Fibroblast Growth Factor Receptor 1 Amplification in Non-Small Cell Lung Cancer by Quantitative Real-Time PCR

et al. (2013) Fibroblast Growth Factor Receptor 1 Amplification in Non-Small Cell Lung Cancer by Quantitative Real-Time PCR. PLoS ONE 8(11): e79820. doi:10.1371/journal.pone.0079820 Fibroblast Growth Factor Receptor 1 Amplification in Non-Small Cell Lung Cancer by Quantitative Real-Time PCR Shirish M. Gadgeel 0 Wei Chen 0 Michele L. Cote 0 Aliccia Bollig-Fischer 0 Susan Land 0 Ann G. Schwartz 0 Gerold Bepler 0 A R M Ruhul Amin, Winship Cancer Institute of Emory University, United States of America 0 Karmanos Cancer Institute & Department of Oncology, Wayne State University , Detroit, Michigan , United States of America Introduction: Amplification of the fibroblast growth factor receptor 1 (FGFR1) gene has been described in tumors of non-small-cell lung cancer (NSCLC) patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance. Materials and Methods: We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application. Results: In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025) for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption) as compared to light smokers. Discussion: Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors. - Funding: Supported by Department of Defense grant W81XWH-11-1-0050. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing interests: The authors have declared no competing interests exist. A paradigm shift in the management of non-small-cell lung cancer (NSCLC) patients has been the identification of therapeutically actionable driver genetic alterations [1]. The number of these genetic alterations is steadily increasing [2]. However, most alterations have been identified in adenocarcinomas of the lung. Therefore, the therapeutic impact of this paradigm shift has been minimal for patients with squamous cell carcinoma of the lung. Recently, amplification of the fibroblast growth factor receptor 1 (FGFR1) gene has been described as an oncogenic alteration in a subgroup of squamous cell carcinomas [3,4]. FGFR1 belongs to the FGFR family of receptors and is involved in inflammation, wound healing and embryonic development. Since the FGFR family of receptors appears to have a role in many cancers, several inhibitors of FGFR are being developed [5]. A single case report has shown that the FGFR inhibitor BGJ398 did demonstrate partial response in a patient with squamous cell lung carcinoma whose tumor was amplified for FGFR1 [6]. An essential aspect for therapeutic targeting of genetic alterations in lung cancer is the rapid, specific, and precise identification of alterations in patient samples. Many investigators have utilized fluorescent in situ hybridization (FISH) to detect FGFR1 amplification [7-11]. The definition of FGFR1 amplification has varied among the various reports. In addition, FISH analysis is laborious, technically complex, and reader dependent. These characteristics limit its clinical applicability. We developed a quantitative, real-time PCR test, which is easier to perform and robust in its interpretation, to evaluate NSCLCs for FGFR1 amplification and assessed the clinical characteristics and prognostic relevance of this genetic alteration. Our ultimate goal is to be able to identify NSCLC patients that can derive clinical benefit from FGFR1 inhibitors utilizing this PCR based test. Patients and Methods Specimen collection and outcomes data Collection of biospecimens and outcomes data complied with the Helsinki Declaration and was approved by the Wayne State University School of Medicine Institutional Review Board. Tumor materials used in this research were from patients who provided written informed consent. Fresh-frozen tumor specimens were collected prospectively from patients who underwent a surgical resection for diagnosed or suspected lung cancer. All patients who were candidates for surgical resection of their lung cancer, either biopsy proven or suspected, were consented for specimen collection. Only patients whose tumors were confirmed to be NSCLC were included in this analysis. Specimens from patients with a final diagnosis of small cell carcinoma (N=16) or a small cell component of NSCLC (N = 2), carcinoid tumors (N = 6), or mesothelioma (N = 3) were excluded. Specimens were kept frozen at -80C in aliquots of approximately 0.1 g. Specimen procurement procedures were developed to reduce the resection to freezing time interval to less than 30 min. The quality of extracted analytes was assured by performing integrity analysis. The overall procurement period ranged from 1985 to 2001. Formalin-fixed and paraffin-embedded specimens were reviewed to verify diagnosis and to determine tumor cell content. Specimens were uniquely identified by laboratory numbers that allowed cross-referencing with clinical data from the tumor registry and chart review without disclosure of patient identity. Demographic and clinical outcomes data collected included the dates of birth, diagnosis (defined as the date of first pathologic verification of malignancy), surgical resection (same as the date of specimen procurement), and date of last follow-up or death; sex, race, tumor histology, pathological and clinical tumor stage (version 6), performance status, weight loss (defined as >5% during the 3 months proceeding surgery), and self-reported smoking history (defined as life-time never smoker for those who had smoked <100 cigarettes, former smoker for those who had quit cigarette smoking for more than one year, and smoker for all others). One of the patients included in this analysis had preoperative chemotherapy and radiation and one had preoperative radiation. None of the patients had adjuvant therapy. A standardized protocol for patients follow-up evaluation was not specified. Specimens were divided into a training and non-overlapping validation cohort. The det (...truncated)