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Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples
  1. Federica Zito Marino1,
  2. Giulio Rossi2,
  3. Immacolata Cozzolino1,
  4. Marco Montella1,
  5. Mariacarolina Micheli3,
  6. Giuseppe Bogina4,
  7. Enrico Munari4,
  8. Matteo Brunelli5,
  9. Renato Franco1
  1. 1 Department of Mental and Physic Health and Preventive Medicine, Pathology Unit, University of Campania Luigi Vanvitelli, Napoli, Italy
  2. 2 Pathology Unit, Ospedale Santa Maria delle Croci, Ravenna, Italy
  3. 3 Pathology Unit, Ospedali dei Colli Monaldi Cotugno CTO, Napoli, Italy
  4. 4 Department of Pathology, Sacro Cuore Don Calabria Hospital, Negrar, Italy, Negrar, Italy
  5. 5 Department of Pathology, University of Verona, Verona, Italy
  1. Correspondence to Professor Renato Franco,University of Campania Luigi Vanvitelli, Napoli, Campania, Italy; RENATO.FRANCO{at}


Aims 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.

Methods 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.

Results 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.

Conclusion 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.

  • FISH
  • lung cancer
  • molecular biology
  • molecular pathology

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  • Handling editor Prof Runjan Chetty.

  • Contributors FZM: has conceived the design of the study and carried out the draft of the manuscript, she was responsible of FISH assay interpretation. GR: was responsible for the revision of cytological diagnosis and immunohistochemistry evaluation. IC and MM: were responsible for provision of biological sample and cytological diagnosis. CM: has helped to carried out FISH assay. GB, EM and MB: have helped to FISH assay interpretation. RF: has participated in the design of the study and the coordination of the manuscript. All authors read and approved the final manuscript.

  • Funding The manuscript was supported by Department of Mental and Physic Health and Preventive Medicine, University of Campania "L. Vanvitelli", Naples, Italy.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.