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

Abstract

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.

Validation of the 4B5 rabbit monoclonal antibody in determining Her2/neu status in breast cancer

“…One study mentions a perfect interlaboratory concordance on a very limited number of cases. 27 Future studies are needed to assess this issue.…”

Delineation of HER2 Gene Status in Breast Carcinoma by Silver in Situ Hybridization is Reproducible among Laboratories and Pathologists

“…Using FISH as the reference standard, it has recently been shown that the anti-HER2 4B5 rabbit monoclonal antibody provides high sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer. 35 In our study, 4B5 demonstrated excellent interlaboratory reproducibility for the detection of HER2 status for each performed comparison (K w ϭ 1.00). The level of agreement between the consensus 4B5 and individual site assessment was satisfactory for all sites (K w ϭ 1.00).…”

HER2 Amplification or Overexpression in Upper GI Tract and Breast Cancer with Clinical Diagnosis and Treatment

“…[85] Clinical assays to assess the HER2 status include IHC, which detects protein overexpression, or FISH, which detects gene amplification. [85][86][87] Both assays have been clinically validated in the above mentioned prospective randomized clinical trials and have received FDA approval for predicting a clinical response and patient benefit from HER2-targeted treatment. Published data from these clinical trials suggest that only those patients whose breast cancer demonstrates protein over-expression and/or gene amplification by the above assays are likely to benefit from therapy with Trastuzumab.…”