Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohistochemistry with FISH confirmation is an effective method to identify this rare target.

To assess the prevalence of ROS1 rearrangements in a retrospective and prospective diagnostic Australian cohort and evaluate the effectiveness of immunohistochemical screening. A retrospective cohort of 278 early stage lung adenocarcinomas and an additional 104 prospective non-small-cell lung cancer (NSCLC) cases referred for routine molecular testing were evaluated. ROS1 immunohistochemistry (IHC) was performed (D4D6 clone, Cell Signaling Technology) on all cases as well as fluorescence in-situ hybridization (FISH) using the ZytoVision and Abbott Molecular ROS1 FISH probes, with ≥15% of cells with split signals considered positive for rearrangement. Eighty-eight cases (32%) from the retrospective cohort showed staining by ROS1 IHC, and one case (0.4%) showed ROS1 rearrangement by FISH. Nineteen of the prospective diagnostic cases showed ROS1 IHC staining, 12 (12%) cases of which were confirmed as ROS1 rearranged by FISH. There were no ROS1 rearranged cases that showed no expression of ROS1 with IHC. The ROS1 rearranged cases in the prospective cohort were all EGFR wild-type and anaplastic lymphoma kinase (ALK) rearrangement-negative. The sensitivity of ROS1 IHC in the retrospective cohort was 100% and specificity was 76%. ROS1 rearrangements are rare events in lung adenocarcinomas. Selection of cases for ROS1 FISH testing, by excluding EGFR/ALK-positive cases and use of IHC to screen for potentially positive cases, can be used to enrich for the likelihood of identifying a ROS1 rearranged lung cancer and prevent the need to undertake expensive and time-consuming FISH testing in all cases.

Selinger CI ，Li BT ，Pavlakis N ，Links M ，Gill AJ ，Lee A ，Clarke S ，Tran TN ，Lum T ，Yip PY ，Horvath L ，Yu B ，Kohonen-Corish MR ，O'Toole SA ，Cooper WA ... -  《-》

On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.

ROS1 proto-oncogene translocations define a new molecular subgroup in non-small cell lung cancers (NSCLC) and are associated with a response to the MET/ALK inhibitor, crizotinib. These rearrangements are described in 0.9-1.7% NSCLC, in wild-type EGFR, KRAS and ALK ("triple negative") lung adenocarcinomas. Rapid and efficient identification of these alterations is thus becoming increasingly important. In this study, 121 triple negative lung adenocarcinomas were screened by both IHC with the ROS1 D4D6 antibody, and FISH using two commercially available ROS1 break-apart probes. To address a possible cross-reactivity of the ROS1 antibody with other protein kinase receptors, we screened 80 additional cases with known EGFR, KRAS, PI3KCA, BRAF, HER2 mutations or ALK-rearrangement. We diagnosed 9 ROS1-rearranged adenocarcinomas, with both a positive FISH result (51-87% rearranged nuclei) and a positive IHC staining (2+/3+ cytoplasmic staining). Only one of the ROS1-positive FISH cases was characterized by a classical split pattern, the others showed a variant pattern, most commonly involving a loss of the 5' telomeric probe. Considering a positivity threshold of 2+ stained cells, the sensitivity of the ROS1 D4D6 antibody compared to FISH was 100% and the specificity 96.9%, as two HER2-mutated tumors were positive with D4D6 antibody, without any translocation in FISH. All the ROS1-positive cases were at an advanced stage, arising in never or light smokers. They were mainly solid cribriform and acinar adenocarcinomas, with signet ring cells noted in 5 cases, and calcifications in 3 cases. One positive case was an invasive mucinous carcinoma. Our results show that a screening algorithm based on an IHC detection of ROS1 fusion proteins, confirmed if positive or doubtful by a ROS1 break-apart FISH assay, is pertinent in advanced "triple negative" lung adenocarcinomas, since the prevalence of ROS1-positive cases in this selected population reaches 7.4% in our series.

Mescam-Mancini L ，Lantuéjoul S ，Moro-Sibilot D ，Rouquette I ，Souquet PJ ，Audigier-Valette C ，Sabourin JC ，Decroisette C ，Sakhri L ，Brambilla E ，McLeer-Florin A ... -  《-》

Concordance of Immunohistochemistry and Fluorescence In Situ Hybridization in the Detection of Anaplastic Lymphoma Kinase (ALK) and Ros Proto-oncogene 1 (ROS1) Gene Rearrangements in Non-Small Cell Lung Carcinoma: A 4.5-Year Experience Highlighting Challe

Nambirajan A ，Sood R ，Khatoon W ，Malik PS ，Mohan A ，Jain D ... -  《-》

Comparison of detection methods and follow-up study on the tyrosine kinase inhibitors therapy in non-small cell lung cancer patients with ROS1 fusion rearrangement.

Wu J ，Lin Y ，He X ，Yang H ，He P ，Fu X ，Li G ，Gu X ... -  《BMC CANCER》

Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples.

Several predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes. We collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring ALK-rearrangement and ROS1-rearrangement. ALK and ROS1 status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 ALK-positive, 2 ROS1-positive and 53 ALK/ROS1-wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision. The dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 ALK-positive and 2 ROS1-positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results. Multiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide in cytological specimens with a small amount of biomaterial.

Zito Marino F ，Rossi G ，Cozzolino I ，Montella M ，Micheli M ，Bogina G ，Munari E ，Brunelli M ，Franco R ... -  《-》