Gene status in Her2 equivocal breast carcinomas: Impact of distinct recommendations and contribution of a polymerase chain reaction-based method

Anna Sapino, Francesca Maletta, Ludovica Verdun Di cantogno, Luigia Macrì, Cristina Botta, Patrizia Gugliotta, Maria Stella Scalzo, Laura Annaratone, Davide Balmativola, Francesca Pietribiasi, Paolo Bernardi, Riccardo Arisio, Laura Viberti, Stefano Guzzetti, Renzo Orlassino, Cristiana Ercolani, Marcella Mottolese, Giuseppe Viale, Caterina Marchiò

Research output: Contribution to journalArticlepeer-review


Background. The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status.

Methods. A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA.

Results. HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/ CEP17 ratio 6.0 signals per cell. In contrast, only one case showing a HER2 copy number 2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas.

Conclusion. The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Original languageEnglish
Pages (from-to)1118-1126
Number of pages9
JournalThe oncologist
Issue number11
Publication statusPublished - 2014


  • Breast cancer
  • Equivocal HER2 status
  • Guidelines
  • HER2 amplification
  • Heterogeneity

ASJC Scopus subject areas

  • Cancer Research
  • Oncology


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