Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

et al. (2014) Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry. PLoS ONE 9(9): e107200. doi:10.1371/ journal.pone.0107200 Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry Esther Conde 0 Ana Sua rez-Gauthier 0 Amparo Benito 0 Pilar Garrido 0 Rosario Garca-Campelo 0 Michele Biscuola 0 Luis Paz-Ares 0 David Hardisson 0 Javier de Castro 0 M. Carmen Camacho 0 Delvys Rodriguez-Abreu 0 Ihab Abdulkader 0 Josep Ramirez 0 Noem Reguart 0 Marta Salido 0 Lara Pijua n 0 Edurne Arriola 0 Julia n Sanz 0 Victoria Folgueras 0 Noem Villanueva 0 Javier Go mez-Roma n 0 Manuel Hidalgo 0 Fernando Lo pez-Ros 0 Renato Franco, Istituto dei tumori Fondazione Pascale, Italy 0 1 Laboratorio de Dianas Terape uticas, Centro Integral Oncolo gico ''Clara Campal'', Hospital Universitario Madrid Sanchinarro, Universidad San Pablo-CEU , Madrid , Spain , 2 Hospital Ramo n y Cajal , Madrid, Spain, 3 C.H.U. A Corun a, La Corun a, Spain , 4 Hospital Virgen del Roc o , Sevilla , Spain , 5 IdiPAZ (Hospital La Paz Institute for Health Research), University Hospital La Paz, Faculty of Medicine, Autonomous University of Madrid , Madrid , Spain , 6 Hospital Insular de Gran Canaria, Las Palmas de Gran Canaria , Spain, 7 C.H.U. Santiago de Compostela, Santiago de Compostela , Spain , 8 Hospital Clinic , Barcelona , Spain , 9 Hospital del Mar-Parc de Salut Mar , Barcelona , Spain , 10 Hospital Cl nico San Carlos , Madrid , Spain , 11 Hospital Central de Asturias , Oviedo , Spain , 12 Hospital Marque s de Valdecilla , Santander , Spain , 13 Oncology Department, Centro Integral Oncol o gico ''Clara Campal'', Hospital Universitario Madrid Sanchinarro, Universidad San Pablo-CEU , Madrid , Spain Background: Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods: Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results: All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions: The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations. - Funding: This work was partially supported by Abbott, Pfizer, Fundacio n Mutua Madrilen a and Fondo de Investigaciones Sanitarias (PI11/02866). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: E. Conde and J. Go mez-Roman: honoraria, Pfizer. M. Salido: Advisory Board, Pfizer. E. Arriola: Advisory Board and Research Funding, Pfizer. F. Lo pez-Ros: Advisory Board and Research Funding, Pfizer. Research funding: Abbott and Ventana Medical Systems. Ventana Medical Systems provided reagents free of charge. There are no further patents, products in development or marketed products to declare. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. In August 2011, crizotinib, a novel ALK tyrosine kinase inhibitor, was approved by the US FDA for the treatment of patients with locally advanced or metastatic non-small-cell lung carcinomas (NSCLCs) that are ALK-positive as detected by an FDA-approved test (i.e. Vysis ALK FISH Break-Apart Probe Kit) [1]. Soon afterwards, the drug was approved by the EMA, with the statement that an accurate and validated ALK assay is necessary for the selection of patients [2]. Based on these excellent results of the crizotinib clinical trials and the development of other ALK inhibitors with consistent efficacy results in this patient population, the importance of accurately identifying ALK positive lung cancer has never been greater [3]. Few areas in cancer biomarkers have been as contentious as HER2 testing in breast cancer patients. Since 1998, we have witnessed a huge clinical advance in this field and, however, a great biomarker conundrum over methods, cut-off points, and algorithms [immunohistochemistry (IHC) versus fluorescence in situ hybridization (FISH) as the primary testing assay] [4,5]. The outcome is a significant percentage of false negative (12%) or false positive results (14%) [6]. This controversy is also entering the field of NSCLC ALK testing [7], with an increasing number of recent publications addressing discordances between in situ hybridization and IHC assays [814], further fuelled by the different regulatory approvals and the arrival of other ALK inhibitors [3,15]. While some groups recommend initial IHC followed by FISH confirmation of some IHC-positive cases [14,16], others believe the detection of ALK rearrangements is improved when using two methodologies [9,17]. This situation prompted us to investigate two IHC antibodies, using a novel ultrasensitive detection-amplification kit, and an automated FISH scanning system in a series of tumor samples to obtain supporting data for an ALK testing algorithm [18]. To our knowledge, there has not been an independent assessment of ALK concordance between these three assays using our strategy (i.e., FDA-cleared automated FISH scanning system) in a large series of ALK positive tumors. Material and Methods Tumor samples Seventy-nine ALK FISH-positive samples from patients with advanced NSCLCs procured at 11 hospitals were used for this study. The Institutional Ethics Committee at Grupo Hospital de Madrid reviewed and approved this study and waived the need for consent. Samples were consecutive ALK positive cases, initially tested as part of routine clinical care. In addition, 77 consecutive ALK FISH-negative samples from advanced NSCLCs diagnosed at the referral institution were included as negative controls. The material available for all tumors had been formali (...truncated)