Abstract
IntroductionHER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.MethodsTissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.ResultsOverall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.ConclusionsThe novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.
Highlights
HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy
We have recently demonstrated a high concordance of silver labeled in situ hybridization (SISH) and FISH results in the detection of EGFR copy number alterations in lung cancer samples and we have provided data that SISH is an accurate method for the evaluation of the Her2 gene amplification status in cytologic breast cancer specimens, in metastatic breast cancer lesions [16] [17]
In our Tissue microarrays (TMAs)-based study, we demonstrate an excellent correlation between IHC score 3+ and gene amplification by SISH using a dual probe for IHC and SISH
Summary
HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. The ASCO guidelines approved several test assays for the HER2 status assessment including immunohistochemistry (IHC) and/or in situ hybridization (ISH) technics with different visualization methods as fluorescence (FISH), silver (SISH) or chromogenic (CISH) labeled probes [2,5,6]. These tests are conducted in a single way, either immunohistochemistry complemented with ISH technology or the ISH technology alone. A novel bright-field assay for concomitant detection of HER2 gene status and protein expression on one tissue section has been described by Tubbs et al [12]. This assay allows the identification of tumor cells with discordant HER2 results, e.g. HER2 protein overexpression without evidence for HER2 gene amplification
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