BACKGROUND: Only a portion of breast cancer patients currently selected for trastuzumab therapy respond. METHODS: Using a novel assay (HERmark) to quantify total human epidermal growth factor receptor 2 (HER2) expression, the authors examined outcomes in 102 trastuzumab-treated metastatic breast cancer patients previously assessed as immunohistochemistry (IHC) 3þ by local but not central IHC, or fluorescence in situ hybridization (FISH) positive, and then retested by central FISH. RESULTS: Of 102 MBC patients previously scored as IHC 3þ or 2þ/FISHpositive and treated with trastuzumab-containing regimens, 98 had both central FISH and HER2 total expression values. Sixty-six of 76 central FISH-positive patients (87%) had high HER2 total expression levels (concordant positive), and 19 of 22 central FISH-negative patients (86%) were HER2 total expression low (concordant negative). Fourteen percent (3 of 22) of central FISH-negative patients were HER2 total expression high (discordant HER2 total expression high), and 13% (10 of 76) of central FISH-positive patients were HER2 total expression low (discordant HER2 total expression low). The concordant positive group had a significantly longer time to progression (TTP, median ¼ 11.3 months) compared with the concordant negative group (median TTP, 4.5 months; hazard ratio [HR] ¼ 0.42, P < .001), and also compared with the discordant HER2 total expression low group (median TTP, 3.7 months; HR ¼ 0.43, P ¼ .01). The discordant HER2 total expression low group behaved similarly compared with concordant negatives (HR ¼ 1, P ¼ .99). In analyses restricted to central FISH-positive patients only (n ¼ 77), Cox proportional hazards multivariate regression identified HER2 total expression as an independent predictor of TTP (HR ¼ 0.29, P ¼ .0015) and overall survival (HR ¼ 0.19, P < .001). CONCLUSIONS: A subset of patients with HER2 gene amplification by FISH express low levels of HER2 protein and have reduced response to trastuzumab-containing therapy, similar to FISH-negative

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

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Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2665-0) contains supplementary material, which is available to authorized users.