Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

// R. Jonas A. Nilsson 1,2,3,* , Niki Karachaliou 4,* , Jordi Berenguer 1 , Ana Gimenez-Capitan 5 , Pepijn Schellen 1,3 , Cristina Teixido 5 , Jihane Tannous 6 , Justine L. Kuiper 7 , Esther Drees 1 , Magda Grabowska 1 , Marte van Keulen 6 , Danielle A. M. Heideman 8 , Erik Thunnissen 8 , Anne-Marie C. Dingemans 9 , Santiago Viteri 4 , Bakhos A. Tannous 6 , Ana Drozdowskyj 10 , Rafael Rosell 4,5,11,12,** , Egbert F. Smit 7,** and Thomas Wurdinger 1,3,6,** 1 Cancer Center Amsterdam, Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Radiation Sciences, Oncology, Umea University, Umea, Sweden 3 ThromboDx B.V., Amsterdam, The Netherlands 4 Translational Research Unit, Dr, Rosell Oncology Institute, Quiron Dexeus University Hospital, Barcelona, Spain 5 Pangaea Biotech SL, Barcelona, Spain 6 Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, Boston, MA, USA 7 Cancer Center Amsterdam, Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands 8 Cancer Center Amsterdam, Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 9 Department of Pulmonary Diseases, Maastricht University Medical Center, Maastricht, The Netherlands 10 Pivotal, Madrid, Spain 11 Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Barcelona, Spain 12 Molecular Oncology Research (MORe) Foundation, Barcelona, Spain * These two authors are co-first authors of the manuscript ** These three authors are co-senior authors of the manuscript Correspondence to: Thomas Wurdinger, email: // Keywords : diagnostics, NSCLC, liquid biopsies, platelets, EML4-ALK Received : August 23, 2015 Accepted : October 06, 2015 Published : November 02, 2015 Abstract Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.