Reevaluating HER2-null breast cancer using an enhanced HER2 immunohistochemistry protocol

Reevaluating HER2-null breast cancer using an enhanced HER2 immunohistochemistry protocol | 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 Reevaluating HER2-null breast cancer using an enhanced HER2 immunohistochemistry protocol Takeshi Ushigusa, Nami Hirakawa, Naoki Kanomata This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6111205/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 May, 2025 Read the published version in Breast Cancer → Version 1 posted 5 You are reading this latest preprint version Abstract Background : Human epidermal growth factor receptor 2 (HER2) status is crucial for the classification of breast cancer and selection of its treatment. Although HER2-low breast cancer is a recognized therapeutic subgroup, the classification of HER2 immunohistochemistry (IHC) 0 remains unclear. Methods : We reassessed 58 HER2-null breast cancer cases using an enhanced HER2 IHC protocol and the VENTANA OptiView detection system. HER2 expression was evaluated based on membrane positivity rate (%) and staining intensity. Microscopic assessment was performed to determine the percentage of HER2-positive tumor cells. Digital image analysis was used to quantify staining intensity. Results : Membrane HER2 positivity was detected to varying degrees in all cases. This indicated that tumors previously classified as HER2-null exhibited HER2 expression when analyzed with the enhanced detection system. Conclusion : These findings suggest that enhanced HER2 IHC protocols can improve detection sensitivity. Further research is needed to determine the clinical significance of HER2 expression detected using enhanced protocols. HER2 HER2-null breast cancer Immunohistochemistry HER2-targeted therapy Trastuzumab deruxtecan Figures Figure 1 Figure 2 Introduction Human epidermal growth factor receptor 2 (HER2) status plays a key role in breast cancer classification and treatment selection. Patients with HER2 positivity, defined as an immunohistochemistry (IHC) score of 3 + or 2+/in situ hybridization (ISH)-positive, are eligible to receive targeted therapies such as trastuzumab. By contrast, patients who are HER2 negative (IHC 0, 1+, or 2+/ISH-negative) have traditionally been considered ineligible because of the limited treatment options. Recent studies have introduced the concept of “HER2-low” (IHC 1 + or IHC 2+/ISH-negative), which accounts for nearly 50% of all breast cancer cases [ 1 , 2 ]. This group has shown responsiveness to HER2-directed antibody–drug conjugates, such as trastuzumab deruxtecan (T-DXd), which increases the clinical relevance of the HER2-low classification [ 3 , 4 ]. HER2 IHC 0 includes two categories: HER2-ultralow and HER2-null. These categories are not defined by guidelines, although HER2-ultralow is typically defined as ≤10% of infiltrating cancer cells showing weak or incomplete membrane staining, and HER2-null refers to no detectable HER2 expression [ 5 ]. Current IHC staining methods are designed primarily to detect high HER2 expression, which raises concerns that lower levels may be underestimated. The choice of the detection system can also affect staining sensitivity. The VENTANA OptiView DAB IHC Detection Kit (OptiView) produces stronger HER2 staining than the VENTANA ultraView Universal DAB Detection Kit (ultraView) in patients with HER2-low expression [ 6 ]. However, the sensitivity of the OptiView in patients previously classified as HER2-null remains unclear. In this study, we applied an enhanced HER2 IHC protocol and the OptiView system to assess HER2-null breast cancer cases. We aimed to determine whether this enhanced protocol could reveal membrane HER2 positivity in cases previously classified as null. By improving HER2 detection sensitivity, this approach may provide a more detailed evaluation of HER2 expression that may have clinical relevance. Patients and methods Patient samples Patient samples were collected from the database of our department between January 2023 and October 2024. Formalin-fixed, paraffin-embedded tissue samples from patients with invasive breast cancer who underwent surgery at St. Luke’s International Hospital (Tokyo, Japan) were included. HER2 IHC staining was assessed based on the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer [7]. Only patients with a HER2 IHC score 0 were selected. Among these, patients with HER2-null status, which is characterized by the complete absence of membrane staining, were included. Patients with HER2-ultralow status, with weak or barely perceptible membrane staining in £10% of tumor cells, or with no invasive foci or with invasion size <1 mm (i.e., classified as pTis) were excluded from the study. In addition to histological evaluation, clinicopathological data—including age, tumor size, histological subtype, hormone receptor status, lymph node involvement, and neoadjuvant chemotherapy history—were collected for all patients. IHC staining procedure HER2 IHC was performed on the BenchMark ULTRA (Roche Diagnostics, Basel, Switzerland) platform using both global and enhanced protocols. The global protocol followed the standard companion diagnostic (CDx) assay, which uses the PATHWAY anti-HER2/neu (4B5) rabbit monoclonal primary antibody and the ultraView [8]. In the enhanced protocol, the same primary antibody was used, but the OptiView was used instead of the ultraView. Additional procedural details are summarized in Table 1. This enhanced protocol was designed to increase HER2 detection sensitivity while maintaining standardized IHC procedures. IHC evaluation HER2 expression was evaluated using both microscopic and image-based assessments of membrane staining in tumor cells. Enhanced HER2 IHC positivity was defined as the presence of partial or circumferential membrane staining, irrespective of staining intensity. For the microscopic evaluation, the entire invasive tumor area was examined at 4´ to 40´ magnification to identify membrane-positive tumor cells. For each patient, three representative regions were selected based on the percentage of membrane-positive tumor cells: one with the highest, one with an intermediate, and one with the lowest value per 100 tumor cells. The mean positivity rate across these regions was calculated and recorded as the final HER2 positivity rate (%). HER2 positivity was scored semiquantitatively using predefined categories: 0%, 1%, 5%, and 10%–100% in 10% increments. Two pathologists evaluated all cases independently, and discrepancies were resolved through consensus discussion. In addition to microscopic evaluation, membrane staining intensity was analyzed using digital image analysis. Microscopic images were captured using an Olympus BX53 microscope (Evident Corporation, Tokyo, Japan) equipped with a Nikon DS-Fi3 digital camera and Nikon NIS-Elements D imaging software (version 5.10; Nikon Corporation, Tokyo, Japan). For each patient, 5–10 images were acquired at 40´ magnification by focusing on 3,3¢-diaminobenzidine (DAB)-positive membrane staining in tumor cells. Images were saved in TIFF format (2880 ´ 2048 pixels, RGB). Regions containing HER2-positive membranes (typically 1–20 cells per region) were cropped, and image analysis was performed using Fiji ImageJ software (version 1.54f; Wayne Rasband, National Institute of Health, Bethesda, MD, USA), following previously reported methods [9]. DAB staining intensity was normalized by dividing the total intensity by the number of cells in the analyzed area. This process was repeated 10 times per patient using randomly selected regions, and the average normalized intensity value was recorded as the representative intensity for each patient. Results Patient characteristics The clinicopathological characteristics of the 58 HER2-null breast cancer patients included in this study are summarized in Table 2. The median age was 56 years (interquartile range: 47–64), and 82.8% (n = 48) of the tumors were estrogen receptor (ER)-positive. Most tumors were invasive carcinoma of no special type (IC-NST; 74.1%, n = 43), followed by mucinous carcinoma (10.3%, n = 6), invasive lobular carcinoma (10.3%, n = 6), and other histological subtypes. As shown in Fig. 1, the samples for these patients exhibited diverse characteristics in terms of ER status, neoadjuvant chemotherapy (NAC) treatment status, and histological subtype. HER2 membrane positivity in patients reevaluated using the enhanced protocol When the 58 patients with HER2-null breast cancer were reevaluated using the enhanced HER2 IHC protocol, membrane-positive tumor cells were observed in all patients (Fig. 1a). The percentage of membrane-positive tumor cells ranged from 0.33% to 90% (overall mean ± standard deviation [SD]: 27.2% ± 23.9%). These findings suggest that, although HER2 expression was undetectable using the global protocol, it was present at varying levels in all patients when the enhanced protocol was applied. Variability in HER2 staining intensity between and within patients Digital image analysis revealed substantial variability in HER2 staining intensity both between patients and within individual tumors (Fig. 1b). The normalized HER2 staining intensity values reflected a broad spectrum of staining intensities from 0.23 to 27.3 (overall mean ± SD: 4.4 ± 4.4). Within individual tumors, some areas exhibited strong staining, whereas others showed weak staining. Some images representative of varying staining intensities are shown in Figure 2. HER2 staining was strong and diffusely positive in one patient (Fig. 2a–c), whereas only weak focal positivity was observed in another patient (Fig. 2d–f). These findings suggest that HER2 expression, as detected using the enhanced HER2 protocol, is heterogeneous both between and within individual tumors. Discussion HER2 IHC scoring has become increasingly important with the expansion of HER2-targeted therapies. The efficacy of T-DXd in the treatment of HER2-low tumors was demonstrated in the DESTINY-Breast04 trial [ 3 ], and the DESTINY-Breast06 trial [ 10 ] found no significant prognostic difference between HER2-low and HER2-ultralow tumors. These trial data suggest that, even within the HER2 IHC 0 category, some tumors may retain HER2 expression at levels that could influence the response to HER2-targeted therapy. The phase 2 DAISY trial provided critical insights into HER2 expression in IHC 0 breast cancer [ 4 ]. In this trial, among the 31 patients reassessed by two pathologists, some were reclassified as having HER2-ultralow or IHC 1+, but 16 remained as having HER2-null. Notably, four of these HER2-null patients showed a response to T-DXd, which suggested that even tumors classified as HER2-null may retain low levels of HER2 expression. Given these earlier reports, our study aimed to determine whether HER2 expression could be detected in patients previously classified as having HER2-null using an enhanced HER2 IHC protocol. Using the VENTANA OptiView system, we found that all 58 patients with HER2-null cancer exhibited membrane HER2 positivity, which suggests that the conventional IHC 0 classification underestimates HER2 expression. To evaluate this more completely, we assessed both the HER2 positivity rate (%) and membrane staining intensity because the current HER2 scoring system considers both parameters. The findings from the DAISY trial highlight the importance of assessing both HER2 positivity and staining intensity. In that trial, three IHC 0 patients reassessed using an enhanced protocol remained classified as having IHC 0, and two of these patients exhibited a partial response to T-DXd [ 4 ]. This underscores the need for more precise HER2 characterization, even with enhanced detection. Our study suggests that incorporating both HER2 positivity and staining intensity into HER2 assessment may help refine the classification and improve the identification of patients who could benefit from HER2-targeted therapies. The HER2 scoring system is a globally recognized tool that continues to evolve alongside advancements in HER2-targeted therapies [ 7 , 11 ]. However, variability in HER2 IHC interpretation remains a challenge, particularly for scores of 1 + and 0. Sakatani et al. recently reported that interobserver agreement was high for IHC 3 + but less consistent for lower scores. In particular, agreement for both IHC 1 + and IHC 0 was slightly lower compared to other IHC score categories [ 12 ]. To address this, the authors suggested that machine learning algorithms and AI-based image analysis might improve scoring accuracy and consistency [ 12 ]. These approaches can also be integrated into future scoring systems for enhanced protocols. The enhanced HER2 IHC protocol used in this study relies on the OptiView detection system, but the clinical significance of this approach remains to be fully established. Although the ultraView-based global protocol is an established CDx tool worldwide, including in Japan [ 8 ], the relationship between HER2 membrane positivity detected using the enhanced protocol and clinical response to T-DXd has yet to be determined. Future research should investigate whether HER2 expression levels identified by this protocol correlate with therapeutic efficacy in IHC 0 breast cancer. In conclusion, our findings suggest that an enhanced HER2 IHC protocol improves the detection of membrane HER2 expression in HER2-null breast cancers and that the cancer in some patients might have previously gone undetected. By refining the HER2 classification by assessing both the positivity rate and staining intensity, this approach might help to optimize patient selection for HER2-targeted therapies. Further research is required to establish a link between HER2 expression and treatment responses to improve patient management. Declarations Acknowledgments We are grateful to the medical technologists at the Department of Pathology, St. Luke’s International Hospital, for their exceptional technical assistance. We acknowledge OnLine English (www.oleng.com.au) for providing English language editing services. Author contributions T.U. designed the study, evaluated the histology, performed data analysis, interpreted the data, and wrote the first draft. N.H. contributed to the pathology review, evaluated the histology, performed data analysis, reviewed the draft, and approved the final manuscript. N.K. conceived and planned the study, contributed to the evaluation of histology and data interpretation, reviewed the draft, and approved the final manuscript. Compliance with ethical standards Conflict of interest statement T.U. has no conflict of interest; N.H. has no conflict of interest; N.K. has received research support from Eiken Chemical Co., Ltd., and is an associate editor of Breast Cancer . Ethical approval This study was conducted in accordance with the principles of the Declaration of Helsinki. Ethical approval was obtained from the institutional review board of St. Luke’s International University, under the approval number 24R-136. Patient consent Informed consent was obtained from all participants included in the study. References Tarantino P, Hamilton E, Tolaney SM, Cortes J, Morganti S, Ferraro E, et al. HER2-Low breast cancer: pathological and clinical landscape. J Clin Oncol 2020;38:1951–62. doi:10.1200/jco.19.02488. Miglietta F, Griguolo G, Bottosso M, Giarratano T, Lo Mele M, Fassan M, et al. Evolution of HER2-low expression from primary to recurrent breast cancer. NPJ Breast Cancer 2021;7:137. doi:10.1038/s41523-021-00343-4. Modi S, Jacot W, Yamashita T, Sohn J, Vidal M, Tokunaga E, et al. Trastuzumab deruxtecan in previously treated HER2-low advanced breast cancer. N Engl J Med 2022;387:9–20. doi:10.1056/NEJMoa2203690. Mosele F, Deluche E, Lusque A, Le Bescond L, Filleron T, Pradat Y, et al. Trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression: the phase 2 DAISY trial. Nat Med 2023;29:2110–20. doi:10.1038/s41591-023-02478-2. Chen Z, Jia H, Zhang H, Chen L, Zhao P, Zhao J, et al. Is HER2 ultra-low breast cancer different from HER2 null or HER2 low breast cancer? A study of 1363 patients. Breast Cancer Res Treat 2023;202:313–23. doi:10.1007/s10549-023-07079-8. Hempenius MA, Eenkhoorn MA, Høeg H, Dabbs DJ, van der Vegt B, Sompuram SR, et al. Quantitative comparison of immunohistochemical HER2-low detection in an interlaboratory study. Histopathology 2024;85:920–8. doi:10.1111/his.15273. Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JMS, 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. J Clin Oncol 2018;36:2105–22. doi:10.1200/jco.2018.77.8738. Hattori M, Honma N, Nagai S, Narui K, Shigechi T, Ozaki Y, et al. Trastuzumab deruxtecan for human epidermal growth factor receptor 2-low advanced or metastatic breast cancer: recommendations from the Japanese Breast Cancer Society Clinical Practice Guidelines. Breast Cancer 2024;31:335–9. doi:10.1007/s12282-024-01550-0. Crowe AR, Yue W. Semi-quantitative determination of protein expression using immunohistochemistry staining and analysis: an integrated protocol. Bio Protoc 2019;9:e3465. doi:10.21769/BioProtoc.3465. Curigliano G, Hu X, Dent RA, Yonemori K, Barrios CH, O’Shaughnessy J, et al. Trastuzumab deruxtecan (T-DXd) vs physician’s choice of chemotherapy (TPC) in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-low or HER2-ultralow metastatic breast cancer (mBC) with prior endocrine therapy (ET): primary results from DESTINY-Breast06 (DB-06). J Clin Oncol 2024;42(17 Suppl):LBA1000. doi:10.1200/JCO.2024.42.17_suppl.LBA1000. Wolff AC, Somerfield MR, Dowsett M, Hammond MEH, Hayes DF, McShane LM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: ASCO–College of American Pathologists guideline update. J Clin Oncol 2023;41:3867–72. doi:10.1200/jco.22.02864. Sakatani T, Tsuda H, Yoshida M, Honma N, Masuda S, Osako T, et al. Current status and challenges in HER2 IHC assessment: scoring survey results in Japan. Breast Cancer Res Treat 2025;210:27–36. doi:10.1007/s10549-024-07532-2. Tables Table 1 Enhanced human epidermal growth factor receptor 2 (HER2) immunohistochemistry protocol Step Staining condition Platform VENTANA BenchMark ULTRA Primary antibody PATHWAY anti-HER2/neu (4B5) rabbit monoclonal primary antibody Detection VENTANA OptiView DAB IHC Detection Kit Deparaffinization 72 ℃, 12 min Cell conditioning time CC1, 100 ℃, 64 min Enzyme None Antibody reaction time 36 ℃, 32 min Linker OptiView HQ Universal Linker, 8 min Multimer OptiView HRP Multimer, 8 min Amplification OptiView Amplification, 8 min Counterstain Hematoxylin Ⅱ, 4 min After counterstain Bluing, 4 min Table 2 Patient background in the human epidermal growth factor receptor 2 (HER2)-null breast cancer group (N = 58) Characteristics Subcategory n (%) Age, years, median (IQR) 56 (47–64) Surgery Mastectomy 13 (22.4%) Breast-conserving surgery 45 (77.6%) Tumor stage (pT) pT1a 2 (3.4%) pT1b 14 (24.1%) pT1c 26 (44.8%) pT2 12 (20.7%) pT3 3 (5.2%) pT4 1 (1.7%) Nodal status (pN) pN0 42 (72.4%) pN1 9 (15.5%) pN2 1 (1.7%) Not assessed 6 (10.3%) Neoadjuvant chemotherapy Yes 5 (8.6%) No 53 (91.4%) Hormone status ER positive 48 (82.8%) ER negative 10 (17.2%) Histology Invasive carcinoma of no special type 43 (74.1%) Invasive lobular carcinoma 6 (10.3%) Mucinous carcinoma 6 (10.3%) Adenoid cystic carcinoma 1 (1.7%) Low-grade adenosquamous carcinoma 1 (1.7%) Squamous cell carcinoma 1 (1.7%) Histological grade Grade 1 26 (44.8%) Grade 2 28 (48.3%) Grade 3 4 (6.9%) IQR: interquartile range; ER: estrogen receptor Cite Share Download PDF Status: Published Journal Publication published 13 May, 2025 Read the published version in Breast Cancer → Version 1 posted Editorial decision: Major Revision 01 Apr, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers invited by journal 19 Mar, 2025 Editor assigned by journal 26 Feb, 2025 First submitted to journal 25 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6111205","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430801147,"identity":"1fef5131-7249-4eee-b09f-b0fb11e20b09","order_by":0,"name":"Takeshi Ushigusa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYJACZgYDBjk+BjYIj7GBgHIeqBZjNhK1MDAktsG0EAT27GeMPxcU1Ka3SaQlPmCosWNgnk3AGh6eHDPpGQbHc4FaDhswHEtmYJxzgJDDcsyYeQyOAbUALWJgO8DAOCOBgBb+N8afgVrS2STS238w/CNGi0SOgTSPQU0Cm0TaMQbGNmK03HhWBtRywLCN51myRGJfMg9Bv7D3J2/+zPOnTp6fPc3ww4dvdnKGhEIMCg5DKKCTeAxnEKWDoQ7BlJcgTssoGAWjYBSMHAAAXi056/8e2RUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5946-3742","institution":"Saint Luke's International Hospital: Sei Roka Kokusai Byoin","correspondingAuthor":true,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Ushigusa","suffix":""},{"id":430801148,"identity":"98a33da9-a40b-447b-8aff-7e4891e4e004","order_by":1,"name":"Nami Hirakawa","email":"","orcid":"","institution":"Saint Luke's International Hospital: Sei Roka Kokusai Byoin","correspondingAuthor":false,"prefix":"","firstName":"Nami","middleName":"","lastName":"Hirakawa","suffix":""},{"id":430801149,"identity":"9629da49-3926-4974-8f30-3cb7ae354509","order_by":2,"name":"Naoki Kanomata","email":"","orcid":"","institution":"Saint Luke's International Hospital: Sei Roka Kokusai Byoin","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Kanomata","suffix":""}],"badges":[],"createdAt":"2025-02-26 08:27:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6111205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6111205/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12282-025-01721-7","type":"published","date":"2025-05-13T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79173030,"identity":"d66cd9c8-64db-4c74-b279-758fbdc9307d","added_by":"auto","created_at":"2025-03-25 09:45:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":158658,"visible":true,"origin":"","legend":"\u003cp\u003eHuman epidermal growth factor receptor 2\u003cstrong\u003e \u003c/strong\u003e(HER2) membrane positivity, staining intensity, and clinicopathological characteristics of patients reassessed using the enhanced HER2 immunohistochemistry protocol.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Bar plot depicting the percentage of HER2 membrane-positive tumor cells in each patient, which ranged from 0.33% to 90% (overall mean ± standard deviation [SD]: 27.2% ± 23.9%). Each blue bar represents the mean percentage of membrane-positive tumor cells, and the black lines indicate the SD. (\u003cstrong\u003eb\u003c/strong\u003e) Bar plot showing the normalized HER2 staining intensity values for all patients, which ranged from 0.23 to 27.3 (overall mean ± SD: 4.4 ± 4.4). Orange bars represent the staining intensity, and black error bars indicate the SD. The bottom panel in (\u003cstrong\u003eb\u003c/strong\u003e) shows a tile map summarizing the clinicopathological characteristics of each patient, which includes estrogen receptor (ER) status (red = ER positive; white = ER negative), neoadjuvant chemotherapy (NAC) treatment status (green = treated with NAC; white = without NAC), and histological subtype (blue = invasive carcinoma of no special type [IC-NST]; white = other histology). The patient numbers are aligned consistently across both panels\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6111205/v1/acc60aacbc9eef096e3876b4.png"},{"id":79173031,"identity":"13ad2a26-b59c-4adf-bbae-8191292d88be","added_by":"auto","created_at":"2025-03-25 09:45:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2119303,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images showing differences in human epidermal growth factor receptor 2 (HER2) immunohistochemistry staining intensity using the enhanced protocol. These patients were initially classified as having HER2-null cancer using the global protocol. (\u003cstrong\u003ea–c\u003c/strong\u003e) Samples from a patient with high staining intensity. (\u003cstrong\u003ea\u003c/strong\u003e) Hematoxylin and eosin (HE) staining of the tumor region (high magnification). (\u003cstrong\u003eb\u003c/strong\u003e) Staining in the same region, showing strong membrane positivity. (\u003cstrong\u003ec\u003c/strong\u003e) Cropped, high-magnification image of the selected region. The normalized intensity value, measured using Fiji, was 25.1. (\u003cstrong\u003ed–f\u003c/strong\u003e) Samples from a patient with low staining intensity. (\u003cstrong\u003ed\u003c/strong\u003e) HE staining of the tumor region. (\u003cstrong\u003ee\u003c/strong\u003e) Staining in the same region, showing partial and weak membrane positivity. (\u003cstrong\u003ef\u003c/strong\u003e) Cropped, high-magnification image of the selected region. The normalized intensity value, measured using Fiji, was 0.03\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6111205/v1/1e128023fef0566050868fac.png"},{"id":83068772,"identity":"3da90d98-61fd-4067-817d-558ec52a749a","added_by":"auto","created_at":"2025-05-19 16:10:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3601638,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6111205/v1/fc4d5705-0057-4c6e-b0b8-1346fa335811.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eReevaluating HER2-null breast cancer using an enhanced HER2 immunohistochemistry protocol\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman epidermal growth factor receptor 2 (HER2) status plays a key role in breast cancer classification and treatment selection. Patients with HER2 positivity, defined as an immunohistochemistry (IHC) score of 3\u0026thinsp;+\u0026thinsp;or 2+/in situ hybridization (ISH)-positive, are eligible to receive targeted therapies such as trastuzumab. By contrast, patients who are HER2 negative (IHC 0, 1+, or 2+/ISH-negative) have traditionally been considered ineligible because of the limited treatment options. Recent studies have introduced the concept of \u0026ldquo;HER2-low\u0026rdquo; (IHC 1\u0026thinsp;+\u0026thinsp;or IHC 2+/ISH-negative), which accounts for nearly 50% of all breast cancer cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This group has shown responsiveness to HER2-directed antibody\u0026ndash;drug conjugates, such as trastuzumab deruxtecan (T-DXd), which increases the clinical relevance of the HER2-low classification [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHER2 IHC 0 includes two categories: HER2-ultralow and HER2-null. These categories are not defined by guidelines, although HER2-ultralow is typically defined as \u0026le;10% of infiltrating cancer cells showing weak or incomplete membrane staining, and HER2-null refers to no detectable HER2 expression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Current IHC staining methods are designed primarily to detect high HER2 expression, which raises concerns that lower levels may be underestimated. The choice of the detection system can also affect staining sensitivity. The VENTANA OptiView DAB IHC Detection Kit (OptiView) produces stronger HER2 staining than the VENTANA ultraView Universal DAB Detection Kit (ultraView) in patients with HER2-low expression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the sensitivity of the OptiView in patients previously classified as HER2-null remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we applied an enhanced HER2 IHC protocol and the OptiView system to assess HER2-null breast cancer cases. We aimed to determine whether this enhanced protocol could reveal membrane HER2 positivity in cases previously classified as null. By improving HER2 detection sensitivity, this approach may provide a more detailed evaluation of HER2 expression that may have clinical relevance.\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003ch2\u003ePatient samples\u003c/h2\u003e\n\u003cp\u003ePatient samples were collected from the database of our department between January 2023 and October 2024. Formalin-fixed, paraffin-embedded tissue samples from patients with invasive breast cancer who underwent surgery at St. Luke\u0026rsquo;s International Hospital (Tokyo, Japan) were included. HER2 IHC staining was assessed based on the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer [7]. Only patients with a HER2 IHC score 0 were selected. Among these, patients with HER2-null status, which is characterized by the complete absence of membrane staining, were included. Patients with HER2-ultralow status, with weak or barely perceptible membrane staining in \u0026pound;10% of tumor cells, or with no invasive foci or with invasion size \u0026lt;1 mm (i.e., classified as pTis) were excluded from the study. In addition to histological evaluation, clinicopathological data\u0026mdash;including age, tumor size, histological subtype, hormone receptor status, lymph node involvement, and neoadjuvant chemotherapy history\u0026mdash;were collected for all patients.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eIHC staining procedure\u003c/h2\u003e\n\u003cp\u003eHER2 IHC was performed on the BenchMark ULTRA (Roche Diagnostics, Basel, Switzerland) platform using both global and enhanced protocols. The global protocol followed the standard companion diagnostic (CDx) assay, which uses the PATHWAY anti-HER2/neu (4B5) rabbit monoclonal primary antibody and the ultraView [8]. In the enhanced protocol, the same primary antibody was used, but the OptiView was used instead of the ultraView. Additional procedural details are summarized in Table 1. This enhanced protocol was designed to increase HER2 detection sensitivity while maintaining standardized IHC procedures.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eIHC evaluation\u003c/h2\u003e\n\u003cp\u003eHER2 expression was evaluated using both microscopic and image-based assessments of membrane staining in tumor cells. Enhanced HER2 IHC positivity was defined as the presence of partial or circumferential membrane staining, irrespective of staining intensity. For the microscopic evaluation, the entire invasive tumor area was examined at 4\u0026acute;\u0026nbsp;to 40\u0026acute;\u0026nbsp;magnification to identify membrane-positive tumor cells. For each patient, three representative regions were selected based on the percentage of membrane-positive tumor cells: one with the highest, one with an intermediate, and one with the lowest value per 100 tumor cells. The mean positivity rate across these regions was calculated and recorded as the final HER2 positivity rate (%). HER2 positivity was scored semiquantitatively using predefined categories: 0%, 1%, 5%, and 10%\u0026ndash;100% in 10% increments. Two pathologists evaluated all cases independently, and discrepancies were resolved through consensus discussion.\u003c/p\u003e\n\u003cp\u003eIn addition to microscopic evaluation, membrane staining intensity was analyzed using digital image analysis. Microscopic images were captured using an Olympus BX53 microscope (Evident Corporation, Tokyo, Japan) equipped with a Nikon DS-Fi3 digital camera and Nikon NIS-Elements D imaging software (version 5.10; Nikon Corporation, Tokyo, Japan). For each patient, 5\u0026ndash;10 images were acquired at 40\u0026acute; magnification by focusing on 3,3\u0026cent;-diaminobenzidine (DAB)-positive membrane staining in tumor cells. Images were saved in TIFF format (2880 \u0026acute; 2048 pixels, RGB). Regions containing HER2-positive membranes (typically 1\u0026ndash;20 cells per region) were cropped, and image analysis was performed using Fiji ImageJ software (version 1.54f; Wayne Rasband, National Institute of Health, Bethesda, MD, USA), following previously reported methods [9]. DAB staining intensity was normalized by dividing the total intensity by the number of cells in the analyzed area. This process was repeated 10 times per patient using randomly selected regions, and the average normalized intensity value was recorded as the representative intensity for each patient.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003ePatient characteristics\u003c/h2\u003e\n\u003cp\u003eThe clinicopathological characteristics of the 58 HER2-null breast cancer patients included in this study are summarized in Table 2. The median age was 56 years (interquartile range: 47\u0026ndash;64), and 82.8% (n = 48) of the tumors were estrogen receptor (ER)-positive. Most tumors were invasive carcinoma of no special type (IC-NST; 74.1%, n = 43), followed by mucinous carcinoma (10.3%, n = 6), invasive lobular carcinoma (10.3%, n = 6), and other histological subtypes. As shown in Fig. 1, the samples for these patients exhibited diverse characteristics in terms of ER status, neoadjuvant chemotherapy (NAC) treatment status, and histological subtype.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eHER2 membrane positivity in patients reevaluated using the enhanced protocol\u003c/h2\u003e\n\u003cp\u003eWhen the 58 patients with HER2-null breast cancer were reevaluated using the enhanced HER2 IHC protocol, membrane-positive tumor cells were observed in all patients (Fig. 1a). The percentage of membrane-positive tumor cells ranged from 0.33% to 90% (overall mean \u0026plusmn; standard deviation [SD]: 27.2% \u0026plusmn; 23.9%). These findings suggest that, although HER2 expression was undetectable using the global protocol, it was present at varying levels in all patients when the enhanced protocol was applied.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eVariability in HER2 staining intensity between and within patients\u003c/h2\u003e\n\u003cp\u003eDigital image analysis revealed substantial variability in HER2 staining intensity both between patients and within individual tumors (Fig. 1b). The normalized HER2 staining intensity values reflected a broad spectrum of staining intensities from 0.23 to 27.3 (overall mean \u0026plusmn; SD: 4.4 \u0026plusmn; 4.4). Within individual tumors, some areas exhibited strong staining, whereas others showed weak staining. Some images representative of varying staining intensities are shown in Figure 2. HER2 staining was strong and diffusely positive in one patient (Fig. 2a\u0026ndash;c), whereas only weak focal positivity was observed in another patient (Fig. 2d\u0026ndash;f). These findings suggest that HER2 expression, as detected using the enhanced HER2 protocol, is heterogeneous both between and within individual tumors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHER2 IHC scoring has become increasingly important with the expansion of HER2-targeted therapies. The efficacy of T-DXd in the treatment of HER2-low tumors was demonstrated in the DESTINY-Breast04 trial [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the DESTINY-Breast06 trial [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] found no significant prognostic difference between HER2-low and HER2-ultralow tumors. These trial data suggest that, even within the HER2 IHC 0 category, some tumors may retain HER2 expression at levels that could influence the response to HER2-targeted therapy. The phase 2 DAISY trial provided critical insights into HER2 expression in IHC 0 breast cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this trial, among the 31 patients reassessed by two pathologists, some were reclassified as having HER2-ultralow or IHC 1+, but 16 remained as having HER2-null. Notably, four of these HER2-null patients showed a response to T-DXd, which suggested that even tumors classified as HER2-null may retain low levels of HER2 expression.\u003c/p\u003e \u003cp\u003eGiven these earlier reports, our study aimed to determine whether HER2 expression could be detected in patients previously classified as having HER2-null using an enhanced HER2 IHC protocol. Using the VENTANA OptiView system, we found that all 58 patients with HER2-null cancer exhibited membrane HER2 positivity, which suggests that the conventional IHC 0 classification underestimates HER2 expression. To evaluate this more completely, we assessed both the HER2 positivity rate (%) and membrane staining intensity because the current HER2 scoring system considers both parameters.\u003c/p\u003e \u003cp\u003eThe findings from the DAISY trial highlight the importance of assessing both HER2 positivity and staining intensity. In that trial, three IHC 0 patients reassessed using an enhanced protocol remained classified as having IHC 0, and two of these patients exhibited a partial response to T-DXd [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This underscores the need for more precise HER2 characterization, even with enhanced detection. Our study suggests that incorporating both HER2 positivity and staining intensity into HER2 assessment may help refine the classification and improve the identification of patients who could benefit from HER2-targeted therapies.\u003c/p\u003e \u003cp\u003eThe HER2 scoring system is a globally recognized tool that continues to evolve alongside advancements in HER2-targeted therapies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, variability in HER2 IHC interpretation remains a challenge, particularly for scores of 1\u0026thinsp;+\u0026thinsp;and 0. Sakatani et al. recently reported that interobserver agreement was high for IHC 3\u0026thinsp;+\u0026thinsp;but less consistent for lower scores. In particular, agreement for both IHC 1\u0026thinsp;+\u0026thinsp;and IHC 0 was slightly lower compared to other IHC score categories [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To address this, the authors suggested that machine learning algorithms and AI-based image analysis might improve scoring accuracy and consistency [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These approaches can also be integrated into future scoring systems for enhanced protocols.\u003c/p\u003e \u003cp\u003eThe enhanced HER2 IHC protocol used in this study relies on the OptiView detection system, but the clinical significance of this approach remains to be fully established. Although the ultraView-based global protocol is an established CDx tool worldwide, including in Japan [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the relationship between HER2 membrane positivity detected using the enhanced protocol and clinical response to T-DXd has yet to be determined. Future research should investigate whether HER2 expression levels identified by this protocol correlate with therapeutic efficacy in IHC 0 breast cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings suggest that an enhanced HER2 IHC protocol improves the detection of membrane HER2 expression in HER2-null breast cancers and that the cancer in some patients might have previously gone undetected. By refining the HER2 classification by assessing both the positivity rate and staining intensity, this approach might help to optimize patient selection for HER2-targeted therapies. Further research is required to establish a link between HER2 expression and treatment responses to improve patient management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe are grateful to the medical technologists at the Department of Pathology, St. Luke\u0026rsquo;s International Hospital, for their exceptional technical assistance. We acknowledge OnLine English (www.oleng.com.au) for providing English language editing services.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eT.U. designed the study, evaluated the histology, performed data analysis, interpreted the data, and wrote the first draft. N.H. contributed to the pathology review, evaluated the histology, performed data analysis, reviewed the draft, and approved the final manuscript. N.K. conceived and planned the study, contributed to the evaluation of histology and data interpretation, reviewed the draft, and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e\n\u003ch2\u003eConflict of interest statement\u003c/h2\u003e\n\u003cp\u003eT.U. has no conflict of interest; N.H. has no conflict of interest; N.K. has received research support from Eiken Chemical Co., Ltd., and is an associate editor of \u003cem\u003eBreast Cancer\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eThis study was conducted in accordance with the principles of the Declaration of Helsinki. Ethical approval was obtained from the institutional review board of St. Luke\u0026rsquo;s International University, under the approval number 24R-136.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePatient consent\u003c/h2\u003e\n\u003cp\u003eInformed consent was obtained from all participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTarantino P, Hamilton E, Tolaney SM, Cortes J, Morganti S, Ferraro E, et al. HER2-Low breast cancer: pathological and clinical landscape. J Clin Oncol 2020;38:1951\u0026ndash;62. doi:10.1200/jco.19.02488.\u003c/li\u003e\n\u003cli\u003eMiglietta F, Griguolo G, Bottosso M, Giarratano T, Lo Mele M, Fassan M, et al. Evolution of HER2-low expression from primary to recurrent breast cancer. NPJ Breast Cancer 2021;7:137. doi:10.1038/s41523-021-00343-4.\u003c/li\u003e\n\u003cli\u003eModi S, Jacot W, Yamashita T, Sohn J, Vidal M, Tokunaga E, et al. Trastuzumab deruxtecan in previously treated HER2-low advanced breast cancer. N Engl J Med 2022;387:9\u0026ndash;20. doi:10.1056/NEJMoa2203690.\u003c/li\u003e\n\u003cli\u003eMosele F, Deluche E, Lusque A, Le Bescond L, Filleron T, Pradat Y, et al. Trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression: the phase 2 DAISY trial. Nat Med 2023;29:2110\u0026ndash;20. doi:10.1038/s41591-023-02478-2.\u003c/li\u003e\n\u003cli\u003eChen Z, Jia H, Zhang H, Chen L, Zhao P, Zhao J, et al. Is HER2 ultra-low breast cancer different from HER2 null or HER2 low breast cancer? A study of 1363 patients. Breast Cancer Res Treat 2023;202:313\u0026ndash;23. doi:10.1007/s10549-023-07079-8.\u003c/li\u003e\n\u003cli\u003eHempenius MA, Eenkhoorn MA, H\u0026oslash;eg H, Dabbs DJ, van der Vegt B, Sompuram SR, et al. Quantitative comparison of immunohistochemical HER2-low detection in an interlaboratory study. Histopathology 2024;85:920\u0026ndash;8. doi:10.1111/his.15273.\u003c/li\u003e\n\u003cli\u003eWolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JMS, 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. J Clin Oncol 2018;36:2105\u0026ndash;22. doi:10.1200/jco.2018.77.8738.\u003c/li\u003e\n\u003cli\u003eHattori M, Honma N, Nagai S, Narui K, Shigechi T, Ozaki Y, et al. Trastuzumab deruxtecan for human epidermal growth factor receptor 2-low advanced or metastatic breast cancer: recommendations from the Japanese Breast Cancer Society Clinical Practice Guidelines. Breast Cancer 2024;31:335\u0026ndash;9. doi:10.1007/s12282-024-01550-0.\u003c/li\u003e\n\u003cli\u003eCrowe AR, Yue W. Semi-quantitative determination of protein expression using immunohistochemistry staining and analysis: an integrated protocol. Bio Protoc 2019;9:e3465. doi:10.21769/BioProtoc.3465.\u003c/li\u003e\n\u003cli\u003eCurigliano G, Hu X, Dent RA, Yonemori K, Barrios CH, O\u0026rsquo;Shaughnessy J, et al. Trastuzumab deruxtecan (T-DXd) vs physician\u0026rsquo;s choice of chemotherapy (TPC) in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-low or HER2-ultralow metastatic breast cancer (mBC) with prior endocrine therapy (ET): primary results from DESTINY-Breast06 (DB-06). J Clin Oncol 2024;42(17 Suppl):LBA1000. doi:10.1200/JCO.2024.42.17_suppl.LBA1000.\u003c/li\u003e\n\u003cli\u003eWolff AC, Somerfield MR, Dowsett M, Hammond MEH, Hayes DF, McShane LM, et al. Human epidermal growth factor receptor 2 testing in breast cancer: ASCO\u0026ndash;College of American Pathologists guideline update. J Clin Oncol 2023;41:3867\u0026ndash;72. doi:10.1200/jco.22.02864.\u003c/li\u003e\n\u003cli\u003eSakatani T, Tsuda H, Yoshida M, Honma N, Masuda S, Osako T, et al. Current status and challenges in HER2 IHC assessment: scoring survey results in Japan. Breast Cancer Res Treat 2025;210:27\u0026ndash;36. doi:10.1007/s10549-024-07532-2.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Enhanced human epidermal growth factor receptor 2 (HER2) immunohistochemistry protocol\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStep\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStaining condition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePlatform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVENTANA BenchMark ULTRA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimary antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePATHWAY anti-HER2/neu (4B5) rabbit monoclonal primary antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDetection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVENTANA OptiView DAB IHC Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDeparaffinization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e72 ℃, 12 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell conditioning time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCC1, 100 ℃, 64 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEnzyme\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAntibody reaction time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e36 ℃, 32 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLinker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOptiView HQ Universal Linker, 8 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMultimer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOptiView HRP Multimer, 8 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAmplification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOptiView Amplification, 8 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCounterstain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHematoxylin Ⅱ, 4 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAfter counterstain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBluing, 4 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Patient background in the human epidermal growth factor receptor 2 (HER2)-null breast cancer group (N = 58)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eSubcategory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eAge, years, median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e56 (47\u0026ndash;64)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 26px;\"\u003e\n \u003cp\u003eSurgery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eMastectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e13 (22.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eBreast-conserving surgery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e45 (77.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 26px;\"\u003e\n \u003cp\u003eTumor stage (pT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT1a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e2 (3.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e14 (24.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e26 (44.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e12 (20.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e3 (5.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 26px;\"\u003e\n \u003cp\u003eNodal status (pN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e42 (72.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e9 (15.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003epN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eNot assessed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e6 (10.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 26px;\"\u003e\n \u003cp\u003eNeoadjuvant chemotherapy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e5 (8.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e53 (91.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 26px;\"\u003e\n \u003cp\u003eHormone status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eER positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e48 (82.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eER negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e10 (17.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 26px;\"\u003e\n \u003cp\u003eHistology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eInvasive carcinoma of no special type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e43 (74.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eInvasive lobular carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e6 (10.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eMucinous carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e6 (10.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eAdenoid cystic carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eLow-grade adenosquamous carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eSquamous cell carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 26px;\"\u003e\n \u003cp\u003eHistological grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eGrade 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e26 (44.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eGrade 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e28 (48.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eGrade 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e4 (6.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIQR: interquartile range; ER: estrogen receptor\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HER2, HER2-null breast cancer, Immunohistochemistry, HER2-targeted therapy, Trastuzumab deruxtecan","lastPublishedDoi":"10.21203/rs.3.rs-6111205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6111205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Human epidermal growth factor receptor 2 (HER2) status is crucial for the classification of breast cancer and selection of its treatment. Although HER2-low breast cancer is a recognized therapeutic subgroup, the classification of HER2 immunohistochemistry (IHC) 0 remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We reassessed 58 HER2-null breast cancer cases using an enhanced HER2 IHC protocol and the VENTANA OptiView detection system. HER2 expression was evaluated based on membrane positivity rate (%) and staining intensity. Microscopic assessment was performed to determine the percentage of HER2-positive tumor cells. Digital image analysis was used to quantify staining intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Membrane HER2 positivity was detected to varying degrees in all cases. This indicated that tumors previously classified as HER2-null exhibited HER2 expression when analyzed with the enhanced detection system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: These findings suggest that enhanced HER2 IHC protocols can improve detection sensitivity. Further research is needed to determine the clinical significance of HER2 expression detected using enhanced protocols.\u003c/p\u003e","manuscriptTitle":"Reevaluating HER2-null breast cancer using an enhanced HER2 immunohistochemistry protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 09:45:27","doi":"10.21203/rs.3.rs-6111205/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-04-01T06:14:24+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-19T04:55:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-19T04:31:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-26T11:04:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer","date":"2025-02-26T03:25:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe180498-0081-47f9-a5cd-7797a038339d","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:09:55+00:00","versionOfRecord":{"articleIdentity":"rs-6111205","link":"https://doi.org/10.1007/s12282-025-01721-7","journal":{"identity":"breast-cancer","isVorOnly":false,"title":"Breast Cancer"},"publishedOn":"2025-05-13 15:57:48","publishedOnDateReadable":"May 13th, 2025"},"versionCreatedAt":"2025-03-25 09:45:27","video":"","vorDoi":"10.1007/s12282-025-01721-7","vorDoiUrl":"https://doi.org/10.1007/s12282-025-01721-7","workflowStages":[]},"version":"v1","identity":"rs-6111205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6111205","identity":"rs-6111205","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}