Next-Generation Sequencing for ALK and ROS1 rearrangement detection in Non-Small Cell Lung Cancer patients: Implications of FISH positive patterns

Sergi Clavé1,2, Natàlia Rodón3, Lara Pijuan1, Olga Díaz3, Marta Lorenzo1,2, Pedro Rocha4,  Álvaro Taus2,4, Remei Blanco5, Joaquim Bosch-Barrera6, Noemí Reguart7, Noelia de la Torre8, Gloria Oliveras6, Blanca Espinet1,2, Beatriz Bellosillo1,2, Xavier Puig3, Edurne Arriola2,4†, Marta Salido1,2†

1 Pathology Department, Hospital del Mar, Barcelona, Spain, 2 Cancer Research Program, Hospital del Mar Medical Research Institute, Barcelona, Spain, 3 BIOPAT Biopatologia Molecular, Grup Assistència, Barcelona, Spain, 4 Medical Oncology Department, Hospital del Mar, Barcelona, Spain, 5 Medical Oncology Department, Consorci Sanitari de Terrassa, Terrassa, Spain, 6 Medical Oncology Department, Catalan Institute of Oncology, Hospital Dr. Josep Trueta, Girona, Spain, 7 Medical Oncology Department, Hospital Clinic, Barcelona, Spain, 8 Pathology Department, Consorci Sanitari de Terrassa, Terrassa, Spain


Detection of ALK and ROS1 gene rearrangements in non-small cell lung cancer (NSCLC) is required for directing patient care. While fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) have been established as gold standard methods, next-generation sequencing (NGS) platforms are called to be at least equally successful. Comparison of these methods for translation into daily use is currently under investigation.

Patients and Methods

Forty NSCLC paraffin-embedded samples with previous ALK (n=33) and ROS1 (n=7) FISH results were examined with the Oncomine Focus Assay and tested for ALK and ROS1 immunoreactivity. Clinical implications of concurrent molecular alterations and concordance between methods were evaluated.


NGS was successful in 32 cases (80%): 25 ALK and seven ROS1. Few concomitant alterations were detected: one ALK rearranged case had an ALK p.L1196M resistant mutation, four had CDK4, MYC and/or ALKamplifications, and one ROS1 rearranged case showed a FGFR4amplification. Comparison between techniques revealed five discordant cases (16%) that had lower progression-free survival than concordant cases: 7.6 [95% CI: 2.2-13] vs. 19.4 [95% CI: 10.1-28.6]. Remarkably, four of these cases had isolated 3′ signal FISH pattern (p= 0.026).


Our data supports that the identification of 3′ isolated signal FISH pattern in ALK and ROS1 cases might suggest a false positive result. NGS seems a reliable technique to assess ALK and ROS1 rearrangements offering the advantage over IHC of detecting other molecular alterations with potential therapeutic implications.


                                                                                                  Clinical Lung Cancer 2019;20(4):421-429