Poor Survival and Cigarette Smoking Dosage Fibroblast Growth Factor Receptor 1 Gene Amplification Is Associated with Poor Survival and Cigarette Smoking Dosage in Resected Squamous Cell Lung Cancer Byoung Chul Cho, M.D., Ph.D. Yonsei Cancer Center Yonsei University College of Medicine JE-UK Laboratory of Molecular Cancer Therapeutics FALCON (Fight Against Lung Cancer Oncology Network) 1

Lung Cancer Mutation Consortium Incidence of Driver Mutations in Adenocarcinoma ROS1 In a recent trial performed by LCMC, targetable driver mutations could be found in about 60% of lung adenocarcinomas. Mutation found in 54% (280/516) of tumors completely tested (CI 50-59%) Kris et al ASCO 2011 2

Squamous Cell Carcinoma of Lung Lung squamous cell carcinoma (SqCC) accounts for ~30% of non-small cell lung cancer ~90% are male smokers (Korean Cancer Registry) Despite advances in personalized treatment of adenocarcinoma, effective targeted therapies for SqCC has remained elusive Lung SqCC accounts for about 30% of NSCLC. 90% are male smokers. In a big contrast to lung ADC, there is no effective targeted therapies in lung SqCC. Therefore, there is an urgent need to identify any druggable target in this disease. Currently, lung SqCC lacks any druggable target

Frequencies of Potential Driver Mutations in Lung Squamous Cell Carcinoma Frequency (%) EGFR <5 ALK HER2 BRAF KRAS PIK3CA AKT1 MAP2K1 MET ~70% unknown You will realize this is really true when you see this Table. Most well-known driver mutations rarely occur in SqCC mostly less than 5% including EGFR mut, which is prevanlent in lung ADC. Therefore, I want the audience to get more interested in gene amp of EGFR and FGFR1 which occur at a relatively high frequency. Recently, our group generated data suggesting that these two genetic alterations may be promising targets, and I will present the data later on. Lancet oncol 2011;12:175

FGFR1 is Amplified in Lung Squamous Cell Carcinoma By FISH, high FGFR1 amplification (≥ 9 copies): 22% (34/153) 8p.12 Recently, investigators identified significant amplification peak in chromosome 8p12 loci, which includes FGFR1. Subsequently analysis revealed frequent FGFR1 amplification in SqCC, but not in other subtypes, and the incidence of FGFR1 amp was 22%. Weiss J. Sci Transl Med 2010

FGFR1 amplifications are associated with FGFR inhibitor activity Treatment with FGFR inhibitor showed induction of apoptosis, downstream inhibition in FGFR1 amplified cell lines, and strong antitumor activity in xenograft model. These data strongly support the utility of FGFR1 amp as a relevant therapeutic target in lung SqCC. Weiss J. Sci Transl Med 2010

Study Purpose prognostic impact of FGFR1 amplification To investigate the frequency and the prognostic impact of FGFR1 amplification in surgically resected lung SqCC To evaluate the association between smoking does and FGFR1-amplification So, we performed the following study to

Patient and Method SqCC patients that underwent radical resection of a primary lung cancer at Severance Hospital between 1998 and 2009. Selection criteria (n= 262): availability of tumor tissue from the primary lung cancer, smoking-data, and survival data Construct a tissue-microarray with 2-mm diameter cores (3 cores per tumor) 262 pts who underwent radical resection for lung SqCC were selected based on availability of tumor tissue, smoking data, and survival data. We constructed tissue microarray with 3 cores per each tumor.

Gene Copy Number FGFR1 FISH assay was performed on the tissue- microarrays using FGFR1-probe that hybridizes to the band 8p12-8p11.23 with Spectrum Orange (red) and CEP 8 with Spectrum Green (Abbott Molecular®) Prespecified Criteria1 “high-amplification” FGFR1 copy number ≥ 9 “low-amplification” FGFR1 copy number >2 or <9 “disomy” FGFR1 copy number = 2 FGFR1 FISH assay was perforemd on tissue microarray using FGFR1 probes supported by Abbott. FGFR1 GCN was classified into 3 strata based on a previous report, and high amp was defined as 9 or more copies of FGFR1. 1Weiss J et al. Sci Transl Med 2010

FGFR1 protein & mRNA Expression IHC analysis was performed using FGFR1 Ab (Epitomics, Burlingame, CA) Only clear membranous staining of the tumor cells was considered positive and cytoplasmic or granular staining was considered negative or trace Scoring system (0-400): % of positive tumor cells (0% to 100%) X dominant staining pattern (1: negative or trace, 2: weak, 3: moderate, 4: intense) mRNA expression analysis was performed by QuantiGene Reagent Systems in FFPE tissue samples To correlate GCN with protein and mRNA expression, we performed IHC using FGFR1 Ab produced by Epitomics, and mRNA expression analysis using QuantiGene reagent system in FFPE tissue. Immunochemical staining was scored from 0 to 400 as previously reported.

Patient characteristics according to FGFR1 gene amplification by FISH By definition, disomy was found in about 50% of pts High FGFR1 amplification : 13% (34/262) Smoking History and dose is strongly correlated with FGFR1 amplification

FGFR1 IHC staining & Gene Copy Number by FISH FISH-assay was performed on the tissue-microarrays using FGFR1-probe that hybridizes to the band 8p12-8p11.23 with Spectrum Orange (red) and CEP 8 with Spectrum Green (Abbott Molecular®) following routine methods 34 (13%) 105 (40.1%) 123 (46.9%)

Association between FGFR1 GCN and FGFR1 protein & mRNA Expression In addition to the analysis of FGFR1 amplification, we also evaluated the correlation between FGFR1 amplification by FISH and FGFR1 expression using mRNA and IHC analyses. Results from mRNA analysis indicated high correlation between FGFR1 copy numbers and FGFR1 mRNA expression levels. As shown in Figure 2, a group with FGFR1 high amplification shows higher mRNA expression levels than those with FGFR1 low amplification and disomy (Mean FGFR1/PPIB mRNA ratio; high amp vs. low amp and disomy, P < 0.0001). Moreover, IHC analysis also demonstrated similar results (Supplementary Figure 1 and 2).

FGFR1 Amplification Is Associated with Poor Survival in Resected Lung SqCC Patients Comparison of survival outcomes between the FGFR1 amp+ ( FGFR1 ≥ 9) and the FGFR1 amp- (FGFR1 < 9) in total patients Survival outcome (DFS, OS) in FGFR1 amp+ is shorter than those in FGFR1 amp– patients. FGFR1 high amp FGFR1 high amp Kim HR, Soo RA, Cho BC. J Clin Oncol 2012

Disease-free survival Multivariate Survival Analyses Variable Category Disease-free survival Overall survival HR 95% CI P Sex Female vs. male 0.68 0.26-1.74 0.42 0.70 0.27-1.79 0.46 Pathologic stage III vs. I+II 2.24 1.45-3.45 <0.0001 2.78 1.76-4.38 Smoking Smoker vs. never smoker 1.60 0.84-3.05 0.15 1.35 0.70-2.58 0.35 Adjuvant therapy Yes vs. no 1.13 0.74-1.73 0.56 1.08 0.68-1.72 0.71 FGFR1 FISH FGFR1 amp+ vs FGFR1 amp- <0.001 1.83 1.15-2.89 0.01

Treatment outcome to EGFR-TKI according to FGFR1 Amplification When we examined the treatment outcome to EGFR-TKIs according to FGFR1 amplification in unresectable lung SqCC, there was no difference in terms of PFS & OS between FGFR1 amp+ and FGFR1 amp-. . This may be due to Small sample size, later line of therapy and mutational evolution of tumors. Independent dataset of unresectable, previously treated lung SqCC (N= 47)

Among patients with > 45pack- years. ~ 20% patients has FGFR1 amp+ FGFR1 Amplification is a Smoking-associated Oncogenic Mutation Another interesting finding in our study was that the incidence of high FGFR1 amplification is significantly higher in current smokers compared with never or former smokers and that FGFR1-amplification is associated with cigarette-smoking in a dose-dependent manner. Among patients with > 45pack- years. ~ 20% patients has FGFR1 amp+ P value was tested by χ2 test for linear trend Kim HR, Soo RA, Cho BC. J Clin Oncol 2012

Intratumoral Heterogeneity? Whole tissue section FISH in 51 randomly selected tumors (31 high FGFR1-amp, 10 low FGFR1-amp and 10 disomy tumors) To further estimate the intratumoral heterogeneity of FGFR1 gene copy number, we analyzed whole tissue sections from a total of 51 randomly selected tumors. We also examined FGFR1 amplification in peritumoral normal tissue.

Summary of Whole Tissue Section FISH Homogenous FGFR1 distribution in high amplified tumors - at least 80% of nuclei per area exhibited ≥ 9 copies of FGFR1 Majority of areas displayed low amplified FGFR1 signals In low amplified tumors Two FGFR1 signals were homogenously distributed in disomy cases No FGFR1 amplification in peritumoral normal tissue High correlation of FGFR1 GCN between primary & metastatic lesion1 We observed…. FGFR1 amplification may be involved not in early tumorigenesis, but in early disease progression 1Friederike Goeke et al. Chest Apr 12, 2012

Conclusion: FGFR1 Amplification- A New “Druggable” Target in SqCC The first high-frequency (13%) therapeutic target of smoking-associated SqCC FGFR1 amplification induced a strong FGFR1 dependency in FGFR1 amplified SqCC FGFR1 amplification is a negative prognostic factor in resected lung SqCC FGFR1-amplification is associated with cigarette-smoking in a dose-dependent manner Strongly implies that FGFR1-amplification is an oncogenic-aberration caused by tobacco-carcinogen 그동안 SCC에서는 therapeutically targettable genetic alteration이없었다. 8P12-specific probe; FGFR1 amplification sensitize tumor to FGFR1 signalling. Knockdown assay나 inhibition시 apoptosis induction; SOX2 TF encoding gene therapeutic targeting uncertain