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

Abstract

Introduction: 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.

Materials and methods: 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.

Results: 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).

Conclusions: 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.

Keywords: ALK; Cytology smear; Lung adenocarcinoma; Multiplex FISH; ROS1.