Fluorescence in Situ Hybridization HER 2 Amplification Status among Immunohistochemistry 2+ (Equivocal) Breast Carcinoma Patients of Bangladesh: A Series of 75 Cases
Authors: Md Zillur Rahman
Institution: Bangladesh Medical University
Introduction
Abstract Background Accurate determination of HER2 status in breast carcinoma is essential for guiding targeted therapy and improving patient outcomes. While immunohistochemistry (IHC) is commonly used as an initial screening method, equivocal (2+) IHC results always require confirmation through fluorescence in situ hybridization (FISH). However, data on Fluorescence in Situ Hybridization HER 2 Amplification status among Immunohistochemistry 2+ (Equivocal) breast carcinoma patients of Bangladesh remain limited, despite its significance in ensuring appropriate treatment planning. Objective: This study aimed to determine the Fluorescence in Situ Hybridization HER 2 Amplification status among Immunohistochemistry 2+ (Equivocal) Breast Carcinoma Patients of Bangladesh.
Methods
Materials and Methods: This retrospective observational study was conducted in the Department of Pathology in Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, Bangladesh, from January 2024 to June 2025. The consecutive sampling technique was used and a total of 75 IHC-tested equivocal or borderline (2+) cases were retrospectively employed in this study. Breast tissue specimens were obtained via mastectomy (n=10), and core needle biopsy (n=65), processed into formalin-fixed paraffin-embedded (FFPE) blocks, and analyzed following ASCO/CAP guidelines (2018). FISH analysis was performed on all cases (N=75) to assess HER2 gene amplification. Then the data were extracted and analyzed using Statistical Package for Social Sciences (SPSS), version 23.0.
Results
Results: A total of 75 cases of invasive breast carcinoma with equivocal (IHC 2+) HER2 expression were analyzed. The mean age of patients was 48.3 ± 11.1 years (median 46; range 28–88 years), with the highest proportion in the 38–47-year age group (36.0%). Most specimens were obtained by core needle biopsy (86.6%), while 13.3% were mastectomy specimens. Histologically, all tumors were invasive ductal carcinoma (NST). Grade II tumors predominated (72.0%), followed by Grade III (17.3%) and Grade I (10.6%). Hormone receptor analysis showed estrogen receptor positivity in 60.0% and progesterone receptor positivity in 45.3% of cases. A high Ki-67 proliferation index (>15%) was observed in 73.3% of tumors. FISH analysis demonstrated HER2 gene amplification in 4 of 75 cases (5.3%), whereas 71 cases (94.7%) were non-amplified despite initial IHC 2+ classification. The exact 95% confidence interval for the amplification rate was 1.5% -13.0%. These findings reinforce the necessity of mandatory hybridization testing for accurate HER2 classification in equivocal cases to ensure appropriate allocation of HER2-targeted therapy.
Conclusion
Conclusion: This study demonstrates that only a small proportion of IHC 2+ breast carcinomas show true HER2 amplification. These findings reinforce the unconditional requirement for hybridization testing in all equivocal cases, particularly in settings where reflex testing may not yet be consistently implemented. Strengthening quality-assured HER2 diagnostics will be essential for advancing precision oncology and optimizing therapeutic resource allocation in Bangladesh.