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 ... -  《-》

Evaluation of a Dual ALK/ROS1 Fluorescent In Situ Hybridization Test in Non-Small-cell Lung Cancer.

Several therapeutics targets have emerged to treat patients with non-small-cell lung carcinoma (NSCLC), with numerous biomarkers available to test for treatment choices. Minimum tumor wastage is necessary to permit the analysis of every potentially relevant target. Searching for targetable ALK and ROS1 rearrangements is now mandatory in NSCLC. In the present study, we evaluated the performance of a dual ALK/ROS1 fluorescent in situ hybridization (FISH) probe that concurrently analyzed the 2 oncogenes on a same FISH slide. We used the FlexISH ALK/ROS1 DistinguISH Probe (Zytovision, Bremerhaven, Germany) to analyze a set of 28 formalin-fixed paraffin-embedded NSCLC tumor samples enriched in tumors with ALK- and ROS1-rearranged status. The dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests (15 ALK and 3 ROS1 rearrangements) without any false-positive results. Dual- and single-probe FISH test results were also concordant regarding the unusual ALK FISH patterns. These included 1 sample that had negative FISH results with diffuse single 5'-ALK signals and positive ALK immunohistochemistry findings in a patient with a response to crizotinib, 2 paired samples with high percentages of ALK FISH-rearranged nuclei despite negative ALK immunohistochemistry findings, and ALK FISH-positive samples from 2 patients lacking a response to crizotinib therapy despite concordant ALK FISH and immunohistochemistry-positive results. The dual ALK/ROS1 FISH probe test is a valuable tool to search concurrently for both ALK and ROS1 rearrangements on a same FISH slide and could help to spare tumor tissue for other biomarkers tests.

Ginestet F ，Lambros L ，Le Flahec G ，Marcorelles P ，Uguen A ... -  《-》

Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in non-small-cell lung cancer.

ALK and ROS1 gene rearrangements are distinct molecular subsets of non-small-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. Study population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. For a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight® SPEC ROS1 Break Apart Probe. Two of the cases were excluded due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.

Lim SM ，Chang H ，Cha YJ ，Liang S ，Tai YC ，Li G ，Pestova E ，Policht F ，Perez T ，Soo RA ，Park WY ，Kim HR ，Shim HS ，Cho BC ... -  《-》

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 ... -  《-》

Multiplex fluorescence in situ hybridization testing for anaplastic lymphoma kinase and c-ros oncogene 1 gene rearrangements on cytology smears in lung adenocarcinomas: comparative study with formalin-fixed paraffin-embedded sections.

Multiplex anaplastic lymphoma kinase (ALK)/c-ros oncogene 1 (ROS1) fluorescence in situ hybridization (FISH) probes conserve tissue by analyzing both ALK and ROS1 gene rearrangements (ALK-R/ROS1-R) in a single test. The positivity cutoffs have been validated on formalin-fixed, paraffin-embedded (FFPE) tissue sections and not tested on non-cell block (CB) cytology preparations. We sought to validate non-CB cytology preparations for the detection of ALK-R/ROS1-R using multiplex ALK/ROS1 FISH probes by comparing the results with matched FFPE results. During the 3.5-year study period, FISH using the FlexISH ALK/ROS1 DistinguISH Probe (ZytoVision) was performed in non-CB cytology preparations of patients for whom FISH on FFPE sections was performed. A total of 20 patients had one or more non-CB cytology preparations (n = 27) suitable for FISH analysis. These comprised direct smears (n = 17), smears from centrifuged effusion pellets (n = 8), cytospin smears (n = 1), and biopsy imprint smears (n = 1). These had been fixed in 95% ethanol (n = 18) or air dried (n = 9), and stained with Papanicolaou (n = 14), May-Grünwald-Giemsa (n = 9), immunocytochemistry (n = 3), or hematoxylin and eosin (n = 1). The median archival time was 1 year. Successful FISH results were achieved in 14 samples (6 with ALK-R, 2 with ROS1-R, 6 negative) and were concordant with the FFPE FISH results for 13 of 14 cases. The single case with discordant results between cytology and FFPE FISH showed ALK-R on cytology concordant with positive ALK D5F3 companion diagnostics assay results and was considered a false-negative FFPE FISH result. FISH failure occurred mainly in the older archived slides because of overdigestion (n = 5), hybridization failure (n = 5), or excessive background fluorescence (n = 3). Non-CB cytology smears are highly suitable for multiplex FISH analysis with 100% concordance with FFPE FISH and/or ALK D5F3 companion diagnostics assay results.

Nambirajan A ，Rana D ，Samant K ，Prabakaran A ，Malik P ，Jain D ... -  《-》