Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project

Igor Letovanec*, Stephen Finn, Panagiota Zygoura, Paul Smyth, Alex Soltermann, Lukas Bubendorf, Ernst Jan M. Speel, Antonio Marchetti, Daisuke Nonaka, Kim Monkhorst, Henrik Hager, Miguel Martorell, Aleksandra Sejda, Richard Cheney, Javier Hernandez-Losa, Eric Verbeken, Walter Weder, Spasenija Savic, Alessia Di Lorito, Atilio NavarroEnriqueta Felip, Arne Warth, Paul Baas, Peter Meldgaard, Fiona Blackhall, Anne Marie Dingemans, Hendrik Dienemann, Rafal Dziadziuszko, Johan Vansteenkiste, Cathal O'Brien, Thomas Geiger, Jon Sherlock, Jeoffrey Schageman, Urania Dafni, Roswitha Kammler, Keith M. Kerr, Erik Thunnissen, Rolf A. Stahel, Solange Peters, Rafael Rosell, Miguel Ángel Molina, Anita Hiltbrunner, Nesa Marti, Zoi Tsourti, Varvara Polydoropoulou, Steven Gray, Isabelle Opitz, Alessandra Curioni, Didier Lardinois, William Mathieson, European Thoracic Oncology Platform Lungscape Consortium

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

65 Citations (Scopus)


Introduction: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. Methods: A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). Results: NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. Conclusion: NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

Original languageEnglish
Pages (from-to)413-425
Number of pages13
JournalJournal of Thoracic Oncology
Issue number3
Publication statusPublished - Mar 2018


  • ALK
  • NGS
  • RT-PCR


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