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Reference standards for gene fusion molecular assays on cytological samples: an international validation study
  1. Umberto Malapelle1,
  2. Francesco Pepe1,
  3. Pasquale Pisapia1,
  4. Annalisa Altimari2,
  5. Claudio Bellevicine1,
  6. Hans Brunnström3,
  7. Rossella Bruno4,
  8. Reinhard Büttner5,
  9. Luis Cirnes6,
  10. Carlos E De Andrea7,
  11. Dario de Biase8,
  12. Catherine I Dumur9,
  13. Kajsa Ericson Lindquist3,
  14. Gabriella Fontanini4,
  15. Eugenio Gautiero10,
  16. David Gentien11,
  17. Paul Hofman12,
  18. Veronique Hofman12,
  19. Antonino Iaccarino1,
  20. Maria Dolores Lozano7,
  21. Clara Mayo-de-Las-Casas13,
  22. Sabine Merkelbach-Bruse5,
  23. Fabio Pagni14,
  24. Ruth Roman13,
  25. Fernando C Schmitt6,
  26. Janna Siemanowski5,
  27. Sinchita Roy-Chowdhuri15,
  28. Giovanni Tallini16,
  29. Francesc Tresserra17,
  30. Sara Vander Borght18,
  31. Philippe Vielh19,
  32. Elena Vigliar1,
  33. Giulia Anna Carmen Vita20,
  34. Birgit Weynand18,
  35. Rafael Rosell21,
  36. Miguel Angel Molina Vila13,
  37. Giancarlo Troncone1
  1. 1 Public Health, University of Naples Federico II, Naples, Italy
  2. 2 Molecular Pathology, University of Bologna Hospital of Bologna Sant'Orsola-Malpighi Polyclinic, Bologna, Italy
  3. 3 Clinical Sciences Lund, Division of Pathology, Lund University, Lund, Sweden
  4. 4 Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy
  5. 5 Pathology, University of Cologne, Koln, Germany
  6. 6 Pathology, IPATIMUP, Porto, Portugal
  7. 7 Pathology, University of Navarra, Pamplona, Spain
  8. 8 Pharmacy and Biotechnology (FaBiT), Molecular Pathology Laboratory, University of Bologna, Bologna, Italy
  9. 9 Aurora Diagnostics, Jacksonville, Florida, USA
  10. 10 Medical Genetics Laboratory, San Gerardo Hospital, Monza, Italy
  11. 11 Translational Research Department, Genomics Platform, Curie Institute Hospital Group, Paris, France
  12. 12 Laboratory of Clinical and Experimental Pathology, University Hospital Centre Nice Pasteur Hospital, Nice, France
  13. 13 Oncology, Quirón Dexeus University Hospital, Barcelona, Spain
  14. 14 Medicine and Surgery, San Gerardo Hospital, Monza, Italy
  15. 15 Pathology, Division of Pathology/Lab Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
  16. 16 Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
  17. 17 Pathology, Quirón Dexeus University Hospital, Barcelona, Spain
  18. 18 Imaging and Pathology, Translational Cell and Tissue Research, KU Leuven, Leuven, Belgium
  19. 19 Pathology, Medipath and American Hospital of Paris, Paris, France
  20. 20 Pathology, IRCCS CROB, Rionero in Vulture, Italy
  21. 21 Oncology, Hospital Municipal de Badalona, Barcelona, Spain
  1. Correspondence to Professor Giancarlo Troncone, Department of Public Health, University of Naples Federico II, Napoli, Italy; giancarlo.troncone{at}


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.

  • pathology
  • molecular
  • 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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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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