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• W1036990384 abstract "We read with much interest the study by Rogers et al. concerning the use of fluorescence in situ (FISH), chromogenic in situ hybridization, and immunohistochemistry (IHC) for the detection of ALK and ROS1 rearrangements in lung cancer.1Rogers TM Russell PA Wright G et al.Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.J Thorac Oncol. 2015; 10: 611-618Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar They concluded that FISH versus IHC showed good correlation in the detection of ALK rearrangements but weak correlation in the detection of ROS1 rearrangements. We would like to comment on this latter conclusion and emphasize the importance of FISH “home-made” probe design. ROS1 (position chr6: 117,609,530-117,747,018 based on the UCSC Genome Browser on Human February 2009 (GRCh37/hg19) Assembly [http://genome.ucsc.edu]) has 43 exons, of which exons 36 to 41 code for the tyrosine kinase domain that is retained in all fusion proteins thus far identified. Breakpoints in the ROS1 gene cluster in introns 31 to 34,2Takeuchi K Soda M Togashi Y et al.RET, ROS1 and ALK fusions in lung cancer.Nat Med. 2012; 18: 378-381Crossref PubMed Scopus (1037) Google Scholar in a 16kb region (position chr6:117,642,538-117,658,284). Commercially available probes are closely located near this breakpoint cluster region (bcr). The ZytoLight SPEC ROS1 Dual Color Break Apart Probe (ZytoVision, Bremerhaven, Germany) is a mixture of two probes, one labeled in green and covering the 3′ part of the gene, the other labeled in orange and covering the 5′ part. Abbott Molecular (Des Plaines, IL) commercializes the Vysis LSI ROS1 (Cen) SpectrumGreen Probe covering the 3′ part of the gene that can be used in a mix with the Vysis LSI ROS1 (Tel) SpectrumOrange Probe that covers the 5′ part of ROS1. In both cases, ROS1 fusion with its gene partner separates both probes. FISH “home-made” probes are constructed from BAC (Bacterial Artificial Chromosome) clones. In each construction, one probe is labeled in green and the other in orange. Five groups, including Rogers et al., have reported their results in the literature (Fig. 1). Two groups used the same construct.1Rogers TM Russell PA Wright G et al.Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.J Thorac Oncol. 2015; 10: 611-618Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,3Bergethon K Shaw AT Ou SH et al.ROS1 rearrangements define a unique molecular class of lung cancers.J Clin Oncol. 2012; 30: 863-870Crossref PubMed Scopus (1265) Google Scholar They mixed RP11-835I21 (chr6: 117,844,792-118,035,413) and RP11-1036C2 (chr6: 117,408,637-117,598,872), centromeric and telomeric of the ROS1 gene, respectively (Fig. 1). Rogers et al. also mentioned an additional clone, RP11-378F24, which is not referenced in databases and is presumably RP11-379F24. Therefore, the gap between both probes covers 246kb and does not cover any part of the ROS1 gene. Two other groups used almost identical homemade kits. Takeuchi et al. prepared a mix of RP1-179P9 (chr6: 117,569,584-117,677,843), covering exons 25 to 43 and sequences flanking the 3′ part of the gene, and RP11-323I17 (chr6: 117,659,043-117,800,093), covering sequences flanking the 5′ part and the first 31 exons of the gene (Fig. 1).2Takeuchi K Soda M Togashi Y et al.RET, ROS1 and ALK fusions in lung cancer.Nat Med. 2012; 18: 378-381Crossref PubMed Scopus (1037) Google Scholar Rikova et al. also used RP1-179P9 as the 3′ probe and RP11-323O17 (chr6: 117,659,115-117,800,072, equivalent to RP11-323I17) and RP1-94G16 (chr6: 117808698-117909505) as the 5′ probe (Fig. 1).4Rikova K Guo A Zeng Q et al.Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1914) Google Scholar With these constructs, RP1-179P9 would be split by breakpoints occurring in the bcr region. Although the probe at the 3′ end will be separated from those at the 5′ end of the gene in case of fusion, separation does not specifically target the bcr. Davies et al. designed a customized ROS1 break-apart probe set made of clones CTD-2314K7 (chr6: 117,338,338-117,438,446) and RP11-59K17 (chr6: 117,448,944-117,627,275), covering exons 42 and 43 and sequences flanking the 3′ part of the gene, and RP11-623N3 (chr6: 117,654,640-117,833,020) and RP11-117O13 (chr6: 117,830,521-117,971,596) covering sequences flanking the 5′ part and the first 31 exons and a small portion of intron 31 of the gene (Fig. 1).5Davies KD Le AT Theodoro MF et al.Identifying and targeting ROS1 gene fusions in non-small cell lung cancer.Clin Cancer Res. 2012; 18: 4570-4579Crossref PubMed Scopus (355) Google Scholar This construct leaves a 27 kb gap in which bcr is located. These FISH “home-made” probe sets differ not only by the BAC clones used to build them but also by the distance between clones, varying from 27 to 246 kb. In fact, the greater the distance from bcr, the more likely false positives could happen. Indeed, breaks could occur outside ROS1 bcr and even outside the gene, without fusion leading to kinase activation. Also, it has been shown that deletion of the 5′ region could be associated with a ROS1 fusion, as it has been reported to happen during fusions of other genes such as those involving ABL1, MLL.6Yoshida A Kohno T Tsuta K et al.ROS1-rearranged lung cancer: A clinicopathologic and molecular study of 15 surgical cases.Am J Surg Pathol. 2013; 37: 554-562Crossref PubMed Scopus (138) Google Scholar In these cases, deletion occurs at the breakpoint site. Therefore, identifying a deletion of RP11-835I21, as used by Rogers et al.1Rogers TM Russell PA Wright G et al.Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.J Thorac Oncol. 2015; 10: 611-618Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar and Bergethon et al.,3Bergethon K Shaw AT Ou SH et al.ROS1 rearrangements define a unique molecular class of lung cancers.J Clin Oncol. 2012; 30: 863-870Crossref PubMed Scopus (1265) Google Scholar does not mean that sequences of the 5′ part of ROS1 were removed. The diversity in probe design could explain, at least partially, the discrepancies between IHC and FISH results. Home-made probes are a good alternative to commercially available probes but they have to be designed carefully. Interpretation of the results requires a good knowledge of the design of the probes being used to enable mechanisms of the chromosomal and molecular rearrangements to be elucidated. In ResponseJournal of Thoracic OncologyVol. 10Issue 8PreviewWe thank Uguen et al. for their interest in our recently published manuscript titled Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.1 We acknowledge their concerns regarding the use of homemade fluorescent in situ hybridization (FISH) probes and the importance of their design. At the time that the study was conducted there were no commercial ROS1 FISH probes available therefore we used the home-made ROS1 FISH probe that was kindly gifted to us from Translational Research Laboratory, Massachusetts General Hospital and previously utilized in the study by Bergethon et al. Full-Text PDF Open Archive" @default.
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• W1036990384 title "Searching for ROS1 Rearrangements in Lung Cancer by Fluorescent In Situ Hybridization: The Importance of Probe Design" @default.
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