Discordance between Equivocal HER2 Immunohistochemistry and Fluorescence in Situ Hybridization Analysis among the Breast Carcinoma Patients in Bangladesh: A Series of 75 Cases

Discordance between Equivocal HER2 Immunohistochemistry and Fluorescence in Situ Hybridization Analysis among the Breast Carcinoma Patients in Bangladesh: A Series of 75 Cases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discordance between Equivocal HER2 Immunohistochemistry and Fluorescence in Situ Hybridization Analysis among the Breast Carcinoma Patients in Bangladesh: A Series of 75 Cases Mohammad Zillur Rahman, Ratim Mir, Md. Ariful Islam, Mohammad Shukur Ali, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8058814/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version 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 often require confirmation through fluorescence in situ hybridization (FISH). However, data on the concordance between IHC and FISH in Bangladesh remain limited, despite its significance in ensuring appropriate treatment planning. Materials and Methods : This retrospective observational cross-sectional 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. 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 : The average age of the patients was 48.3 ± 11.1 years, with a range of 28 to 88 years. Most patients were aged 38 to 47 years (36.0%), and specimens were predominantly acquired by core needle biopsy (86.6%). In tumor grading, cases (core needle biopsy, n = 65), 75.3% were classified as Grade II tumors. FISH analysis revealed HER2 amplification in only 4 cases (5.3%), while the majority (94.6%) were non-amplified despite initial IHC 2 + scoring. The difference between the IHC and FISH findings was statistically significant (p < 0.001), highlighting the risk of misclassification when relying solely on IHC. Conclusion : This study demonstrates substantial discordance between equivocal IHC and FISH findings in determining HER2 status among breast carcinoma patients in Bangladesh. These results reinforce the critical need for routine molecular confirmation using FISH in IHC 2 + cases to ensure accurate diagnosis and guide effective HER2-targeted therapy, ultimately improving clinical decision-making and patient care. Breast carcinoma HER2 immunohistochemistry fluorescence in situ hybridization discordance Bangladesh diagnostic accuracy targeted therapy Figures Figure 1 Figure 2 Figure 3 Introduction Breast cancer continues to represent the most frequently diagnosed malignancy among women worldwide, contributing substantially to the global public health burden due to its rising incidence and biological complexity ( 1 ). In South Asia, and notably in Bangladesh, the incidence of breast cancer has been increasing steadily over the past two decades. Unfortunately, cases are often detected at more advanced stages, a consequence of limited diagnostic resources, inadequate screening programs, and delays in seeking medical care ( 2 , 3 ). Among the several genetic subtypes of breast cancer, the overexpression or amplification of the human epidermal growth factor receptor 2 (HER2) gene is essential in assessing tumour aggressiveness, therapy responsiveness, and patient prognosis ( 4 ). Accurate evaluation of HER2 status is essential for guiding the use of targeted treatments, particularly trastuzumab, which has significantly improved survival rates for patients with HER2-positive breast cancer ( 5 ). In standard procedures, HER2 status is initially evaluated using immunohistochemistry (IHC), a cost-effective and widely available method that evaluates protein overexpression at the tumor cell membrane ( 6 ). However, IHC, especially in cases scored as 2+ (equivocal), may yield ambiguous results due to subjective interpretation, technical variability, and tumor heterogeneity ( 7 ). For such equivocal cases, Fluorescence in situ hybridisation (FISH) identifies HER2 gene amplification at the DNA level, is recommended to ensure accurate classification and guide appropriate treatment decisions ( 8 ). Although a high concordance rate is generally reported between IHC 0/1 + and negative FISH, or IHC 3 + and positive FISH, discrepancies are frequently observed in 2 + cases and even in a subset of 3 + cases ( 9 ). Such discordance may arise from pre-analytical and analytical variability, differences in interpretation criteria, or the biological complexity of tumors, including HER2 gene heterogeneity or chromosome 17 polysomy ( 10 ). In Bangladesh, there remains a scarcity of research investigating the concordance and discordance between IHC and FISH in HER2 testing, despite the significant clinical implications. Given the limitations in molecular diagnostic infrastructure in many low- and middle-income countries, understanding these patterns is vital for developing accurate, cost-effective diagnostic strategies ( 11 ). This study, therefore, aims to assess the concordance and discordance of equivocal HER2 IHC and FISH results among 75 breast carcinoma cases in Bangladesh. The findings will help refine HER2 testing protocols and support evidence-based therapeutic decisions in resource constrained settings. Materials and methods Study Design and Setting A retrospective observational cross-sectional study was conducted at the pathology department of Ahsania Mission Cancer and General Hospital (AMCGH) in Uttara, Dhaka, Bangladesh. Study Duration The research was conducted from January − 2024 to June- 2025. Sample Size and Population A total of 75 IHC tested equivocal, or borderline (2+) cases were retrospectively enrolled in this study. Sampling technique: All cases were collected consecutively from the pathology department of Ahsania Mission Cancer & General Hospital (AMCGH) during the study period. Inclusion criteria Inclusion criteria consisted of patients with sufficient tissue samples for both IHC and FISH analyses and a confirmed diagnosis of invasive breast carcinoma. Exclusion Criteria Cases with inadequate tissue for FISH testing or incomplete clinical data were excluded from this study. Specimen Collection and Processing A total of 75 FFPE tissue blocks of breast carcinoma patients who experienced equivocal or borderline (2+) in IHC stain for HER2 were collected from the histopathology laboratory of Ahsania Mission Cancer & General Hospital (AMCGH) and their recorded data were also collected for the analysis of this study. Fluorescence in Situ Hybridization (FISH) Analysis FISH analysis was done on 75 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from breast cancer patients who had demonstrated equivocal (2+) HER2 expression using immunohistochemistry (IHC). Dual-color FISH was performed using HER2 (labeled with a red fluorophore) and CEP17 (chromosome 17 centromere, labeled with a green fluorophore) probes. The HER2 gene amplification status was assessed according to ASCO/CAP guidelines by evaluating the HER2/CEP17 signal ratio in tumor cell nuclei under a fluorescence microscope. Data Collection Tools: A pre-structured questionnaire and a case-record form were used to collect the data from the patients’ hospital records. Data Analysis The extracted data were analyzed using the Statistical Package for Social Sciences (SPSS) software, version-23.0. Descriptive statistical analysis were performed and the results were presented in the tables as frequency and percentage. Chi-square test was applied to find out the proportional difference between the IHC and FISH analysis where, p < 0.05 considered as the level of significance with 95% CI. Research Ethics The Managing Director and Registrar of Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, Bangladesh, gave legal approval for data access because this was a retrospective study. Results This study encompassed 75 instances of equivocal (2+) HER2 Immunohistochemistry (IHC) breast cancer. The ages of the cases varied from 28 to 88 years, with a median age of 46 years and a mean age of 48.3 ± 11.1 years. The age group of 38–47 years old accounted for the majority of patients (27 cases, 36.0%), followed by 48–57 years (22 cases, 29.3%), 28–37 years (13 cases, 17.3%), 58–67 years (10 cases, 13.3%), and ≥ 68 years (3 cases, 4.0%) (Table-1). Regarding specimen site, 37 cases (49.3%) were from the left breast, 34 cases (45.3%) from the right breast, 3 cases (4.0%) from the axillary tail of the left breast, and 1 case (1.3%) from the chest wall (recurrent)(Table-2). The majority of specimens were obtained via core needle biopsy (65 cases, 86.6%), while mastectomy specimens accounted for 10 cases (13.3%)(Table-3). Tumor grading was performed in 65 patients (n = 65, core needle biopsy). Most tumors were Grade II (49 cases, 75.3%), followed by Grade III (13 instances, 20.0%) and Grade I (3 cases, 4.6%) (Table-4). Tumor staging was performed on 10 cases (n = 10 mastectomy), indicated that pT2N1Mx and pT4NxMx stages were most common (2 cases each, 20.0% respectively). The remaining stages included pT2N0Mx (1 case, 10.0%), pT1N1Mx (1 case, 10.0%), pT1cNxMx (2 cases, 20.0%), pT2N3Mx (1 case, 10.0%), and pT3N2bM (1 case, 10.0%)(Table-5). Fluorescence In Situ Hybridization (FISH) Analysis revealed that only 4 cases (5.3%) were HER2 amplified, whereas 71 cases (94.6%) were not amplified. This finding highlights the predominance of HER2-negative tumors within equivocal IHC cases in this cohort. According to a comparison of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), all 75 cases had an initial HER2 IHC 2+ (equivocal) score, but after FISH confirmation, 71 cases (94.6%) were HER2 non-amplified and 4 cases (5.3%) were HER2 amplified. The observed discordance between IHC 2 + results and the results for FISH analysis were statistically significant (p < 0.001) (Table-6). This significant discrepancy underscores the essential function of molecular confirmation in precisely directing HER2-targeted therapeutic choices. Discussion This study assessed 75 Bangladeshi women with invasive breast cancer who initially exhibited ambiguous (2+) HER2 IHC scores for concordance and discordance between HER2 immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) analysis. The results show that there is a lot of variation in diagnosis, which is essential for making treatment decisions in places like Bangladesh where resources are limited. Only 5.3% of IHC 2 + cases demonstrated HER2 gene amplification on FISH, while the overwhelming majority (94.6%) were HER2 non-amplified. hese approaches and interpretation thresholds are codified in ASCO/CAP guideline updates to improve diagnostic accuracy and interlaboratory reproducibility ( 12 , 13 ). The low amplification rate in equivocal cases in our study aligns with earlier findings from Bangladesh. Such discordance is not unique to Bangladesh; studies across South Asia reported comparable variability. In breast cancer, 14.6% of IHC 2 + cases were found to be HER2-positive by FISH, indicating a notable discrepancy and misclassification can lead to inappropriate therapeutic decisions, as patients may be denied effective HER2-targeted therapies ( 14 , 15 ). These findings differ significantly from data in high-income countries, where strict adherence to standardized protocols ensures higher concordance rates. In North America, concordance between IHC 3 + and FISH positivity exceeds 95%, supported by automated scoring systems and robust quality assurance measures ( 11 , 16 ). The discordance observed in this study may be attributed to multiple factors, including pre-analytical variables (such as fixation time and tissue processing), subjective interpretation of IHC, and the inherent biological heterogeneity of breast tumors ( 17 ). Polysomy 17, a condition where multiple copies of chromosome 17 are present, can result in elevated HER2 protein expression without corresponding gene amplification, contributing to falsely high IHC scores ( 7 ). Moreover, study highlights that core needle biopsies (CNBs) present challenges in capturing tumor heterogeneity, which can lead to increased variability and sampling errors ( 18 ). This sampling error can lead to significant issues, including misclassification of the tumor type, as these lesions are often large and heterogeneous ( 19 ). Interestingly, our cohort predominantly comprised Grade II tumors (75.3%), with infiltrating ductal carcinoma as the most common histologic type. This pattern is consistent with national and regional epidemiological trends, where luminal subtypes are more frequent than HER2-enriched or triple-negative breast cancers ( 2 ). However, despite the higher grade often correlating with HER2 overexpression, this data indicate that histological grade alone does not reliably predict HER2 gene amplification. Recent reports highlight persistent challenges in reproducibility, especially within the emerging HER2-low category, emphasizing the need for standardized scoring and confirmatory molecular techniques ( 20 ). This finding further supports the need for molecular confirmation in equivocal cases to prevent both overtreatment with expensive targeted therapies and under treatment of patients who may benefit from them ( 21 ). From a clinical and public health perspective, the results of this present study reinforce the imperative for scaling up access to reliable molecular diagnostic tools such as FISH in Bangladesh. Relying solely on IHC 2 + results risks inappropriate treatment decisions and without reflex ISH confirmation can lead to misclassification of HER2 status, resulting in inappropriate treatment decisions and potentially harming patients, while also misallocating healthcare resources ( 22 – 24 ). Even in low- and middle-income nations, international standards like the ASCO/CAP guidelines for HER2 testing are being put into place, is crucial to ensure accurate diagnosis and optimal patient outcomes. Implementing these guidelines necessitates training healthcare professionals and ensuring access to necessary diagnostic resources, which can be challenging in resource-limited settings ( 25 ). This will require investment in laboratory infrastructure, training of pathologists and technicians, and the development of cost-effective diagnostic algorithms tailored to local settings. Finally, this study adds to the growing evidence that HER2 IHC 2 + results require molecular confirmation by FISH or equivalent techniques to guide appropriate treatment. As Bangladesh strengthens its cancer care infrastructure, prioritizing quality-assured HER2 testing will be essential for the success of precision oncology Limitations of the Study The primary limitation of this study is its retrospective design that depend on existing medical records and pathology reports. This may have introduced selection bias and limited the availability of certain clinical and pathological data, such as detailed staging information or long-term patient outcomes. Furthermore, the results may not be entirely applicable to all breast cancer populations in Bangladesh or other areas because the study was limited to a single center. Recommendations Considering the significant discordance observed between IHC 2 + results and FISH analysis, it is recommended that FISH or an equivalent molecular confirmatory test be routinely implemented for all breast carcinoma cases showing equivocal (2+) IHC scores. This will help ensure accurate HER2 status determination and guide appropriate HER2-targeted therapy, thereby improving patient outcomes. Furthermore, national guidelines should consider integrating mandatory confirmatory testing for borderline cases to standardize diagnostic practice. Future multicenter, prospective studies with larger and more diverse populations are encouraged to validate these findings and explore their impact on therapeutic response and survival. Conclusion This study demonstrates substantial discordance between equivocal IHC and FISH findings in determining HER2 status among breast carcinoma patients in Bangladesh. These results reinforce the critical need for routine molecular confirmation using FISH in IHC 2 + cases to ensure accurate diagnosis and guide effective HER2-targeted therapy, ultimately improving clinical decision-making and patient care. Declarations Ethics approval and consent to participate This study was based on a retrospective review of anonymized data and did not involve any direct patient intervention. According to the institutional policy, ethical approval and informed consent were waived by the Ethics Committee of the Histopathology Department, Ahsania Mission Cancer and General Hospital, Dhaka, Bangladesh. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Additional datasets that support the findings of this study are stored in controlled institutional data archives at Ahsania Mission Cancer and General Hospital (AMCGH) and are not publicly available due to institutional data protection policies. However, the data are available from the corresponding author on reasonable request and with permission from AMCGH. Competing interest The authors declare no competing interests. Financial support and sponsorship None Funding None Authors’ contributions MZR conceptualized and supervised the study. MZR and SRAS developed the research protocol and contributed to data collection, data entry, data analysis, report writing, and article formatting. RM conducted the literature review, contributed to questionnaire development, and participated in data collection. MAI contributed to data entry, data analysis, and report writing. MSA performed literature review support and language editing. MZR, MAI, and MSA prepared the original manuscript draft. MZR reviewed and edited the final manuscript. All authors read and approved the final manuscript. Acknowledgement The authors would like to express their sincere gratitude to the Department of Pathology at Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, for providing the necessary facilities and support to conduct this study. Special thanks are extended to the laboratory technicians and staff for their assistance in specimen processing and data collection. The authors also acknowledge the patients whose data made this research possible. Authors’ Information 1* MZR is Professor and Chairman, Department of Pathology, Bangladesh Medical University (BMU), Shahbag, Dhaka, Bangladesh, and Senior Consultant, Department of Histopathology, Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0000-0003-3222-1118 2 RM is Assistant Professor, Department of Pathology, Shaheed M. Monsur Ali Medical College, Sirajganj, and Consultant, Ahsania Mission Cancer and General Hospital, Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0006-6393-1891 3 MAI is Assistant Professor, Department of Pathology, International Medical College, Dhaka, and Junior Consultant, Ahsania Mission Cancer and General Hospital, Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0008-0504-0104 4 MSA is CEO of NINO Explore (Research & Development), Khilgaon, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0002-9158-1156 5 SRAS is Associate Professor, Department of Biochemistry, Chittagong Medical College, Chattogram, Bangladesh. ORCID: https://orcid.org/0009-0002-2553-8491 References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J Clin. 2018;68(6):394–424. Hossain MS, Ferdous S, Karim-Kos HE. Breast cancer in South Asia: a Bangladeshi perspective. Cancer Epidemiol. 2014;38(5):465–70. Lim YX, Lim ZL, Ho PJ, Li J. Breast cancer in Asia: incidence, mortality, early detection, mammography programs, and risk-based screening initiatives. Cancers. 2022;14(17):4218. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177–82. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005;353(16):1659–72. Wolff AC, Hammond MEH, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013. Ross JS, Fletcher JA. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Oncologist. 1998;3(4):237–52. Wolff AC, Hammond MEH, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2006;25(1):118–45. Hanna WM, Kwok K. Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm. Mod Pathol. 2006;19(4):481–7. Perez EA, Cortés J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: current status and future directions. Cancer Treat Rev. 2014;40(2):276–84. Press MF, Sauter G, Bernstein L, Villalobos IE, Mirlacher M, Zhou J-Y, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 2005;11(18):6598–607. Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. Arch Pathol Lab Med. 2018;142(11):1364–82. Farshid G, Dhatrak D, Gilhotra A, Koszyca B, Nolan J. The impact of 2018 ASCO-CAP HER2 testing guidelines on breast cancer HER2 results. An audit of 2132 consecutive cases evaluated by immunohistochemistry and in situ hybridization. Mod Pathol. 2020;33(9):1783–90. Podany P, Golastani R, Zhan H, Liang Y, Harigopal M, Krishnamurti U. HER2 amplification in cases with low HER2 expression by immunohistochemistry (IHC): A Single Institutional Experience. Am J Clin Pathol. 2023;160(Supplement1):S9–10. Ayatollahi H, Jafarian AH, Moghadam ZP, Ayatollahi Y, Hassankhani GG, Mehrad-Majd H. Clinicopathological Characteristics of Breast Cancer Patients with Equivocal Immunohistochemistry: A Prevalence-Based Statistical Analysis. Iran J Pathol. 2025;20(3):273. Hanna WM, Rüschoff J, Bilous M, Coudry RA, Dowsett M, Osamura RY, et al. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol. 2014;27(1):4–18. Viale G. The current state of breast cancer classification. Ann Oncol. 2012;23:x207–10. Scribano C, Adstamongkonkul P, Caenepeel S, Dana N, Derrick J, Flietner E, et al. A sequential treatment strategy for ex vivo profiling of live tumor fragments that mitigates tumor heterogeneity and tissue scarcity from core needle biopsies to characterize response to immunotherapies. Cancer Res. 2025;85(8Supplement1):3301. Thavikulwat AC, Wu JS, Chen X, Anderson ME, Ward A, Kung J. Image-guided core needle biopsy of adipocytic tumors: diagnostic accuracy and concordance with final surgical pathology. Am J Roentgenol. 2021;216(4):997–1002. Sajjadi E, Venetis K, Ivanova M, Fusco N. Improving HER2 testing reproducibility in HER2-low breast cancer. Cancer Drug Resist. 2022;5(4):882. Gutierrez C, Schiff R. HER2: biology, detection, and clinical implications. Arch Pathol Lab Med. 2011;135(1):55–62. Alsebai T, Swied MY, Cossyleon R, Robinson K, Delfino K, Gao J, et al. H-score as a diagnostic tool in HER2 equivocal (IHC 2+) breast cancer. Cancer Res. 2025;85(8Supplement1):677. Tchrakian N, Flanagan L, Harford J, Gannon J, Quinn C. New ASCO/CAP guideline recommendations for HER2 testing increase the proportion of reflex in situ hybridization tests and of HER2 positive breast cancers. Virchows Arch. 2016;468(2):207–11. Curado M, Caramelo AS, Eloy C, Polonia A. What to expect from the 2018 ASCO/CAP HER2 guideline in the reflex in situ hybridization test of immunohistochemically equivocal 2 + cases? Virchows Arch. 2019;475(3):303–11. Ahuja S, Khan AA, Zaheer S. Understanding the spectrum of HER2 status in breast cancer: From HER2-positive to ultra-low HER2. Pathology-Research Pract. 2024;262:155550. Tables Table-1: Age distribution of the study patients with breast carcinoma (N=75). Age groups(years) Frequency N % 28-37 13 17.3 38-47 27 36.0 48-57 22 29.3 \58-67 10 13.3 ≥68 3 4.0 Total 75 100 Mean age (years) 48.30±11.10 Median 46 Mode 45 Range 28-88 Table-2: Specimen site distribution of the study patients with breast carcinoma (N=75). Specimen Sites Frequency N % Tissue from left breast 37 49.3 Tissue from right breast 34 45.3 Axillary tail of left breast 3 4.0 Chest wall (Recurrent) 1 1.3 Total 75 100 Table 3 Distribution of the study patients by specimen type (N=75). Types of specimen Frequency N % Core needle biopsy 65 86.6 Mastectomy 10 13.3 Total 75 100 Table 4 Grade distribution of breast carcinoma with the study patients (n=65). Grades of breast carcinoma Frequency N % Grade-1 3 4.6 Grade-II 49 75.3 Grade-III 13 20.0 Total 65 100.0 Table 5 Stage distribution of breast carcinoma with the study patients (n=10). Stage distribution Frequency Percent pT2N0Mx 1 10 pT2N1Mx 2 20 pT4NxMx 2 20 pT1N1Mx 1 10 pT1cNxMx 2 20 pT2N3Mx 1 10 pT3N2bM 1 10 Total 10 100 Table 6 Expression of HER2_ in FISH analysis of breast carcinoma with the study patients (N=75). Expression of HER2_ in FISH analysis Frequency Percent N % Not amplified 71 94.6 Amplified 4 5.3 Total 75 100 Table-7 : Discordance status of IHC and FISH analysis of the breast carcinoma (n=75). HER2_IHC analysis N (%) HER2_FISH analysis P-value Not amplified n (%) Amplified n (%) Equivocal (2+) 75(100) 71(94.6) 4(5.3) < 0.001 Chi-square test was performed, where p<0.05 considered as the level of significance with 95%CI. Additional Declarations No competing interests reported. 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Ariful Islam","email":"","orcid":"","institution":"International Medical College","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Ariful","lastName":"Islam","suffix":""},{"id":542757106,"identity":"bd872b75-bb07-4e7a-817b-bceca60233ae","order_by":3,"name":"Mohammad Shukur Ali","email":"","orcid":"","institution":"NINO Explore (Research \u0026 Development)","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Shukur","lastName":"Ali","suffix":""},{"id":542757107,"identity":"727a0488-d3dc-4c95-9f08-7770b5bc02b2","order_by":4,"name":"Syeda Rumman Aktar Siddiqui","email":"","orcid":"","institution":"Chittagong Medical College","correspondingAuthor":false,"prefix":"","firstName":"Syeda","middleName":"Rumman Aktar","lastName":"Siddiqui","suffix":""}],"badges":[],"createdAt":"2025-11-07 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17:17:56","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99957,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8058814/v1/4762b8f0506f846eb94291da.html"},{"id":95798346,"identity":"dd4928f2-6f34-47c0-be53-752740e20c46","added_by":"auto","created_at":"2025-11-13 08:16:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1513639,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of IHC tested Equivocal (2+) case.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8058814/v1/e57d96c904b5bfced2fcc24d.jpeg"},{"id":95670252,"identity":"b3a3da47-39d8-4ab6-b85f-54cdf0c94d65","added_by":"auto","created_at":"2025-11-11 17:17:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":563936,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of Amplified case in FISH analysis.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8058814/v1/378319003445f65b09e7139e.jpeg"},{"id":95670258,"identity":"12a3a1a2-f611-4301-8cd5-b0a2e8840ead","added_by":"auto","created_at":"2025-11-11 17:17:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":411798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAn image of Not amplified case in FISH analysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8058814/v1/aaf7bbbd31dc10d2e71b079e.jpeg"},{"id":95804751,"identity":"560ad9cb-a985-48f5-a2c3-fdb06dbe160f","added_by":"auto","created_at":"2025-11-13 08:39:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3354242,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8058814/v1/a72e86cf-8df2-4512-afc4-080a3d267d1e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discordance between Equivocal HER2 Immunohistochemistry and Fluorescence in Situ Hybridization Analysis among the Breast Carcinoma Patients in Bangladesh: A Series of 75 Cases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer continues to represent the most frequently diagnosed malignancy among women worldwide, contributing substantially to the global public health burden due to its rising incidence and biological complexity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In South Asia, and notably in Bangladesh, the incidence of breast cancer has been increasing steadily over the past two decades. Unfortunately, cases are often detected at more advanced stages, a consequence of limited diagnostic resources, inadequate screening programs, and delays in seeking medical care (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Among the several genetic subtypes of breast cancer, the overexpression or amplification of the human epidermal growth factor receptor 2 (HER2) gene is essential in assessing tumour aggressiveness, therapy responsiveness, and patient prognosis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Accurate evaluation of HER2 status is essential for guiding the use of targeted treatments, particularly trastuzumab, which has significantly improved survival rates for patients with HER2-positive breast cancer (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In standard procedures, HER2 status is initially evaluated using immunohistochemistry (IHC), a cost-effective and widely available method that evaluates protein overexpression at the tumor cell membrane (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, IHC, especially in cases scored as 2+ (equivocal), may yield ambiguous results due to subjective interpretation, technical variability, and tumor heterogeneity (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). For such equivocal cases, Fluorescence in situ hybridisation (FISH) identifies HER2 gene amplification at the DNA level, is recommended to ensure accurate classification and guide appropriate treatment decisions (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Although a high concordance rate is generally reported between IHC 0/1\u0026thinsp;+\u0026thinsp;and negative FISH, or IHC 3\u0026thinsp;+\u0026thinsp;and positive FISH, discrepancies are frequently observed in 2\u0026thinsp;+\u0026thinsp;cases and even in a subset of 3\u0026thinsp;+\u0026thinsp;cases (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Such discordance may arise from pre-analytical and analytical variability, differences in interpretation criteria, or the biological complexity of tumors, including HER2 gene heterogeneity or chromosome 17 polysomy (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In Bangladesh, there remains a scarcity of research investigating the concordance and discordance between IHC and FISH in HER2 testing, despite the significant clinical implications. Given the limitations in molecular diagnostic infrastructure in many low- and middle-income countries, understanding these patterns is vital for developing accurate, cost-effective diagnostic strategies (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This study, therefore, aims to assess the concordance and discordance of equivocal HER2 IHC and FISH results among 75 breast carcinoma cases in Bangladesh. The findings will help refine HER2 testing protocols and support evidence-based therapeutic decisions in resource constrained settings.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design and Setting\u003c/h2\u003e\u003cp\u003eA retrospective observational cross-sectional study was conducted at the pathology department of Ahsania Mission Cancer and General Hospital (AMCGH) in Uttara, Dhaka, Bangladesh.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy Duration\u003c/h3\u003e\n\u003cp\u003eThe research was conducted from January \u0026minus;\u0026thinsp;2024 to June- 2025.\u003c/p\u003e\n\u003ch3\u003eSample Size and Population\u003c/h3\u003e\n\u003cp\u003eA total of 75 IHC tested equivocal, or borderline (2+) cases were retrospectively enrolled in this study.\u003c/p\u003e\n\u003ch3\u003eSampling technique:\u003c/h3\u003e\n\u003cp\u003eAll cases were collected consecutively from the pathology department of Ahsania Mission Cancer \u0026amp; General Hospital (AMCGH) during the study period.\u003c/p\u003e\n\u003ch3\u003eInclusion criteria\u003c/h3\u003e\n\u003cp\u003eInclusion criteria consisted of patients with sufficient tissue samples for both IHC and FISH analyses and a confirmed diagnosis of invasive breast carcinoma.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eExclusion Criteria\u003c/h2\u003e\u003cp\u003eCases with inadequate tissue for FISH testing or incomplete clinical data were excluded from this study.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSpecimen Collection and Processing\u003c/h3\u003e\n\u003cp\u003eA total of 75 FFPE tissue blocks of breast carcinoma patients who experienced equivocal or borderline (2+) in IHC stain for HER2 were collected from the histopathology laboratory of Ahsania Mission Cancer \u0026amp; General Hospital (AMCGH) and their recorded data were also collected for the analysis of this study.\u003c/p\u003e\n\u003ch3\u003eFluorescence in Situ Hybridization (FISH) Analysis\u003c/h3\u003e\n\u003cp\u003eFISH analysis was done on 75 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from breast cancer patients who had demonstrated equivocal (2+) HER2 expression using immunohistochemistry (IHC). Dual-color FISH was performed using HER2 (labeled with a red fluorophore) and CEP17 (chromosome 17 centromere, labeled with a green fluorophore) probes. The HER2 gene amplification status was assessed according to ASCO/CAP guidelines by evaluating the HER2/CEP17 signal ratio in tumor cell nuclei under a fluorescence microscope.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eData Collection Tools:\u003c/h2\u003e\u003cp\u003eA pre-structured questionnaire and a case-record form were used to collect the data from the patients\u0026rsquo; hospital records.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eThe extracted data were analyzed using the Statistical Package for Social Sciences (SPSS) software, version-23.0. Descriptive statistical analysis were performed and the results were presented in the tables as frequency and percentage. Chi-square test was applied to find out the proportional difference between the IHC and FISH analysis where, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered as the level of significance with 95% CI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eResearch Ethics\u003c/h2\u003e\u003cp\u003e The Managing Director and Registrar of Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, Bangladesh, gave legal approval for data access because this was a retrospective study.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThis study encompassed 75 instances of equivocal (2+) HER2 Immunohistochemistry (IHC) breast cancer. The ages of the cases varied from 28 to 88 years, with a median age of 46 years and a mean age of 48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1 years. The age group of 38\u0026ndash;47 years old accounted for the majority of patients (27 cases, 36.0%), followed by 48\u0026ndash;57 years (22 cases, 29.3%), 28\u0026ndash;37 years (13 cases, 17.3%), 58\u0026ndash;67 years (10 cases, 13.3%), and \u0026ge;\u0026thinsp;68 years (3 cases, 4.0%) (Table-1). Regarding specimen site, 37 cases (49.3%) were from the left breast, 34 cases (45.3%) from the right breast, 3 cases (4.0%) from the axillary tail of the left breast, and 1 case (1.3%) from the chest wall (recurrent)(Table-2). The majority of specimens were obtained via core needle biopsy (65 cases, 86.6%), while mastectomy specimens accounted for 10 cases (13.3%)(Table-3). Tumor grading was performed in 65 patients (n\u0026thinsp;=\u0026thinsp;65, core needle biopsy). Most tumors were Grade II (49 cases, 75.3%), followed by Grade III (13 instances, 20.0%) and Grade I (3 cases, 4.6%) (Table-4). Tumor staging was performed on 10 cases (n\u0026thinsp;=\u0026thinsp;10 mastectomy), indicated that pT2N1Mx and pT4NxMx stages were most common (2 cases each, 20.0% respectively). The remaining stages included pT2N0Mx (1 case, 10.0%), pT1N1Mx (1 case, 10.0%), pT1cNxMx (2 cases, 20.0%), pT2N3Mx (1 case, 10.0%), and pT3N2bM (1 case, 10.0%)(Table-5). Fluorescence In Situ Hybridization (FISH) Analysis revealed that only 4 cases (5.3%) were HER2 amplified, whereas 71 cases (94.6%) were not amplified. This finding highlights the predominance of HER2-negative tumors within equivocal IHC cases in this cohort. According to a comparison of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), all 75 cases had an initial HER2 IHC 2+ (equivocal) score, but after FISH confirmation, 71 cases (94.6%) were HER2 non-amplified and 4 cases (5.3%) were HER2 amplified. The observed discordance between IHC 2\u0026thinsp;+\u0026thinsp;results and the results for FISH analysis were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table-6). This significant discrepancy underscores the essential function of molecular confirmation in precisely directing HER2-targeted therapeutic choices.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed 75 Bangladeshi women with invasive breast cancer who initially exhibited ambiguous (2+) HER2 IHC scores for concordance and discordance between HER2 immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) analysis. The results show that there is a lot of variation in diagnosis, which is essential for making treatment decisions in places like Bangladesh where resources are limited. Only 5.3% of IHC 2\u0026thinsp;+\u0026thinsp;cases demonstrated HER2 gene amplification on FISH, while the overwhelming majority (94.6%) were HER2 non-amplified. hese approaches and interpretation thresholds are codified in ASCO/CAP guideline updates to improve diagnostic accuracy and interlaboratory reproducibility (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The low amplification rate in equivocal cases in our study aligns with earlier findings from Bangladesh. Such discordance is not unique to Bangladesh; studies across South Asia reported comparable variability. In breast cancer, 14.6% of IHC 2\u0026thinsp;+\u0026thinsp;cases were found to be HER2-positive by FISH, indicating a notable discrepancy and misclassification can lead to inappropriate therapeutic decisions, as patients may be denied effective HER2-targeted therapies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These findings differ significantly from data in high-income countries, where strict adherence to standardized protocols ensures higher concordance rates. In North America, concordance between IHC 3\u0026thinsp;+\u0026thinsp;and FISH positivity exceeds 95%, supported by automated scoring systems and robust quality assurance measures (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The discordance observed in this study may be attributed to multiple factors, including pre-analytical variables (such as fixation time and tissue processing), subjective interpretation of IHC, and the inherent biological heterogeneity of breast tumors (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Polysomy 17, a condition where multiple copies of chromosome 17 are present, can result in elevated HER2 protein expression without corresponding gene amplification, contributing to falsely high IHC scores (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Moreover, study highlights that core needle biopsies (CNBs) present challenges in capturing tumor heterogeneity, which can lead to increased variability and sampling errors (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This sampling error can lead to significant issues, including misclassification of the tumor type, as these lesions are often large and heterogeneous (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Interestingly, our cohort predominantly comprised Grade II tumors (75.3%), with infiltrating ductal carcinoma as the most common histologic type. This pattern is consistent with national and regional epidemiological trends, where luminal subtypes are more frequent than HER2-enriched or triple-negative breast cancers (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, despite the higher grade often correlating with HER2 overexpression, this data indicate that histological grade alone does not reliably predict HER2 gene amplification. Recent reports highlight persistent challenges in reproducibility, especially within the emerging HER2-low category, emphasizing the need for standardized scoring and confirmatory molecular techniques (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This finding further supports the need for molecular confirmation in equivocal cases to prevent both overtreatment with expensive targeted therapies and under treatment of patients who may benefit from them (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). From a clinical and public health perspective, the results of this present study reinforce the imperative for scaling up access to reliable molecular diagnostic tools such as FISH in Bangladesh. Relying solely on IHC 2\u0026thinsp;+\u0026thinsp;results risks inappropriate treatment decisions and without reflex ISH confirmation can lead to misclassification of HER2 status, resulting in inappropriate treatment decisions and potentially harming patients, while also misallocating healthcare resources (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Even in low- and middle-income nations, international standards like the ASCO/CAP guidelines for HER2 testing are being put into place, is crucial to ensure accurate diagnosis and optimal patient outcomes. Implementing these guidelines necessitates training healthcare professionals and ensuring access to necessary diagnostic resources, which can be challenging in resource-limited settings (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). This will require investment in laboratory infrastructure, training of pathologists and technicians, and the development of cost-effective diagnostic algorithms tailored to local settings. Finally, this study adds to the growing evidence that HER2 IHC 2\u0026thinsp;+\u0026thinsp;results require molecular confirmation by FISH or equivalent techniques to guide appropriate treatment. As Bangladesh strengthens its cancer care infrastructure, prioritizing quality-assured HER2 testing will be essential for the success of precision oncology\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLimitations of the Study\u003c/h2\u003e\u003cp\u003eThe primary limitation of this study is its retrospective design that depend on existing medical records and pathology reports. This may have introduced selection bias and limited the availability of certain clinical and pathological data, such as detailed staging information or long-term patient outcomes. Furthermore, the results may not be entirely applicable to all breast cancer populations in Bangladesh or other areas because the study was limited to a single center.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRecommendations\u003c/h2\u003e\u003cp\u003eConsidering the significant discordance observed between IHC 2\u0026thinsp;+\u0026thinsp;results and FISH analysis, it is recommended that FISH or an equivalent molecular confirmatory test be routinely implemented for all breast carcinoma cases showing equivocal (2+) IHC scores. This will help ensure accurate HER2 status determination and guide appropriate HER2-targeted therapy, thereby improving patient outcomes. Furthermore, national guidelines should consider integrating mandatory confirmatory testing for borderline cases to standardize diagnostic practice. Future multicenter, prospective studies with larger and more diverse populations are encouraged to validate these findings and explore their impact on therapeutic response and survival.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates substantial discordance between equivocal IHC and FISH findings in determining HER2 status among breast carcinoma patients in Bangladesh. These results reinforce the critical need for routine molecular confirmation using FISH in IHC 2\u0026thinsp;+\u0026thinsp;cases to ensure accurate diagnosis and guide effective HER2-targeted therapy, ultimately improving clinical decision-making and patient care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was based on a retrospective review of anonymized data and did not involve any direct patient intervention. According to the institutional policy, ethical approval and informed consent were waived by the Ethics Committee of the Histopathology Department, Ahsania Mission Cancer and General Hospital, Dhaka, Bangladesh.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epublication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edata\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. Additional datasets that support the findings of this study are stored in controlled institutional data archives at Ahsania Mission Cancer and General Hospital (AMCGH) and are not publicly available due to institutional data protection policies. However, the data are available from the corresponding author on reasonable request and with permission from AMCGH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;sponsorship\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMZR conceptualized and supervised the study. MZR and SRAS developed the research protocol and contributed to data collection, data entry, data analysis, report writing, and article formatting. RM conducted the literature review, contributed to questionnaire development, and participated in data collection. MAI contributed to data entry, data analysis, and report writing. MSA performed literature review support and language editing. MZR, MAI, and MSA prepared the original manuscript draft. MZR reviewed and edited the final manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to the Department of Pathology at Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, for providing the necessary facilities and support to conduct this study. Special thanks are extended to the laboratory technicians and staff for their assistance in specimen processing and data collection. The authors also acknowledge the patients whose data made this research possible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eInformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e1*\u003c/sup\u003e\u003c/strong\u003eMZR is Professor and Chairman, Department of Pathology, Bangladesh Medical University (BMU), Shahbag, Dhaka, Bangladesh, and Senior Consultant, Department of Histopathology, Ahsania Mission Cancer and General Hospital (AMCGH), Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0000-0003-3222-1118\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eRM is Assistant Professor, Department of Pathology, Shaheed M. Monsur Ali Medical College, Sirajganj, and Consultant, Ahsania Mission Cancer and General Hospital, Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0006-6393-1891\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eMAI is Assistant Professor, Department of Pathology, International Medical College, Dhaka, and Junior Consultant, Ahsania Mission Cancer and General Hospital, Uttara, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0008-0504-0104\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eMSA is CEO of NINO Explore (Research \u0026amp; Development), Khilgaon, Dhaka, Bangladesh. ORCID: https://orcid.org/0009-0002-9158-1156\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003eSRAS is Associate Professor, Department of Biochemistry, Chittagong Medical College, Chattogram, Bangladesh. ORCID: https://orcid.org/0009-0002-2553-8491\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J Clin. 2018;68(6):394\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain MS, Ferdous S, Karim-Kos HE. Breast cancer in South Asia: a Bangladeshi perspective. Cancer Epidemiol. 2014;38(5):465\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim YX, Lim ZL, Ho PJ, Li J. Breast cancer in Asia: incidence, mortality, early detection, mammography programs, and risk-based screening initiatives. Cancers. 2022;14(17):4218.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePiccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005;353(16):1659\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWolff AC, Hammond MEH, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997\u0026ndash;4013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoss JS, Fletcher JA. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Oncologist. 1998;3(4):237\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWolff AC, Hammond MEH, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2006;25(1):118\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHanna WM, Kwok K. Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm. Mod Pathol. 2006;19(4):481\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerez EA, Cort\u0026eacute;s J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: current status and future directions. Cancer Treat Rev. 2014;40(2):276\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePress MF, Sauter G, Bernstein L, Villalobos IE, Mirlacher M, Zhou J-Y, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 2005;11(18):6598\u0026ndash;607.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. Arch Pathol Lab Med. 2018;142(11):1364\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarshid G, Dhatrak D, Gilhotra A, Koszyca B, Nolan J. The impact of 2018 ASCO-CAP HER2 testing guidelines on breast cancer HER2 results. An audit of 2132 consecutive cases evaluated by immunohistochemistry and in situ hybridization. Mod Pathol. 2020;33(9):1783\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePodany P, Golastani R, Zhan H, Liang Y, Harigopal M, Krishnamurti U. HER2 amplification in cases with low HER2 expression by immunohistochemistry (IHC): A Single Institutional Experience. Am J Clin Pathol. 2023;160(Supplement1):S9\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyatollahi H, Jafarian AH, Moghadam ZP, Ayatollahi Y, Hassankhani GG, Mehrad-Majd H. Clinicopathological Characteristics of Breast Cancer Patients with Equivocal Immunohistochemistry: A Prevalence-Based Statistical Analysis. Iran J Pathol. 2025;20(3):273.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHanna WM, R\u0026uuml;schoff J, Bilous M, Coudry RA, Dowsett M, Osamura RY, et al. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol. 2014;27(1):4\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViale G. The current state of breast cancer classification. Ann Oncol. 2012;23:x207\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScribano C, Adstamongkonkul P, Caenepeel S, Dana N, Derrick J, Flietner E, et al. A sequential treatment strategy for ex vivo profiling of live tumor fragments that mitigates tumor heterogeneity and tissue scarcity from core needle biopsies to characterize response to immunotherapies. Cancer Res. 2025;85(8Supplement1):3301.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThavikulwat AC, Wu JS, Chen X, Anderson ME, Ward A, Kung J. Image-guided core needle biopsy of adipocytic tumors: diagnostic accuracy and concordance with final surgical pathology. Am J Roentgenol. 2021;216(4):997\u0026ndash;1002.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSajjadi E, Venetis K, Ivanova M, Fusco N. Improving HER2 testing reproducibility in HER2-low breast cancer. Cancer Drug Resist. 2022;5(4):882.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGutierrez C, Schiff R. HER2: biology, detection, and clinical implications. Arch Pathol Lab Med. 2011;135(1):55\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlsebai T, Swied MY, Cossyleon R, Robinson K, Delfino K, Gao J, et al. H-score as a diagnostic tool in HER2 equivocal (IHC 2+) breast cancer. Cancer Res. 2025;85(8Supplement1):677.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTchrakian N, Flanagan L, Harford J, Gannon J, Quinn C. New ASCO/CAP guideline recommendations for HER2 testing increase the proportion of reflex in situ hybridization tests and of HER2 positive breast cancers. Virchows Arch. 2016;468(2):207\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCurado M, Caramelo AS, Eloy C, Polonia A. What to expect from the 2018 ASCO/CAP HER2 guideline in the reflex in situ hybridization test of immunohistochemically equivocal 2\u0026thinsp;+\u0026thinsp;cases? Virchows Arch. 2019;475(3):303\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhuja S, Khan AA, Zaheer S. Understanding the spectrum of HER2 status in breast cancer: From HER2-positive to ultra-low HER2. Pathology-Research Pract. 2024;262:155550.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable-1:\u003c/strong\u003e\u0026nbsp; Age distribution of the study patients with breast carcinoma (N=75).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAge groups(years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 294px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e28-37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e17.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e38-47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e36.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e48-57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e29.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\\58-67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u0026ge;68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean age (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e48.30\u0026plusmn;11.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRange\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e28-88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable-2:\u003c/strong\u003e Specimen site distribution of the study patients with breast carcinoma (N=75).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003eSpecimen Sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60.6383%;\"\u003e\n \u003cp\u003eFrequency\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003eTissue from left breast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e49.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003eTissue from right breast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e45.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003eAxillary tail of left breast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003eChest wall (Recurrent)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.3617%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.8511%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.7872%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eDistribution of the study patients by specimen type (N=75).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTypes of specimen\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 348px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eCore needle biopsy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e86.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eMastectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e100\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Grade distribution of breast carcinoma with the study patients (n=65).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"570\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGrades of breast carcinoma\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 348px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u0026nbsp; Grade-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003eGrade-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e75.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003eGrade-III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Stage distribution of breast carcinoma with the study patients (n=10).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage distribution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT2N0Mx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT2N1Mx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT4NxMx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT1N1Mx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT1cNxMx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT2N3Mx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003epT3N2bM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u0026nbsp;\u003c/strong\u003e Expression of\u003cstrong\u003e\u0026nbsp;HER2_ in FISH analysis of\u003c/strong\u003e breast carcinoma with the study patients (N=75).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"583\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eExpression of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHER2_ in FISH analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eNot amplified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e94.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eAmplified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable-7\u003c/strong\u003e: Discordance status of IHC and FISH analysis of the breast carcinoma (n=75).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"558\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 169px;\"\u003e\n \u003cp\u003eHER2_IHC analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003eHER2_FISH analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eNot amplified\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eAmplified\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 169px;\"\u003e\n \u003cp\u003eEquivocal (2+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e75(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e71(94.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e4(5.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eChi-square test was performed, where p\u0026lt;0.05 considered as the level of significance with 95%CI.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast carcinoma, HER2, immunohistochemistry, fluorescence in situ hybridization, discordance, Bangladesh, diagnostic accuracy, targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-8058814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8058814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAccurate 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 often require confirmation through fluorescence in situ hybridization (FISH). However, data on the concordance between IHC and FISH in Bangladesh remain limited, despite its significance in ensuring appropriate treatment planning.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e: This retrospective observational cross-sectional 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. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The average age of the patients was 48.3 ± 11.1 years, with a range of 28 to 88 years. Most patients were aged 38 to 47 years (36.0%), and specimens were predominantly acquired by core needle biopsy (86.6%). In tumor grading, cases (core needle biopsy, n = 65), 75.3% were classified as Grade II tumors. FISH analysis revealed HER2 amplification in only 4 cases (5.3%), while the majority (94.6%) were non-amplified despite initial IHC 2 + scoring. The difference between the IHC and FISH findings was statistically significant (p \u0026lt; 0.001), highlighting the risk of misclassification when relying solely on IHC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study demonstrates substantial discordance between equivocal IHC and FISH findings in determining HER2 status among breast carcinoma patients in Bangladesh. These results reinforce the critical need for routine molecular confirmation using FISH in IHC 2 + cases to ensure accurate diagnosis and guide effective HER2-targeted therapy, ultimately improving clinical decision-making and patient care.\u003c/p\u003e","manuscriptTitle":"Discordance between Equivocal HER2 Immunohistochemistry and Fluorescence in Situ Hybridization Analysis among the Breast Carcinoma Patients in Bangladesh: A Series of 75 Cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 17:17:51","doi":"10.21203/rs.3.rs-8058814/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-03T14:49:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-03T02:20:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-12T09:52:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256627996063004552900167022189408595701","date":"2026-01-10T14:50:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T11:46:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T11:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289964068575878530859608143683287891122","date":"2026-01-02T04:41:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88810684733002759729557819289746379647","date":"2025-12-31T03:41:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35606764829286080869639179912536592626","date":"2025-12-31T01:31:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88647194316296533830088622030048106694","date":"2025-12-30T18:07:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-30T16:37:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-14T15:47:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T07:47:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-14T07:45:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-11-07T15:57:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ee09522-c083-467a-804e-e3b025766123","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-24T05:08:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 17:17:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8058814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8058814","identity":"rs-8058814","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}