A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.

The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.

The expression of the cytoskeletal focal adhesion protein paxillin in breast cancer correlates with HER2 overexpression and may help predict response to chemotherapy: a retrospective immunohistochemical study.

Paxillin, a cytoskeletal focal adhesion adaptor protein, has been shown to be transcriptionally up-regulated and phosphorylated by human epidermal growth factor receptor-2 (HER2) signaling in vitro. Paxillin expression may also correlate with HER2 amplification in breast cancer patients. In the current study, we sought to explore the relationship further between paxillin expression and clinicopathologic features and clinical outcome in breast cancer. A total of 314 primary invasive breast carcinomas were assessed for paxillin expression via immunohistochemistry. Paxillin immunoreactivity was compared with estrogen receptor/progesterone receptor status, HER2 status by silver in situ hybridization, age, tumor size, stage, Bloom-Richardson grade, nodal status, disease-free survival (DFS), and overall survival (OS). Paxillin expression was identified in 27.7% of breast carcinomas as diffuse cytoplasmic staining and the expression correlated with HER2 overexpression (p < 0.001). The influence of paxillin on clinical outcome, in particular the response to chemotherapy, appeared to differ depending on the HER2 status of the tumor. For the subset of HER2 nonamplified cases treated with chemotherapy, patients whose tumor showed a loss of paxillin expression demonstrated a significantly lengthened DFS and OS. In contrast, loss of paxillin expression in the HER2 amplified subset of patients who received chemotherapy correlated with a significantly worse outcome. These data suggest that paxillin up-regulation may be a part of the HER2 pathway in some breast cancers and, furthermore, paxillin expression may also influence the clinical response to chemotherapy, depending upon the HER2 status of a given patient's tumor. Further study of a role for paxillin expression in predicting response to cytotoxic regimens or targeted treatments is warranted.