Building up a high-throughput screening platform to assess the heterogeneity of HER2 gene amplification in breast cancers

Giulia Ercoli, Gianluca Lopez, Camilla Ciapponi, Chiara Corti, Luca Despini, Donatella Gambini, Letterio Runza, Concetta Blundo, Amedeo Sciarra, Nicola Fusco

Research output: Contribution to journalArticlepeer-review


Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2- positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Original languageEnglish
Article numbere56686
JournalJournal of Visualized Experiments
Issue number130
Publication statusPublished - Dec 5 2017


  • Breast cancer
  • Heterogeneity
  • High-throughput molecular analysis
  • Human epidermal growth factor receptor 2 (HER2)
  • Immunology
  • In situ hybridization
  • Issue 130
  • Tissue microarray

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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