Abstract 812: Lack of intra-tumoral heterogeneity in lung adenocarcinoma supports gene fusions involving ALK as early clonal events
Authors: Varella-Garcia Marileila , Flacco Antonella , Cho Yong Gon , Tan Aik-Choon , Kono Scott , Franklin Wilbur , Cappuzzo Federico , Crino Lucio , Doebele Robert , Camidge D. Ross 
Published: February 2011

Abstract
Adenocarcinomas account for half of the non-small cell lung carcinomas (NSCLC) in the US. Lately, molecularly distinct subtypes of lung adenocarcinomas have been described with EGFR, KRAS and ALK genes as major drivers. Gene fusions activating ALK tyrosine kinase function have been described in approx 3% of unselected NSCLC; but in over 20% of adenocarcinomas without mutations in RAS and EGFR from never /light smokers. The most frequent ALK partner in lung cancer is EML4; other reported partners are KIF5B and TFG. There are recent compelling reports that ALK inhibitors are especially efficacious in advanced NSCLC carrying ALK rearrangements. However, questions have been raised regarding the role of ALK fusions in lung cancer development because (a) EML4-ALK fusions were reported in non-tumor cells of NSCLC specimens by PCR-based assays and (b) not all tumor cells harbored the rearrangement in FISH-based assays, which was taken to indicate that ALK fusions were either late-stage aberrations and/or not primary cancer drivers. To test these hypotheses, we investigated the patterns of fluorescence signals generated by the ALK Break Apart FISH probe (Abbott Molecular) in tumor and non-tumor cells of multiple lung areas from 50 patients diagnosed with adenocarcinoma (25 positive and 25 negative for ALK rearrangements). The FISH probe includes sequences contiguous to the 5’ end of ALK (promoter) and the 3’ end of ALK (tyrosine kinase domain). A fused 3’/5’ signal is observed when ALK gene is in native status and split or single signals are observed when a rearrangement involving ALK occurs. However, split or single signals may be due to technical artifacts such as nuclear truncation. Cells carrying split signals or single 3’ ALK were considered positive for rearrangement and when representing &gt;20% of scored cells the tumor was classified as ALK positive. Among the 25 ALK negative patients, there was no difference in the frequencies of cells positive for rearrangement between tumor and non-tumor areas, all ranging between 0 to 20%. Similar results were found among the non-tumor areas of the 25 ALK positive patients. Conversely, these patients showed &gt;30% of cells positive for rearrangement in &gt;90% of tumor areas. Five tumor areas were required to correctly detect positive and negative specimens. The positive cells were always diffusely distributed along the tumor areas and lower frequencies in some areas were likely due to nuclear truncation and scoring criteria (gap of ≥2 signal diameters between the 3’ and 5’ signals required to be called split). When the minimum gap was decreased to 1 signal diameter, &gt;10% cells were additionally called positive. In conclusion, our findings (a) support the hypothesis that ALK rearrangement is not a focal phenomenon in advanced adenocarcinoma but rather an early tumorigenesis event, and (b) highlight the importance of immediate standardization of technical procedures.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 812.