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Standardisation of EGFR FISH in colorectal cancer: results of an international interlaboratory reproducibility ring study
  1. Andrea Sartore-Bianchi1,
  2. Steffen Fieuws2,
  3. Silvio Veronese3,
  4. Mauro Moroni1,
  5. Nicola Personeni4,
  6. Milo Frattini5,
  7. Valter Torri6,
  8. Federico Cappuzzo4,
  9. Sara Vander Borght7,
  10. Vittoria Martin5,
  11. Margaret Skokan8,
  12. Armando Santoro4,
  13. Marcello Gambacorta3,
  14. Sabine Tejpar9,
  15. Marileila Varella-Garcia8,
  16. Salvatore Siena1
  1. 1The FalcK Division Of Medical Oncology, Ospedale Niguarda Ca' Granda, Milan, Italy
  2. 2I-Biostat, Katholieke Universiteit Leuven, Belgium
  3. 3Division of Pathology, Ospedale Niguarda Ca' Granda, Milan, Italy
  4. 4Department of Oncology and Hematology, Humanitas Cancer Center, Rozzano, Italy
  5. 5Laboratory of Molecular Diagnostic, Istituto Cantonale di Patologia, Locarno, Switzerland
  6. 6Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
  7. 7Department of pathology,University Hospital Gasthuisberg, Leuven, Belgium
  8. 8University of Colorado School of Medicine, Aurora, CO, USA
  9. 9Gastroenterology and Center for Human Genetics, University Hospital Gasthuisberg, Leuven, Belgium
  1. Correspondence to Dr Andrea Sartore-Bianchi, Falck Division of Medical Oncology, Ospedale Niguarda Ca' Granda, Piazza Ospedale Maggiore, 3, 20162 Milan, Italy; andrea.sartorebianchi{at}


Aims Epidermal growth factor receptor (EGFR) gene copy number evaluated by fluorescence in situ hybridisation (FISH) can discriminate among KRAS wild-type patients those with better outcome to EGFR-targeted therapy in metastatic colorectal cancer, further enhancing selection of patients. Nevertheless, enumeration of gene copies is challenging and the lack of analytical standardisation has limited incorporation of the test into the clinical practice. We therefore assessed EGFR FISH interlaboratory consensus among five molecular diagnostic reference centres.

Methods A set of 12 colorectal cancer samples circulated among laboratories, and samples were scored according to commonly agreed guidelines. Reproducibility was quantified using the standard error of measurement (SEM).

Results A SEM of 0.865 and a within-subject coefficient of variation (WSCV) of 26.8% for mean EGFR gene/nuclei and a SEM of 0.235 and a WSCV of 19.4% for the mean EGFR gene/CEP7 ratio were observed. Measurement of the fraction of cells displaying chromosome 7 polysomy showed WSCV of 46.6%, 34.0% and 51.0% for percentage of cells displaying ≤2, ≥3 and ≥4 EGFR signals, respectively. Among different slides of the same specimen, the WSCV was 6.1% for mean EGFR gene/nuclei and 3.9% for mean of EGFR gene/CEP7 ratios.

Conclusions Molecular diagnosis of EGFR gene copy number by FISH varied largely among pathology centres, with fluctuations covering the whole range of proposed cut-offs of predictive usefulness from literature. Definition of a detailed scoring system and implementation of comprehensive training programmes for laboratories are therefore necessary before including the test into clinical practice.

  • Colorectal cancer
  • FISH
  • EGFR
  • diagnosis
  • oncology
  • cancer research
  • molecular oncology
  • molecular pathology
  • molecular genetics
  • breast cancer
  • brain tumours
  • cancer genetics
  • tumour markers
  • melanocytic lesions
  • lung cancer
  • oncogenes
  • gene amplification
  • neoplasms

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  • AS-B and SF equally contributed as first authors.

  • MVG and SS equally contributed as senior authors.

  • Funding This work was supported by Oncologia Ca' Granda ONLUS Fondazione and Associazione Italiana per la Ricerca sul Cancro (AIRC): Investigator Grant 4412 and 2010 Special Program Molecular Clinical Oncology 5 × 1000, project 9970.

  • Competing interests AS-B: following relevant financial activities outside the submitted work: payment for board membership (AMGEN); payment for lectures including service on speakers bureaus (AMGEN, Merck-Serono). FC: following relevant financial activities outside the submitted work: payment for consultancy (Roche). MV-G: following relevant financial activities outside the submitted work: payment to my Institution (University of Colorado) as co-inventor in a patent to use EGFR biomarker for selection of non-small-cell lung cancer patients to EGFR inhibitors therapy. SS: following relevant financial activities outside the submitted work: payment for Board membership (Roche, Merck-Serono, AMGEN). ST: following relevant financial activities outside the submitted work: payment to my Institution for research grants (Merck-Serono). Other co-authors: none declared.

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

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