Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients Pablo Martinez 1 *, Javier Herna ´ ndez-Losa 2 ,M a A ´ ngeles Montero 2 , Susana Cedre ´s 1 , Josep Castellvı´ 2 , Alex Martinez-Marti 1 , Natalia Tallada 2 , Nuria Murtra-Garrell 1 , Alejandro Navarro-Mendivill 1 , Victor Rodriguez-Freixinos 1 , Mercedes Canela 3 , Santiago Ramon y Cajal 2 , Enriqueta Felip 1 1 Medical Oncology Department, Vall d’Hebron University Hospital, Universidad Auto ´ noma de Barcelona, Barcelona, Spain, 2 Pathology Department, Vall d’Hebron University Hospital, Universidad Auto ´ noma de Barcelona, Barcelona, Spain, 3 Thoracic Oncology Department, Vall d’Hebron University Hospital, Universidad Auto ´ noma de Barcelona, Barcelona, Spain Abstract Background: Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients. Methods: NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored. Results: ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative. Conclusions: ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients. Citation: Martinez P, Herna ´ndez-Losa J, Montero MA ´ , Cedre ´s S, Castellvı ´ J, et al. (2013) Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients. PLoS ONE 8(1): e52261. doi:10.1371/journal.pone.0052261 Editor: Mohammad O. Hoque, Johns Hopkins University, United States of America Received June 25, 2012; Accepted October 31, 2012; Published January 24, 2013 Copyright: ß 2013 Martinez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no funding or support to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pamartinez@vhebron.net Introduction Lung cancer is the most frequent cause of cancer-related death worldwide, accounting for more than 1 million deaths per year. [1] Although cytotoxic chemotherapy remains the mainstay of treatment for the majority of patients with advanced non-small cell lung cancer (NSCLC), [2] the identification of specific genetic lesions which drive proliferation of cancer cells has led to the development of new target therapies in a subset of patients with NSCLC [3,4]. In recent years, anaplastic lymphoma kinase (ALK) rearrangement, predominantly with the echinoderm microtubule- associated protein like 4 (EML4) gene, has been identified as an oncogenic event in a subset of NSCLC patients [4]. ALK translocation results in the constitutive expression of the tyrosine kinase domain of ALK protein, which results in tumor develop- ment and growth. The oncogenic dependence of this event is demonstrated on the basis that removal ALK kinase activity reverses the malignant pattern and growth [5]. Recently, results of a phase 1 trial evaluating an ALK inhibitor, Crizotinib, in patients with ALK positive NSCLC demonstrated encouraging results [6]. Clinical trials with Crizotinib and other ALK inhibitors in this subset population of ALK positive NSCLC patients are ongoing. Initial reports have shown that ALK positive NSCLC patients tend to be younger, predominantly non/light smokers with an adenocarcinoma histology than the overall NSCLC patients population [7]. These clinicopathological features are also frequent in patients with EGFR mutations, but both genetic events seem to be mutually exclusive [8]. In unselected patients with NSCLC the prevalence of ALK positivity range from 1% to 7% [9], but more than 30% in patients selected for EGFR Wild- Type (WT), adenocarcinoma and no smoking history [7]. Its PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e52261