Research Papers: Pathology:

Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization

Laura Annaratone, Michele Simonetti, Erik Wernersson, Caterina Marchiò, Silvano Garnerone, Maria Stella Scalzo, Magda Bienko, Roberto Chiarle, Anna Sapino and Nicola Crosetto _

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Oncotarget. 2017; 8:18680-18698. https://doi.org/10.18632/oncotarget.15727

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Laura Annaratone1, Michele Simonetti2,*, Erik Wernersson2,*, Caterina Marchiò1,3, Silvano Garnerone2, Maria Stella Scalzo1, Magda Bienko2, Roberto Chiarle4,5, Anna Sapino1,6 and Nicola Crosetto2

1 Department of Medical Sciences, University of Turin, Turin, Italy

2 Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Karolinska Institutet, Stockholm, Sweden

3 Department of Laboratory Medicine, Pathology Unit, Azienda Ospedaliera Città della Salute e della Scienza di Torino, Turin, Italy

4 Department of Pathology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

5 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy

6 Candiolo Cancer Institute, FPO, IRCCS, Candiolo, Italy

* These authors have contributed equally to this work

Correspondence to:

Nicola Crosetto, email:

Roberto Chiarle, email:

Anna Sapino, email:

Keywords: single-molecule RNA FISH (smFISH); breast cancer; intra-tumor heterogeneity; epidermal growth factor receptor 2 (HER2); estrogen receptor 1 (ER); Pathology Section

Received: July 13, 2016 Accepted: February 06, 2017 Published: February 25, 2017


Intra-tumor heterogeneity is a pervasive property of human cancers that poses a major clinical challenge. Here, we describe the characterization, at the transcriptional level, of the intra-tumor topography of two prominent breast cancer biomarkers and drug targets, epidermal growth factor receptor 2 (HER2) and estrogen receptor 1 (ER) in 49 archival breast cancer samples. We developed a protocol for single-molecule RNA FISH in formalin-fixed, paraffin-embedded tissue sections (FFPE-smFISH), which enabled us to simultaneously detect and perform absolute quantification of HER2 and ER mature transcripts in single cells and multiple tumor regions. We benchmarked our method with standard diagnostic techniques, demonstrating that FFPE-smFISH is able to correctly classify breast cancers into well-established molecular subgroups. By counting transcripts in thousands of single cells, we identified different expression modes and levels of inter-cellular variability. In samples expressing both HER2 and ER, many cells co-expressed both genes, although expression levels were typically uncorrelated. Finally, we applied diversity metrics from the field of ecology to assess the intra-tumor topography of HER2 and ER gene expression, revealing that the spatial distribution of these key biomarkers can vary substantially even among breast cancers of the same subtype. Our results demonstrate that FFPE-smFISH is a reliable diagnostic assay and a powerful method for quantification of intra-tumor transcriptional heterogeneity of selected biomarkers in clinical samples.

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