Abstract P1-09-18: A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH
Authors: Gunn S , Yaziji H , Sims C , Govender S , Moore M , Cotter P , Jones S 
Published: March 2017

Abstract
Background: In all stages of breast cancer, the HER2 status of a patient's tumor is critically important as both a prognostic indicator, and for predicting response to targeted anti-HER2 therapies. CAP/ASCO 2013 guidelines recommend that newly diagnosed, recurrent, and metastatic breast tumors be evaluated for HER2 positivity by protein-based immunohistochemistry (IHC) and/or chromosome-based fluorescence in situ hybridization (FISH). In the majority of cases, these testing modalities provide a clearly actionable “positive” or “negative” answer. However, in an estimated 10- to 20% of breast cancers, both tests are reported as “equivocal” leaving the clinician with a treatment decision dilemma and no definitive alternative testing method. Here we report validation of an IHC-targeted DNA microarray comparative genomic hybridization (array CGH) assay for HER2 equivocal breast cancer, and definition of a new genomic subtype of HER2 status in high-risk breast cancer with equivocal IHC and FISH results.
Methods: IHC-targeted HER2 receptor “hot spot” DNA samples extracted from 25 formalin fixed paraffin embedded (FFPE) breast tumor tissue samples previously characterized by IHC and FISH, were analyzed by array CGH. Eight tumors were known to be highly HER2 positive, seven tumors had IHC scores of 0 with negative FISH, and ten tumors had HER2 receptor staining by IHC (1-2+) and equivocal results by FISH (4-6 HER2 gene copies.) Tumor DNA (test) and human genomic DNA (reference) were fluorescently labeled, and competitively hybridized to a custom-designed genomic DNA microarray with high-density probe coverage of the HER2 amplicon on chromosome 17 (Agilent Technologies, Santa Clara CA). The array design includes over 4,600 chromosome 17 probes representing the p arm, q arm, telomeric and centromeric regions with 66 tiling probes over the HER2 (ERBB2) gene. Following hybridization, average HER2 gene copy number was calculated for each tumor sample by converting mean log2 signal intensity ratio value into genomic region copy number adjusted for % clonal fraction and experimentally established log2 ratio compression of the assay.
Results: 25/25 (100%) of samples yielded adequate DNA for analysis and all highly HER2 positive and HER2 negative results were confirmed by array CGH. In 10/10 IHC equivocal cases with HER2 gene copy number 4-6 by FISH, CGH results confirmed HER2-Low gene copy number.
Results for 25 Validation SamplesNumber of CasesIHC ScoreFISHCGH Copy NumberResult83+Positive&gt; 6HER2-Positive101-2+Equivocal4-6HER2-Low70Negative&lt; 6HER2-Negative
Conclusions: High-resolution HER2 testing by IHC-targeted DNA microarray analysis accurately classifies HER2 status in breast cancer and better defines the HER2-Low genomic subtype most often called “equivocal” by standard IHC and FISH testing. This subcategory is characterized at the protein level by IHC evidence of anti-HER2 therapy target receptor expression on the surface of the cells, and at the genomic level by HER2 gene copy number &lt; 6. Results of the NSABP-B47 clinical trial and further studies with larger numbers of HER2-Low patients are needed to determine if these patients benefit from anti-HER2 therapy.
Citation Format: Gunn S, Yaziji H, Sims C, Govender S, Moore M, Cotter P, Jones S. A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-18.