Abstract P6-05-04: Stratification of breast carcinomas with double-equivocal HER2 status
Authors: Marchio C , Trisolini E , Maletta F , Annaratone L , Scalzo MS , Mascali D , Verdun di Cantogno L , Medico E , Sapino A 
Published: May 2016

BACKGROUND AND RATIONALE: Immunohistochemistry (IHC) and in situ hybridization (ISH) represent the cornerstone of HER2 evaluation in breast cancer (BC). We have demonstrated that up to 13% of BCs with equivocal HER2 expression (score 2+ in IHC) harbor an equivocal HER2 gene status (HER2/CEP17 &lt;2 and mean HER2 copy number between 4 and 6), leading to the definition of the double-equivocal category. When we assessed HER2 gene levels in these cases by a PCR-based method we observed copy gain in 25%. When we tested HER2 protein levels by an in situ quantitative assay, HER2 levels ranged from those of score 0/ISH- to those observed in score 2+/ISH+ BCs. These data suggest that, rather than exploring alternative methods to assess HER2 status, a complementary functional approach may be beneficial. Our aim was to stratify double-equivocal BCs using global transcriptomics.

METHODS & RESULTS: We retrieved a series of 27 formalin fixed paraffin embedded double-equivocal BCs and two control groups matched for oestrogen receptor and histological grade: 22 HER2- (IHC score 0/ISH-) and 22 HER2+ (IHC score 3+/ISH+) BCs. RNA was extracted following microdissection to enrich for tumor cell content &gt;80% and subjected to whole-Genome DASL (cDNA-mediated Annealing, Selection, extension and Ligation; Illumina).

We first identified genes with differential expression in HER2+ versus HER2- BCs based on T-test significance (p&lt;0.01) and on mean gene expression variations higher than +/- 2-fold. To best characterize the signature we performed a cluster analysis with the GEDAS software using the "Fuzzy Self-organizing Maps" algorithm and the cosenic distance to generate the clusters. The classifier (24 genes) tested on double-equivocal cases led to a separation in "HER2+ like" and "HER2- like" BCs. More in details, three main clusters emerged, showing a significantly different distribution of cases with distinct HER2 status (p&lt;0.0001, Chi square). Cluster A included all HER2+ and 5 double-equivocal cases and was associated to high expression of genes that pertain to the HER2 amplicon. Cluster B, composed of 9 HER2- and 12 double-equivocal cases, showed low expression of HER2 and HER2 amplicon-related genes and high levels of expression of TPRG1, NOVA1, AGTR1, SEZ6L, MAPT, GSTM1, SORCS1, DSCR6, NPY1R genes. Cluster C, formed by 13 HER2- and 10 double-equivocal cases, showed low expression of HER2 and HER2 amplicon-related genes but a non-homogeneous expression of the other genes of the classifier.

The expression of best performing genes of the classifier (TPRG1, NOVA1, AGTR1) was investigated in The Cancer Genome Atlas (TCGA) dataset of breast cancer (Breast Invasive Carcinoma, TCGA provisional from cbioportal.org) and a striking mutually exclusive pattern with HER2 expression was observed.

CONCLUSION: By resolving HER2 double-equivocal BCs into HER2 likely-positive or likely-negative, this approach may pave the way to an informed therapeutic decision in this controversial category of patients.

Citation Format: Marchio C, Trisolini E, Maletta F, Annaratone L, Scalzo MS, Mascali D, Verdun di Cantogno L, Medico E, Sapino A. Stratification of breast carcinomas with double-equivocal HER2 status. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-04.