitial del(6)(q21q21) Glioblastoma cell line U118MG Charest et al., 2003 likely deletionCholangiocarcinoma 3631 Gu et al., 2011 PLOS likely deletionCholangiocarcinoma 3571 Gu et al., 2011 PLOS kb interstitial deletion Low Malignant Potential Serous Ovarian Carcinoma 3571 Birsch et al., 2011 LRIG3-ROS1t(6;12)(q22;q14.1) Lung adenocarcinoma35161 Takeuchi et al., 2012 SLC34A2-ROS1t(4;6)(p15.2;q22) NSCLC cell line HCC78 Rikova et al., 2007 Lung adenocarcinomaNR 1 Bergethon et al., 2012 Lung adenocarcinoma Takeuchi et al., 2012 NSCLC Doebele 2012 SDC4-ROS1t(6;20)(q22;q12) Lung adenocarcinoma3223 Takeuchi et al., 2012 NSCLC Doebele 2012 TPM3-ROS1 t(1;6)(q21.2;q22)Lung adenocarcinoma3582 Takeuchi et al., 2012 Unknown Lung adenocarcinomaNR 12 Bergethon et al., 2012 Lung adenocarcinomaNR 1 Takeuchi et al., lung cancer clinical cases + HCC78 cell line 7 distinct fusions (in 26) + unknown (in 13) Novel Target in NSCLC: ROS1

15 3’ 5’ ROS1 FISH probe for NSCLC TMA ROS1 FISH Positive = Split 3’-5’ or Single 3’ ROS1 FISH+ are likely to benefit from Crizotinib Bergethon et al, 2012; Doebele et al., under review

16 Novel Target in NSCLC: RET Reference KIF5B- RET Fusion Number of Patients Frequency in Tested Cohorts Ju et al, 2012 K16R12 1proband K15R12 11 /5 ADC (20%) K23R12 11 /15 ADC (6.7%) Kohno et al, 2012 K15R12 3 6/319 ADC (1.9%) K16R12 1 K23R12 1 K24R8 1 Takeuchi et al, 2012 K15R /1121 ADC (1.0%) K16R12 1 K22R12 1 K23R22 1 K24R11 1 Lipson et al, 2012 K15R12 8* 12/585 ADC (2.0%) K16R12 3 K22R12 1 K15R11 1* Total Patients33 Activated RET is a target for Vandetanib Ponatinib Sorafinib

17 Negative KIF5B-RET fusion detected by FISH Positive

18 Novel FISH Reagents FGFR1/FGFR2 4-target Enumeration FGFR1/CEP8FGFR2/CEP10 FGFR1/CEP8 FGFR2/CEP10

19 Novel FISH Reagents FGFR1/FGFR2 Enumeration FGFR1/CEP8 FGFR2/CEP10 FGFR1/FGFR2

20 FGFR1/CEP8 FGFR2/CEP10 FGFR1/CEP8 FGFR2/CEP10 FGFR2/CEP10 Novel FISH Reagents Multiplex FGFR1/FGFR2 Enumeration FGFR1/CEP8 FGFR2/CEP10

21 Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart (~10% ADC) 3’ALK/5’ALK 3’ROS1/5’ROS1 Positive for ALK Negative for ROS1

22 Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart (~10% ADC) 3’ALK/5’ALK 3’ROS1/5’ROS1 3’ROS1/5’ROS1 Positive for ALK rearrangement Negative for ROS1 rearrangement normal Single 3’

23 3’ALK/5’ALK 3’ROS1/5’ROS1 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 Novel FISH Reagents Multiplex ALK-ROS1 Break-Apart Time is over, for updates follow us on and normal Single 3’

24 CONCLUSIONS Multiplexing is highly recommended due to scarcity of tumor material in advanced NSCLC 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

25 THANKS 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 Margaret Skokan Mariana Theodoro Adelita Mendoza Yong Gon Cho Antonella Flacco Severine Kako Tara O’Brien Liang-Guo Xu Nathan Schulte


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