br Study approval br Author contributions br Conflicts of

Study approval
Author contributions
Conflicts of interest
Acknowledgements This work was supported by grants of the Youth Backbone Program (to Jianming Ying) of Cancer Hospital, CAMS, Beijing, Beijing Hope Run Special Fund of Cancer Foundation of China (LC2015A06) and the National Natural Science Foundation of China (NSFC No. 81650024).
Introduction Lung cancer remains a major cause of cancer deaths. Treatment of non–small-cell lung cancer (NSCLC) has improved by our better understanding of the molecular mechanisms involved in tumor initiation and progression, mainly in adenocarcinoma. The discovery of EGFR activating mutations (∼15% of NSCLC), ALK rearrangements (∼5% of NSCLC), and ROS1 rearrangements (∼1% of NSCLC) led to major changes in the therapeutic strategy.2, 3, 4 Tyrosine kinase inhibitor therapies targeting EGFR, ALK, or ROS1 have resulted in disease regression and survival improvement in some patients.2, 3, 4 Thus, the detection of EGFR, ALK, and ROS1 molecular alterations has become mandatory to screen for patients who could benefit from targeted therapies. The determination of EGFR mutational status has mainly been performed using DNA-based molecular Irsogladine methods and next generation sequencing (NGS). However, the current guidelines still recommend morphologic methods for the detection of ALK and ROS1 rearrangements in NSCLC tumor samples such as fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC).5, 6, 7, 8 The Vysis LSI ALK Dual Color Break Apart FISH probe (Abbott Molecular, Rungis, France) was the first molecular test approved by the Food and Drug Administration for searching for an ALK rearrangement and is considered the reference standard in this field.7, 8, 9 Nevertheless, ALK IHC (clone D5F3; Ventana) was also approved by the Food and Drug Administration as a companion diagnostic method to detect patients eligible for anti-ALK crizotinib therapy. New guidelines from France in 2017 recommended the use of first-line ALK IHC for determining treatment choices, followed by ALK FISH only in the case of weak (score, 1+) to moderate (score, 2+) IHC staining to differentiate between ALK-rearranged and non-rearranged tumors (ie, no additional FISH test is recommended for scores of 0, indicating negative IHC findings and scores of 3+, indicating strongly positive IHC results). A first-line IHC test has also been recommended for determining the presence of ROS1-rearranged NSCLC. However, although a score of is considered to indicate a negative IHC result and does not require further FISH testing, a weak to strong ROS1 IHC result (ie, score 1+, 2+ and 3+) requires additional ROS1 FISH testing to confirm the ROS1 rearrangement, which will allow for the use of targeted therapy. For both ALK and ROS1, the same cutoff of ≥ 15% of tumor nuclei with rearranged signals using FISH testing is used to consider a tumor as having ALK or ROS1 rearrangement. Nevertheless, the FISH results can sometimes remain ambiguous around the 15% cutoff, and some investigators have proposed that performing additional FISH tests with different probes could help to interpret these ambiguous statuses.11, 12, 13, 14 The number of analyses to perform on NSCLC tumor samples in an intent-to-treat strategy has continued to increase with the identification of several new clinically relevant and potentially targetable molecular events. In addition to EGFR, ALK, and ROS1, other genetic alterations (eg, RET or NTRK1 rearrangements, BRAF, PIK3CA, C-MET, and HER2 mutations) or immune checkpoints (programmed cell death 1/programmed cell death ligand 1 axis) are promising targets and require specific testing.2, 3, 4, 15, 16, 17, 18, 19, 20 Nevertheless, the diagnostic strategy must consider the problem of tiny biopsy specimens at risk of rapid cell depletion. The tissue handling, processing, and sectioning must be optimal to minimize tumor wastage and to permit analysis of every potentially relevant target.9, 10 Thus, tissues could be interesting to use FISH tests that concurrently detect the rearrangements of several molecular targets. In the present study, we evaluated the performance of a certified for in vitro diagnosis (CE-IVD) procedure using a dual ALK and ROS1 FISH probe on a set of NSCLC samples enriched in tumors with ALK- and ROS1-rearranged status.