Igor Letovanec1, Stephen Finn2, Panagiota Zygoura3, Paul Smyth2, Alex Soltermann4, Lukas Bubendorf5, Ernst-Jan Speel6, Antonio Marchetti7, Daisuke Nonaka8, Kim Monkhorst9, Henrik Hager10, Miguel Martorell11, Aleksandra Sejda12, Richard Cheney13, Javier Hernandez-Losa14, Eric Verbeken15, Walter Weder16, Spasenija Savic5, Alessia Di Lorito7, Atilio Navarro11, Enriqueta Felip17, Arne Warth18, Paul Baas19, Peter Meldgaard20, Fiona Blackhall21, Anne-Marie Dingemans22, Hendrik Dienemann23, Rafal Dziadziuszko24, Johan Vansteenkiste25, Cathal O'Brien2, Thomas Geiger26, Jon Sherlock27, Jeoffrey Schageman28, Urania Dafni29, Roswitha Kammler26, Keith Kerr30, Erik Thunnissen31, Rolf Stahel32, Solange Peters33. 1. Institute of Pathology, Centre Hospitalier Universitaire Vaudois CHUV, Lausanne, Switzerland. Electronic address: Igor.Letovanec@chuv.ch. 2. Department of Histopathology, St James's Hospital and Trinity College, Dublin, Ireland. 3. Frontier Science Foundation-Hellas, Athens, Greece. 4. Institute of Pathology and Molecular Pathology, University Hospital Zürich, Zürich, Switzerland. 5. Institute of Pathology, University Hospital Basel, Basel, Switzerland. 6. Department of Pathology, Maastricht University Medical Center, Maastricht, The Netherlands. 7. Center of Predicitve Molecular Medicine, CeSI, University of Chieti-Pescara, Chieti, Italy. 8. Department of Histopathology, The Christie National Health Service Foundation Trust, Manchester, United Kingdom. 9. Division of Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. 10. Department of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark. 11. Department of Pathology, Consorcio Hospital General Universitario de Valencia, Valencia, Spain. 12. Department of Pathomorphology, Medical University of Gdansk, Gdansk, Poland. 13. Department of Pathology, State University of New York at Buffalo, Buffalo, New York. 14. Department of Pathology, Vall d'Hebron University Hospital, Barcelona, Spain. 15. Department of Pathology, University Hospital KU Leuven, Leuven, Belgium. 16. Department of Thoracic Surgery, University Hospital Zürich, Zürich, Switzerland. 17. Medical Oncology Department, Vall d'Hebrone University Hospital, Barcelona, Spain. 18. Department of Pathology, Heidelberg University Hospital, Heidelberg, Germany. 19. Department of Thoracic Oncology, The Netherlands Cancer Institute-Antoni Van Leeuwenhoek Hospital, Amsterdam, The Netherlands. 20. Department of Oncology, Aarhus University Hospital, Aarhus, Denmark. 21. Deparment of Medical Oncology, The Chrisite NHS Foundation Trust, Manchester, United Kingdom. 22. Department of Pulmonology, Maastricht University Medical Center, Maastricht, The Netherlands. 23. Department of Surgery, Thoraxklinik at Heidelberg University, Heidelberg, Germany. 24. Department of Oncology and Radiotherapy, Medical University of Gdansk, Poland. 25. Department of Respiratory Oncology, University Hospital KU Leuven, Leuven, Belgium. 26. European Thoracic Oncology Platform, Bern, Switzerland. 27. Thermo Fisher Scientific, Paisley, United Kingdom. 28. Thermo Fisher Scientific, Austin, Texas. 29. Frontier Science Foundation-Hellas & University of Athens, Athens, Greece. 30. Department of Pathology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom. 31. Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. 32. Clinic of Oncology, University Hospital Zürich, Zürich, Switzerland. 33. Department of Oncology, Centre Hospitalier Universitaire Vaudois CHUV, Lausanne, Switzerland.
Abstract
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.
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.