AbstractBackgroundSimultaneous genotyping has advantages in turnaround time and detecting the real mutational prevalence in unresectable non‐small‐cell lung cancer (NSCLC), a group not previously genetically characterized.MethodsWe developed simultaneous panel of screening EGFR and KRAS mutations by direct sequencing or PNA clamping, and ALK rearrangement by fluorescent in situ hybridization (FISH) in multicenter manner.ResultsOf 510 NSCLC Korean patients, simultaneous genotyping identified mutations of EGFR (29.0%) and KRAS (8.6%) and rearrangement of ALK (9.2%). Seven patients had overlaps in mutations. Although several well‐known associations between genotypes and clinical characteristics were identified, we found no relationship between ALK rearrangement and sex or smoking history. Unlike the other genotype mutations, ALK rearrangement was associated with advanced disease. Among the ALK‐negative group, patients with 10–15% of ALK FISH split shared characteristics, such as younger age and advanced stage disease, more with the ALK‐positive group (>15% ALK FISH split) than <10% ALK FISH split group.ConclusionsSimultaneous panel genotyping revealed more prevalent ALK rearrangements than reported in previous studies and their strong association with advanced stage irrespective of sex or smoking history. ALK rearrangement seems to be a marker for aggressive tumor biology and should be assessed in advanced disease. J. Surg. Oncol. 2014 110:245–251. © 2014 Wiley Periodicals, Inc.