Abstract B45: Analysis of c-Met gene copy number by dual-color silver in situ hybridization (SISH) in non-small cell lung cancer (NSCLC)
Authors: Wynes Murry W. , Dziadziuszko Rafal , Singh Shalini , Ranger-Moore James , Szostakiewicz Barbara , Dziadziuszko Katarzyna , Jassem Jacek , Hirsch Fred R. 
Published: June 2012

Background: c-Met is a receptor capable of activating downstream proliferative, survival and invasive pathways. Numerous human malignancies, including NSCLC, show over-expressed and amplified c-Met. Elevated expression of c-Met and its ligand, HGF, have been associated with inferior prognosis. The aim of this study was to evaluate the prognostic relationship of c-Met gene copy number in surgically-treated NSCLC patients (pts) using dual-color silver in situ hybridization. SISH is advantageous because bright field microscopy allows visualization of intact malignant and non-malignant cell morphology.

Methods: Experimental SISH probes, Ventana Medical Systems, Inc. (Tucson, AZ), were used to evaluate c-MET and CH 7 copy numbers on a tissue microarray containing triplicate samples from 189 pts (median follow-up 5.3 yrs and 5-yr survival probability 38%). Evaluable results, at least one core with valid c-Met and CH 7 counts, were obtained for 140 pts. There were 109 males; 80 squamous cell carcinomas; and 37 adenocarcinomas. The pathological stages were I: 50, II: 33 and III: 46, IV: 8 and unknown: 3. A certified pathologist counted signals (1 for individual signal, 6 for small cluster and 12 for big cluster) in 50 nuclei for each core and the mean number of c-Met and CH 7 copies/nucleus/core were determined, along with the ratio. The triplicate cores were averaged and compared to the core with the highest score.

Results: There was very high correlation between the highest core and the average of the triplicate cores: c-Met (r=0.98), CH 7 (r=0.96), and ratio (r=0.98). The highest core value was used for further analysis. The median copy numbers were: c-Met 3.1 (1.7–11.8), CH7 2.4 (1.5–2.4), and ratio 1.3 (0.6–6.3). There were 14 pts with greater than or equal to five c-Met genes/nucleus and 3 pts with clusters. Using the median as a cut-off there was no statistical difference related to age, gender, pathological stage, histology, grade or smoking. Kaplan-Meier analysis revealed no significant differences in PFS or OS using the median scores, however there was a trend for increased PFS and OS in patients with high copy numbers. Cox regression analysis examining copy number as a continuous variable, and accounting for other variables, showed a similar trend of protection but statistical significance was not achieved: c-Met (PFS- HR 0.88 (0.72–1.07, p=0.19, OS- HR 0.90 (0.73–1.10), p=0.29); CH7 (PFS- HR 0.88 (0.57–1.34, p=0.54, OS- HR 1.02 (0.66–1.57), p=0.95): ratio (PFS- HR 0.84 (0.54–1.30, p=0.43, OS- HR 0.79 (0.49–1.28), p=0.33). Analysis of the data using a cut-off of 5 for c-Met gave similar results.

Conclusions: This study shows the clinical feasibility of SISH technology for c-Met gene copy number assessment. There was a trend for increased PFS and OS in pts with increased c-Met, CH 7 or the ratio between the two. Further studies are needed to determine the predictive value of c-Met gene copy number in pts receiving molecular targeted therapeutics.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B45.