The current guidelines are designed

The current guidelines are designed to optimize the detection of IHC+FISH+ cases, most of which (but not all encountered in our study) being classically good responders to crizotinib therapy (resistance mechanisms to crizotinib therapy were not investigated in our study). In addition to the current proposed guidelines, performing the dual FISH test in the first line (eg, in patients without a smoking history with frequent oncogenic rearrangements, even in the case of negative IHC test results) could also permit the detection of some patients with ALK IHC−FISH+ who could also be treated using crizotinib anti-ALK therapy (eg, patient 18 in our study). In our experience and in the reported data, the response rates were not negligible in ALK FISH+IHC− patients (∼46% of response) and using both ALK IHC and ALK FISH could remain useful for maximizing the detection rate of patients treatable by anti-ALK–targeted therapies to detect and treat not only patients with ALK IHC+FISH+ but also ALK IHC−FISH+ and ALK IHC+FISH− status (eg, patient 8 in our study with a response to crizotinib despite ALK FISH− status).11, 26, 27 Additional oncogenic rearrangements or amplifications could consist of valuable druggable targets in NSCLC. Some of them, such as MET amplifications (not tested in our study) could also be predictive markers of response to crizotinib therapy (and could perhaps explain the response to crizotinib of some cases with discrepant ALK IHC and FISH findings). In terms of oncogenic rearrangements, albeit rare (< 1% of NSCLC), RET and NTRK1 rearrangements are also promising in this casr field .15, 20, 28 Nevertheless, integrating additional probes labeled with different fluorochromes into a multiplexed FISH approach could be limited by the overlapping wavelengths of the fluorochromes used for FISH application. In a reasonable approach, 4 different fluorochromes would be combined in commercially available FISH probes (eg, red, green, aqua, and gold fluorochromes in the Vysis Melanoma FISH probe; Abbott Molecular). Thus, in addition to the 3 fluorochromes combined in the dual ALK and ROS1 FISH test we studied, in the case of future approval of targeted therapies for NSCLC harboring a new oncogenic rearrangement (eg, involving RET or NTRK1), it would seem feasible to design a new multiplex FISH test to search concurrently for several rearrangements. In 3 different oncogenes using a combination of 4 fluorochromes (ie, red- and green-labeled break apart probes for the 3 oncogenes with co-labeling with an aqua fluorochrome for 1 oncogene [as designed for the ROS1 part of the dual ALK and ROS1 FISH test] and a gold fluorochrome for another; the third targeted oncogene would not have had additional co-labeling [eg, as the ALK part of the dual ALK and ROS1 FISH test]). Orange fluorochrome, detected in the red and gold wavelength spectrum, would not be appropriate for such a multiplex FISH test. To the best of our knowledge, no FISH probe, to date, allows for the detection of an additional oncogenic rearrangement in a concurrent multiplexed analysis in addition to ALK and ROS1, which are still the only 2 rearrangements for which access to approved targeted therapies for NSCLC is conditional on their presence.
Conclusion Multiplexed FISH testing is a valuable approach to search for both ALK and ROS1 rearrangements in a single slide of a NSCLC tissue sample. Its use as a second-line tool, if required by the results of first-line ALK IHC (ie, in cases of a score of 1+ or 2+) or ROS1 IHC (ie, a score of 1+, 2+, or 3+ in accordance with the updated guidelines) or as a first-line test without considering previous IHC findings seems suitable for the detection of tyrosine kinase inhibitor–susceptible oncogenic rearrangements. Multiplexed testing is also suitable for minimizing tissue section wastage in NSCLC samples, which are often small biopsy specimens, sometimes with poor cells, to allow for the determination of an increasing number of theranostic parameters required for determining the best treatment for patients with advanced NSCLC. In addition, this multiplexed FISH approach could consist of a more cost-effective and rapid method than new sequencing technologies whose superiority against morphologic methods (ie, FISH and IHC) has still not been clearly demonstrated for the search for oncogenic rearrangements in the field of NSCLC.