Aims Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.
Methods Cell lines harbouring EML4(13)–ALK(20) and SLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.
Results Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.
Conclusions Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.
- molecular biology
- cytological techniques
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
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Handling editor Runjan Chetty.
Twitter @UmbertoMalapel1, @PasqualePisapia, @Sinchita_Roy
UM, FPe and PP contributed equally.
MAMV and GTr contributed equally.
Contributors UM, FPe, PP, MAMV and GTr contributed to conceptualisation, writing–original draft preparation; all authors contributed to methodology, software, validation, formal analysis, investigation, resources, data curation, writing–review & editing, visualisation; UM, RRos, MAMV and GTr contributed to supervision, project administration; GTr contributed to funding acquisition.
Funding Monitoraggio ambientale, studio ed approfondimento della salute della popolazione residente inaree a rischio—In attuazione della D.G.R. Campanian. 180/2019. POR Campania FESR 2014–2020 Progetto “Sviluppo di Approcci Terapeutici Innovativi per patologie Neoplastiche resistenti ai trattamenti—SATIN”.
Competing interests UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, GSK, Merck and Astra Zeneca, unrelated to the current work. Lukas Bubendorf has a consulting or advisory role with Astra Zeneca, AbbVie, Bayer, Boehringer Ingelheim, Eli Lilly, MSD, Pfizer, Takeda and F. Hoffmann-La Roche and has received research funding (institution) from F. Hoffmann-La Roche, MSD and Sanofi. PH reports personal fees (as advisor) from Roche, Astrazeneca, BMS, MSD, Pfizer, Bayer, Amgen, Illumina, Qiagen, Thermo Fisher Scientific, Biocartis, Ed Lilly, unrelated to the current work. SM-B has received personal fees (as consultant and/or speaker bureau) from Astra Zeneca, Roche, BMS, Novartis, GSK, MSD, Targos, Merck, unrelated to the current work. Spasenija Savic Prince received personal fees from MSD, Astra Zeneca, Boehringer Ingelheim, Roche, Pfizer, Bristol-Myers Squibb and Thermo Fisher Scientific, unrelated to the submitted work. EV has received personal fees (as consultant and/or speaker bureau) from Diaceutics, unrelated to the current work. GTr reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini, AstraZeneca, Amgen and Bayer, unrelated to the current work. The other authors have nothing to disclose.
Provenance and peer review Not commissioned; externally peer reviewed.
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