Comparative Analysis of HER2 Immunohistochemistry (IHC) and Brightfield Dual-Color In Situ Hybridization (DISH) in Lymph Node Metastasized Breast Cancer

Comparative Analysis of HER2 Immunohistochemistry (IHC) and Brightfield Dual-Color In Situ Hybridization (DISH) in Lymph Node Metastasized Breast Cancer | 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 Comparative Analysis of HER2 Immunohistochemistry (IHC) and Brightfield Dual-Color In Situ Hybridization (DISH) in Lymph Node Metastasized Breast Cancer Kroonpong Iampenkhae, Adiluck Pisutpunya, Sompon Apornvirat This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4830804/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The human epidermal growth factor receptor 2 (HER2) gene encodes a tyrosine kinase receptor, implicated in 15–20% of breast cancers. HER2 amplification is both prognostic and predictive of response to HER2-targeted therapy. IHC is the preferred initial test due to its cost-effectiveness and simplicity. While FISH is the gold standard test for HER2 amplification, it has several limitations, prompting consideration of DISH as an alternative. This study aims to evaluate the concordance between HER2 IHC and DISH in lymph node metastasized breast cancer and compares the results to existing data on HER2 IHC and FISH. Methods This retrospective study included 1,307 cases of pathologically confirmed lymph node metastasized invasive breast carcinoma with HER2 IHC scores of 2+ (481 cases) and 3+ (826 cases). Interpretation of HER2 IHC was performed using the 2023 ASCO/CAP guideline. HER2 DISH was conducted and evaluated by the HER2/Chromosome 17 signals ratio. Results HER2 amplification was detected in 933 cases, including 92% (760) of IHC 3 + cases and 36% (173) of IHC 2 + cases. Comparison with the meta-analysis data of IHC and FISH showed no significant differences, indicating that DISH is a reliable alternative to FISH. Conclusion Our study demonstrates no significant difference in the positive rates of HER2 IHC to DISH when compared with prior data of IHC to FISH, reaffirming the use of HER2 DISH as an effective and more accessible alternative to FISH in breast cancer. The high concordant rate between HER2 IHC and DISH in the IHC 3 + group and low positive rate in the IHC 2 + group support the recommendation to proceed with HER2-targeted therapy in IHC 3 + cases and to perform ISH in IHC 2 + cases. HER2 Breast neoplasms Immunohistochemistry In situ hybridization Concordance analysis Figures Figure 1 Introduction The human epidermal growth factor receptor 2 (HER2) gene, locating on band q21 of chromosome 17 (Chr17), encodes a type of tyrosine kinase receptor. Approximately 15–20% of breast cancers exhibit HER2 gene amplification and/or protein overexpression, and this HER2 status is both prognostic and predictive of response to HER2-targeted therapy [ 1 – 4 ]. The 2023 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommends utilizing immunohistochemistry (IHC), brightfield in situ hybridization, or fluorescent in situ hybridization (FISH) as detection tests. The new guideline reaffirms the use of HER2 targeted therapy in patients with HER2 IHC 3 + and IHC 2 + with amplified in situ hybridization (ISH) while acknowledging the benefits of newer generation HER2 combined drugs in HER2-low patients [ 3 ]. Due to its cost-effectiveness and simplicity in performing and interpreting, many laboratories prefer using IHC as a screening test and perform ISH only if the IHC result is 2 + or 3+. For ISH tests, FISH is still considered a gold standard test for HER2 amplification. However, FISH has several limitations when compared to brightfield dual-color in situ hybridization (DISH), including the necessity for a fluorescent microscope, the fast degradation time of fluorescent stains, and the limitation of visualization of cell features [ 5 ]. Several studies have demonstrated very high to almost perfect agreement on HER2 amplification between FISH and DISH, suggesting that DISH can be used as a more accessible alternative to FISH [ 6 – 19 ]. While there are extensive studies on the concordance of HER2 IHC and FISH, as shown in the meta-analysis by Bahreini et al., 2015 [ 20 ], the extant literature on direct comparison between IHC and DISH is still lacking. Hence, the objectives of our study are to provide additional information on the concordance rate between HER2 IHC and DISH in a specific setting of lymph node metastasized breast cancer, and to compare the result to the existing data on the concordance rate between HER2 IHC and FISH. Materials and Methods Sample All cases of lymph node metastasized invasive breast carcinoma with HER2 IHC scores of 2 + and 3 + in King Chulalongkorn Memorial Hospital (KCMH) and Biobridge project, which received specimens from multiple hospitals across Thailand from 2015 to 2021, were included. As per the project protocol, all samples had appropriate pre-analytical factors, pathologically documented lymph node metastasis, and validated IHC assays were used to evaluate HER2. The formalin-fixed paraffin-embedded (FFPE) tissue blocks, H&E slides, and the initial HER2 IHC slides were retrieved. Two pathologists (KI and AP) reviewed the HER2 IHC slides using the 2023 ASCO/CAP HER2 scoring guideline, and their consensus was used. If any problems interfered with slide interpretation, re-staining would be performed in KCMH, and the slides would be reevaluated. The case would be excluded if the issues were unresolved, a consensus could not be reached, or there was inadequate remaining tumor quantity. Ultimately, a total of 1,307 cases remained in the study and HER2 DISH was then performed. Of these, 826 cases had a score of 3+, and 481 cases had a score of 2+. IHC The FFPE tissue blocks were sectioned into 3-µm-thick sections. Anti-HER2/Neu (4b5) antibody (Ventana Medical Systems, Inc, Tuscon, Arizona, USA, catalog number 05278368001) was applied as per the manufacturer’s protocol, using the automated Benchmark Ultra system (Ventana Medical Systems, Inc, Tuscon, Arizona, USA). The interpretation of repeated HER2 IHC slides was done in the same way as the IHC slides received from the Biobridge project. DISH The INFORM HER2 Dual ISH probe cocktail (Ventana Medical Systems, Inc, Tuscon, Arizona, USA, catalog number 760–6072) was performed as per the manufacturer’s protocol on 4-µm-thick tissue sections, using the automated Benchmark Ultra system (Ventana Medical Systems, Inc, Tuscon, Arizona, USA). Quality assessment for HER2 and Chr17 signals in both internal control and tumor cells, as well as background staining, was conducted. If any interference was detected, repeat staining with proper adjustment was done. If the issue persisted, we switched to performing DISH on lymph node specimen. The interpretation of HER2 DISH slides was performed independently by two pathologists (KI and AP), using light microscopes with 40x and 60x objective lens and their consensus was used. The HER2 and chromosome 17 (Chr17) signals were enumerated in 20 invasive tumor cell nuclei. If the HER2/Chr17 ratio was within the range of 1.8 to 2.2, additional 20 nuclei would be counted. The final HER2/Chr17 ratio of equal to or greater than 2.0 was regarded as positive or amplified, while HER2/Chr17 ratio of less than 2.0 was interpreted as negative or non-amplified. Statistical Analysis The data were statistically analyzed using ChatGPT-4o. Metrics calculated were the concordant rate of positive IHC and DISH tests, the positive rate of HER2 DISH in the equivocal (2+) IHC group, 95% Confidence Intervals (CI) for the concordant rate and positive rate, and Fisher’s exact test to compare the significance of associations between concordant rate and positive rate of IHC and DISH to IHC and FISH. Results Concordance of HER2 IHC and DISH Of the 1,307 cases, 933 samples showed HER2 amplification by DISH. HER2 amplifications were detected in 92% of IHC 3 + cases (95% CI = 90.2%-93.9%) and 36% of IHC 2 + cases (95% CI = 31.7%-40.3%). Fisher’s exact test demonstrated non-random association in the data (p < 0.001). The result is summarized in Table 1 . Table 1 Result of HER2 IHC and DISH HER2 DISH result HER2 IHC score Positive Negative Total 3+ 760 66 826 2+ 173 308 481 Total 933 374 1307 P-value < 0.001 Additionally, tumor heterogeneity showing equal or less than 30% positive area by DISH were observed in 11 cases (0.84%). Four of these had an IHC score of 2 + and seven had an IHC score of 3+. One example of such cases is demonstrated in Fig. 1 . Comparison between IHC to DISH and IHC to FISH Comparison of our IHC and DISH data to the data of IHC and FISH from the meta-analysis [ 20 ]. was performed. Fisher’s exact test indicated no statistically significant differences in either HER2 IHC score 2 + and 3 + groups. The comparative data was shown in Table 2 . Table 2 Comparison of HER2 IHC and DISH to IHC and FISH ISH Positive rate Sample size P-value HER2 IHC score 3+ Our IHC to DISH data 92% 826 0.394 IHC to FISH data [ 20 ] 91% 1434 HER2 IHC score 2+ Our IHC to DISH data 36% 481 1 IHC to FISH data [ 20 ] 36% 1346 Discussion Our study aimed to evaluate the concordance between HER2 IHC and DISH in lymph node metastasized breast cancer, providing a comparative perspective against the well-established concordance between HER2 IHC and FISH. The findings could contribute to the ongoing discourse on optimizing HER2 testing methodologies to enhance diagnostic accuracy and therapeutic outcomes. The results demonstrate a very high concordance between HER2 DISH and IHC in the IHC 3 + group and a low positive rate of HER2 IHC 2 + when compared to HER2 DISH. This data aligns well with the current recommendations on HER2 testing, which emphasize the reliability of IHC as a primary screening method, followed by ISH confirmation in equivocal cases [ 3 ]. Although there are minor discrepancies between our findings and the results of some prior studies, which are presented in Table 3 , we believe these slight differences do not affect the testing recommendation. Table 3 Prior studies comparing HER2 IHC 2 + and 3 + to DISH in breast cancer Year ISH Positive rate Sample size HER2 IHC score 3+ Lim et al. [ 11 ] 2013 94.1% 17 Gao et al [ 13 ] 2014 100% 20 Layfield et al. [ 17 ] 2017 52% 31 Rathi et al. [ 18 ] 2022 97.4% 39 HER2 IHC score 2+ Gao et al. [ 13 ] 2014 25.5% 102 Layfield et al. [ 17 ] 2017 6% 32 Rathi et al. [ 18 ] 2022 20.2% 74 The strength of this study lies in its large sample size. The 1,307-sample size is the largest study on this topic in the current English literature especially when considering the problematic 2 + IHC score group. Additionally, this study included the samples from the Biobridge project, which collected specimens from multiple hospitals across Thailand. While this subjected the study to increased heterogeneity in pre-analytic factors, the strong agreement in the IHC 3 + group validates its applicability in wider settings. One limitation of this study is the lack of the gold standard FISH test. Nonetheless, as previously discussed, prior investigations show excellent agreement between DISH and FISH tests [ 6 – 19 ]. We also find no significant difference when comparing our result to the result of IHC and FISH, thereby justifying the use of DISH as a substitute for FISH. Another limitation of this study is the lack of cases with IHC scores of 0 and 1+. This limits the ability to precisely calculate some statistical values such as sensitivity, specificity, and Cohen’s kappa agreement. Conclusion Our study adds valuable evidence to the body of literature supporting the use of DISH as an effective alternative to FISH for confirming HER2 status in breast cancer. It reveals an expected correlation between HER2 IHC and DISH in lymph node metastasized breast carcinomas, comparable to prior studies between HER2 IHC and FISH in breast cancers. The concordance rate of the IHC score 3+ group is 92%, and the positive rate of the IHC score 2+ group is 36%, reinforcing the recommendation to perform ISH test in the IHC score of 2+ group and proceed to HER2 targeted therapy in the IHC score of 3+ group especially in resource-limited settings. Declarations Ethics declaration This retrospective study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, reference number 938/64. Consent to participate declaration Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding statement This research received no external funding. Publication fee was partially supported by the author’s university. Acknowledgement Biobridge Project Thailand was funded by Roche Thailand Ltd. to encourage the utilization of HER2 DISH. Competing interests The first author was involved with the project and obtained permission to use the data but did not receive any incentive or obligation to publish the result in academic journal. The remaining authors declare no conflict of interest. Author Contributions Kroonpong Iampenkhae: Conception and design, manuscript writing and editing, data collection, analysis, and interpretation Adiluck Pisutpanya: Manuscript editing, data collection, analysis and interpretation Sompon Apornvirat: Manuscript writing and editing, data analysis and interpretation References 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 Jan 9;235(4785):177-82. doi: 10.1126/science.3798106. PMID: 3798106. Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004 Apr;5(1):63-9. doi: 10.3816/cbc.2004.n.011. PMID: 15140287. Wolff AC, Somerfield MR, Dowsett M, Hammond MEH, Hayes DF, McShane LM, Saphner TJ, Spears PA, Allison KH. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: ASCO-College of American Pathologists Guideline Update. J Clin Oncol. 2023 Aug 1;41(22):3867-3872. doi: 10.1200/JCO.22.02864. Epub 2023 Jun 7. PMID: 37284804. Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002 Feb 1;20(3):719-26. doi: 10.1200/JCO.2002.20.3.719. Corrected and republished in: J Clin Oncol. 2023 Mar 20;41(9):1638-1645. PMID: 11821453. Chrzanowska NM, Kowalewski J, Lewandowska MA. Use of Fluorescence In Situ Hybridization (FISH) in Diagnosis and Tailored Therapies in Solid Tumors. Molecules. 2020 Apr 17;25(8):1864. doi: 10.3390/molecules25081864. PMID: 32316657; PMCID: PMC7221545. Pedersen M, Rasmussen BB. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer. Diagn Mol Pathol. 2009 Jun;18(2):96-102. doi: 10.1097/PDM.0b013e31817f5227. PMID: 19430295. Koh YW, Lee HJ, Lee JW, Kang J, Gong G. Dual-color silver-enhanced in situ hybridization for assessing HER2 gene amplification in breast cancer. Mod Pathol. 2011 Jun;24(6):794-800. doi: 10.1038/modpathol.2011.9. Epub 2011 Feb 11. PMID: 21317877. Bartlett JM, Campbell FM, Ibrahim M, O'Grady A, Kay E, Faulkes C, Collins N, Starczynski J, Morgan JM, Jasani B, Miller K. A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay. Am J Clin Pathol. 2011 Jan;135(1):157-62. doi: 10.1309/AJCPVPRKK1ENEDGQ. PMID: 21173138. Brügmann A, Lelkaitis G, Nielsen S, Jensen KG, Jensen V. Testing HER2 in breast cancer: a comparative study on BRISH, FISH, and IHC. Appl Immunohistochem Mol Morphol. 2011 May;19(3):203-11. doi: 10.1097/PAI.0b013e3181f7118e. PMID: 21475037. Mollerup J, Henriksen U, Müller S, Schønau A. Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization. BMC Clin Pathol. 2012 Feb 14;12:3. doi: 10.1186/1472-6890-12-3. PMID: 22333181; PMCID: PMC3305592. Lim SJ, Cantillep A, Carpenter PM. Validation and workflow optimization of human epidermal growth factor receptor 2 testing using INFORM HER2 dual-color in situ hybridization. Hum Pathol. 2013 Nov;44(11):2590-6. doi: 10.1016/j.humpath.2013.07.005. Epub 2013 Sep 25. PMID: 24075600. Jacquemier J, Spyratos F, Esterni B, Mozziconacci MJ, Antoine M, Arnould L, Lizard S, Bertheau P, Lehmann-Che J, Fournier CB, Krieger S, Bibeau F, Lamy PJ, Chenard MP, Legrain M, Guinebretière JM, Loussouarn D, Macgrogan G, Hostein I, Mathieu MC, Lacroix L, Valent A, Robin YM, Revillion F, Triki ML, Seaume A, Salomon AV, de Cremoux P, Portefaix G, Xerri L, Vacher S, Bièche I, Penault-Llorca F. SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases. BMC Cancer. 2013 Jul 22;13:351. doi: 10.1186/1471-2407-13-351. PMID: 23875536; PMCID: PMC3729815. Gao FF, Dabbs DJ, Cooper KL, Bhargava R. Bright-field HER2 dual in situ hybridization (DISH) assay vs fluorescence in situ hybridization (FISH): focused study of immunohistochemical 2+ cases. Am J Clin Pathol. 2014 Jan;141(1):102-10. doi: 10.1309/AJCP6CXS8OSRHXIR. PMID: 24343743. Mansfield AS, Sukov WR, Eckel-Passow JE, Sakai Y, Walsh FJ, Lonzo M, Wiktor AE, Dogan A, Jenkins RB. Comparison of fluorescence in situ hybridization (FISH) and dual-ISH (DISH) in the determination of HER2 status in breast cancer. Am J Clin Pathol. 2013 Feb;139(2):144-50. doi: 10.1309/AJCP13GJAOJAYJMW. PMID: 23355198. Horii R, Matsuura M, Iwase T, Ito Y, Akiyama F. Comparison of dual-color in-situ hybridization and fluorescence in-situ hybridization in HER2 gene amplification in breast cancer. Breast Cancer. 2014 Sep;21(5):598-604. doi: 10.1007/s12282-012-0436-0. Epub 2013 Jan 12. PMID: 23307494. Tang SM, Soong IS, Luk MY, Suen DT, Hioe F, Man EP, Tsun OK, Khoo US. Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong. Hong Kong Med J. 2016 Apr;22(2):144-51. doi: 10.12809/hkmj144458. PMID: 26823385. Layfield LJ, Wallander ML, Tripp SR, Redpath S, Banks PM. Comparison of Dual-ISH (DISH) With Fluorescence In Situ Hybridization (FISH) and Correlation With Immunohistochemical Findings for HER2/Neu Status in Breast Carcinoma. Appl Immunohistochem Mol Morphol. 2017 Apr;25(4):231-236. doi: 10.1097/PAI.0000000000000304. PMID: 26766122. Rathi A, Sahay A, Shet TM, Patil A, Desai SB. Validation of Dual-Color Dual In Situ Hybridization for HER2/neu Gene in Breast Cancer. Arch Pathol Lab Med. 2023 Jul 25. doi: 10.5858/arpa.2022-0543-OA. Epub ahead of print. PMID: 37490416. Gajaria PK, Tambe S, Pai T, Patil A, Desai SB, Shet TM. Dual-color dual-hapten in situ hybridization (D-DISH) - Comparison with fluorescence in situ hybridization (FISH) for HER2/neu testing in breast cancer. Indian J Pathol Microbiol. 2020 Apr-Jun;63(2):194-199. doi: 10.4103/IJPM.IJPM_861_19. PMID: 32317514. Bahreini F, Soltanian AR, Mehdipour P. A meta-analysis on concordance between immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to detect HER2 gene overexpression in breast cancer. Breast Cancer. 2015 Nov;22(6):615-25. doi: 10.1007/s12282-014-0528-0. Epub 2014 Apr 10. PMID: 24718809. Additional Declarations Competing interest reported. The first author was involved with the project and obtained permission to use the data but did not receive any incentive or obligation to publish the result in academic journal. The remaining authors declare no conflict of interest. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4830804","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":344860039,"identity":"752a0528-3fc5-4172-8f4a-dbf2c2f8e161","order_by":0,"name":"Kroonpong Iampenkhae","email":"","orcid":"","institution":"Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society","correspondingAuthor":false,"prefix":"","firstName":"Kroonpong","middleName":"","lastName":"Iampenkhae","suffix":""},{"id":344860043,"identity":"f814799e-472f-4b7a-a8bf-ffecb20803df","order_by":1,"name":"Adiluck Pisutpunya","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Adiluck","middleName":"","lastName":"Pisutpunya","suffix":""},{"id":344860045,"identity":"4ccd63f9-218a-4178-9202-08a565af539e","order_by":2,"name":"Sompon Apornvirat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBAC+QYogx8mYkBIi8EBKEOyDUJLENYCZxwjWgt7d9qDjzvs8ozv9xgw/KhhqDMnpEW+5+x2w5lnkovNjvEYMPYcY5CwbCCk50buNmneNubEbUAtDLwNQIcdIE5LfeLmNqAtf0nQcjhxAxuPATNRthicAfml7XixxLG0gsMyxyQkNxDSIt/eu+3Bx7bqPP7mwxsfvqmx4SfsMAYGNhCRACKAiiUIq0fRMgpGwSgYBaMAKwAA5LE+/6QUV/kAAAAASUVORK5CYII=","orcid":"","institution":"Chulabhorn International College of Medicine, Thammasat University","correspondingAuthor":true,"prefix":"","firstName":"Sompon","middleName":"","lastName":"Apornvirat","suffix":""}],"badges":[],"createdAt":"2024-07-30 19:00:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4830804/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4830804/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64004971,"identity":"b26c25d5-1e42-4148-ba4a-6080f6965f1b","added_by":"auto","created_at":"2024-09-04 21:34:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":670118,"visible":true,"origin":"","legend":"\u003cp\u003eTumor heterogeneity in HER2 gene amplification and expression (A) Hematoxylin and eosin stain showing invasive breast carcinoma of no special type. (B) HER2 IHC revealing a positive 3+ result on the left and negative 1+ result on the right. (C) HER2 DISH displaying positivity in area corresponding to the HER2 IHC positive region. (D) HER2 DISH showing negativity in area corresponding to the HER2 IHC negative region.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4830804/v1/28a5ddaa5f0626e0275affb0.png"},{"id":83654758,"identity":"ca142932-e036-43fb-a40d-9ec413433977","added_by":"auto","created_at":"2025-05-30 08:17:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1175624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4830804/v1/c2fb9448-e495-4406-83cb-db588adf475d.pdf"}],"financialInterests":"Competing interest reported. The first author was involved with the project and obtained permission to use the data but did not receive any incentive or obligation to publish the result in academic journal.\nThe remaining authors declare no conflict of interest.","formattedTitle":"Comparative Analysis of HER2 Immunohistochemistry (IHC) and Brightfield Dual-Color In Situ Hybridization (DISH) in Lymph Node Metastasized Breast Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human epidermal growth factor receptor 2 (HER2) gene, locating on band q21 of chromosome 17 (Chr17), encodes a type of tyrosine kinase receptor. Approximately 15\u0026ndash;20% of breast cancers exhibit HER2 gene amplification and/or protein overexpression, and this HER2 status is both prognostic and predictive of response to HER2-targeted therapy [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 2023 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommends utilizing immunohistochemistry (IHC), brightfield in situ hybridization, or fluorescent in situ hybridization (FISH) as detection tests. The new guideline reaffirms the use of HER2 targeted therapy in patients with HER2 IHC 3\u0026thinsp;+\u0026thinsp;and IHC 2\u0026thinsp;+\u0026thinsp;with amplified in situ hybridization (ISH) while acknowledging the benefits of newer generation HER2 combined drugs in HER2-low patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to its cost-effectiveness and simplicity in performing and interpreting, many laboratories prefer using IHC as a screening test and perform ISH only if the IHC result is 2\u0026thinsp;+\u0026thinsp;or 3+. For ISH tests, FISH is still considered a gold standard test for HER2 amplification. However, FISH has several limitations when compared to brightfield dual-color in situ hybridization (DISH), including the necessity for a fluorescent microscope, the fast degradation time of fluorescent stains, and the limitation of visualization of cell features [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Several studies have demonstrated very high to almost perfect agreement on HER2 amplification between FISH and DISH, suggesting that DISH can be used as a more accessible alternative to FISH [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile there are extensive studies on the concordance of HER2 IHC and FISH, as shown in the meta-analysis by Bahreini et al., 2015 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the extant literature on direct comparison between IHC and DISH is still lacking. Hence, the objectives of our study are to provide additional information on the concordance rate between HER2 IHC and DISH in a specific setting of lymph node metastasized breast cancer, and to compare the result to the existing data on the concordance rate between HER2 IHC and FISH.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample\u003c/h2\u003e \u003cp\u003eAll cases of lymph node metastasized invasive breast carcinoma with HER2 IHC scores of 2\u0026thinsp;+\u0026thinsp;and 3\u0026thinsp;+\u0026thinsp;in King Chulalongkorn Memorial Hospital (KCMH) and Biobridge project, which received specimens from multiple hospitals across Thailand from 2015 to 2021, were included. As per the project protocol, all samples had appropriate pre-analytical factors, pathologically documented lymph node metastasis, and validated IHC assays were used to evaluate HER2. The formalin-fixed paraffin-embedded (FFPE) tissue blocks, H\u0026amp;E slides, and the initial HER2 IHC slides were retrieved. Two pathologists (KI and AP) reviewed the HER2 IHC slides using the 2023 ASCO/CAP HER2 scoring guideline, and their consensus was used. If any problems interfered with slide interpretation, re-staining would be performed in KCMH, and the slides would be reevaluated. The case would be excluded if the issues were unresolved, a consensus could not be reached, or there was inadequate remaining tumor quantity. Ultimately, a total of 1,307 cases remained in the study and HER2 DISH was then performed. Of these, 826 cases had a score of 3+, and 481 cases had a score of 2+.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIHC\u003c/h2\u003e \u003cp\u003eThe FFPE tissue blocks were sectioned into 3-\u0026micro;m-thick sections. Anti-HER2/Neu (4b5) antibody (Ventana Medical Systems, Inc, Tuscon, Arizona, USA, catalog number 05278368001) was applied as per the manufacturer\u0026rsquo;s protocol, using the automated Benchmark Ultra system (Ventana Medical Systems, Inc, Tuscon, Arizona, USA).\u003c/p\u003e \u003cp\u003eThe interpretation of repeated HER2 IHC slides was done in the same way as the IHC slides received from the Biobridge project.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDISH\u003c/h2\u003e \u003cp\u003eThe INFORM HER2 Dual ISH probe cocktail (Ventana Medical Systems, Inc, Tuscon, Arizona, USA, catalog number 760\u0026ndash;6072) was performed as per the manufacturer\u0026rsquo;s protocol on 4-\u0026micro;m-thick tissue sections, using the automated Benchmark Ultra system (Ventana Medical Systems, Inc, Tuscon, Arizona, USA). Quality assessment for HER2 and Chr17 signals in both internal control and tumor cells, as well as background staining, was conducted. If any interference was detected, repeat staining with proper adjustment was done. If the issue persisted, we switched to performing DISH on lymph node specimen.\u003c/p\u003e \u003cp\u003eThe interpretation of HER2 DISH slides was performed independently by two pathologists (KI and AP), using light microscopes with 40x and 60x objective lens and their consensus was used. The HER2 and chromosome 17 (Chr17) signals were enumerated in 20 invasive tumor cell nuclei. If the HER2/Chr17 ratio was within the range of 1.8 to 2.2, additional 20 nuclei would be counted. The final HER2/Chr17 ratio of equal to or greater than 2.0 was regarded as positive or amplified, while HER2/Chr17 ratio of less than 2.0 was interpreted as negative or non-amplified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were statistically analyzed using ChatGPT-4o. Metrics calculated were the concordant rate of positive IHC and DISH tests, the positive rate of HER2 DISH in the equivocal (2+) IHC group, 95% Confidence Intervals (CI) for the concordant rate and positive rate, and Fisher\u0026rsquo;s exact test to compare the significance of associations between concordant rate and positive rate of IHC and DISH to IHC and FISH.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConcordance of HER2 IHC and DISH\u003c/h2\u003e \u003cp\u003eOf the 1,307 cases, 933 samples showed HER2 amplification by DISH. HER2 amplifications were detected in 92% of IHC 3\u0026thinsp;+\u0026thinsp;cases (95% CI\u0026thinsp;=\u0026thinsp;90.2%-93.9%) and 36% of IHC 2\u0026thinsp;+\u0026thinsp;cases (95% CI\u0026thinsp;=\u0026thinsp;31.7%-40.3%). Fisher\u0026rsquo;s exact test demonstrated non-random association in the data (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The result is summarized in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResult of HER2 IHC and DISH\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHER2 DISH result\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHER2 IHC score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e826\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e481\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eP-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAdditionally, tumor heterogeneity showing equal or less than 30% positive area by DISH were observed in 11 cases (0.84%). Four of these had an IHC score of 2\u0026thinsp;+\u0026thinsp;and seven had an IHC score of 3+. One example of such cases is demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eComparison between IHC to DISH and IHC to FISH\u003c/h2\u003e \u003cp\u003eComparison of our IHC and DISH data to the data of IHC and FISH from the meta-analysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. was performed. Fisher\u0026rsquo;s exact test indicated no statistically significant differences in either HER2 IHC score 2\u0026thinsp;+\u0026thinsp;and 3\u0026thinsp;+\u0026thinsp;groups. The comparative data was shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of HER2 IHC and DISH to IHC and FISH\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISH Positive rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2 IHC score 3+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOur IHC to DISH data\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIHC to FISH data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1434\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHER2 IHC score 2+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOur IHC to DISH data\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIHC to FISH data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1346\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study aimed to evaluate the concordance between HER2 IHC and DISH in lymph node metastasized breast cancer, providing a comparative perspective against the well-established concordance between HER2 IHC and FISH. The findings could contribute to the ongoing discourse on optimizing HER2 testing methodologies to enhance diagnostic accuracy and therapeutic outcomes.\u003c/p\u003e \u003cp\u003eThe results demonstrate a very high concordance between HER2 DISH and IHC in the IHC 3\u0026thinsp;+\u0026thinsp;group and a low positive rate of HER2 IHC 2\u0026thinsp;+\u0026thinsp;when compared to HER2 DISH. This data aligns well with the current recommendations on HER2 testing, which emphasize the reliability of IHC as a primary screening method, followed by ISH confirmation in equivocal cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although there are minor discrepancies between our findings and the results of some prior studies, which are presented in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we believe these slight differences do not affect the testing recommendation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrior studies comparing HER2 IHC 2\u0026thinsp;+\u0026thinsp;and 3\u0026thinsp;+\u0026thinsp;to DISH in breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eISH Positive rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2 IHC score 3+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLim et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGao et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLayfield et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRathi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHER2 IHC score 2+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGao et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLayfield et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRathi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe strength of this study lies in its large sample size. The 1,307-sample size is the largest study on this topic in the current English literature especially when considering the problematic 2\u0026thinsp;+\u0026thinsp;IHC score group. Additionally, this study included the samples from the Biobridge project, which collected specimens from multiple hospitals across Thailand. While this subjected the study to increased heterogeneity in pre-analytic factors, the strong agreement in the IHC 3\u0026thinsp;+\u0026thinsp;group validates its applicability in wider settings.\u003c/p\u003e \u003cp\u003eOne limitation of this study is the lack of the gold standard FISH test. Nonetheless, as previously discussed, prior investigations show excellent agreement between DISH and FISH tests [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We also find no significant difference when comparing our result to the result of IHC and FISH, thereby justifying the use of DISH as a substitute for FISH. Another limitation of this study is the lack of cases with IHC scores of 0 and 1+. This limits the ability to precisely calculate some statistical values such as sensitivity, specificity, and Cohen\u0026rsquo;s kappa agreement.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study adds valuable evidence to the body of literature supporting the use of DISH as an effective alternative to FISH for confirming HER2 status in breast cancer. It reveals an expected correlation between HER2 IHC and DISH in lymph node metastasized breast carcinomas, comparable to prior studies between HER2 IHC and FISH in breast cancers. The concordance rate of the IHC score 3+ group is 92%, and the positive rate of the IHC score 2+ group is 36%, reinforcing the recommendation to perform ISH test in the IHC score of 2+ group and proceed to HER2 targeted therapy in the IHC score of 3+ group especially in resource-limited settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, reference number 938/64.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003cbr\u003e This research received no external funding. Publication fee was partially supported by the author’s university.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiobridge Project Thailand was funded by Roche Thailand Ltd. to encourage the utilization of HER2 DISH. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first author was involved with the project and obtained permission to use the data but did not receive any incentive or obligation to publish the result in academic journal.\u003c/p\u003e\n\u003cp\u003eThe remaining authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKroonpong Iampenkhae: Conception and design, manuscript writing and editing, data collection, analysis, and interpretation\u003c/p\u003e\n\u003cp\u003eAdiluck Pisutpanya: Manuscript editing, data collection, analysis and interpretation\u003c/p\u003e\n\u003cp\u003eSompon Apornvirat: Manuscript writing and editing, data analysis and interpretation\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\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 Jan 9;235(4785):177-82. doi: 10.1126/science.3798106. PMID: 3798106.\u003c/li\u003e\n\u003cli\u003eOwens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004 Apr;5(1):63-9. doi: 10.3816/cbc.2004.n.011. PMID: 15140287.\u003c/li\u003e\n\u003cli\u003eWolff AC, Somerfield MR, Dowsett M, Hammond MEH, Hayes DF, McShane LM, Saphner TJ, Spears PA, Allison KH. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: ASCO-College of American Pathologists Guideline Update. J Clin Oncol. 2023 Aug 1;41(22):3867-3872. doi: 10.1200/JCO.22.02864. Epub 2023 Jun 7. PMID: 37284804.\u003c/li\u003e\n\u003cli\u003eVogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002 Feb 1;20(3):719-26. doi: 10.1200/JCO.2002.20.3.719. Corrected and republished in: J Clin Oncol. 2023 Mar 20;41(9):1638-1645. PMID: 11821453.\u003c/li\u003e\n\u003cli\u003eChrzanowska NM, Kowalewski J, Lewandowska MA. Use of Fluorescence In Situ Hybridization (FISH) in Diagnosis and Tailored Therapies in Solid Tumors. Molecules. 2020 Apr 17;25(8):1864. doi: 10.3390/molecules25081864. PMID: 32316657; PMCID: PMC7221545.\u003c/li\u003e\n\u003cli\u003ePedersen M, Rasmussen BB. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer. Diagn Mol Pathol. 2009 Jun;18(2):96-102. doi: 10.1097/PDM.0b013e31817f5227. PMID: 19430295.\u003c/li\u003e\n\u003cli\u003eKoh YW, Lee HJ, Lee JW, Kang J, Gong G. Dual-color silver-enhanced in situ hybridization for assessing HER2 gene amplification in breast cancer. Mod Pathol. 2011 Jun;24(6):794-800. doi: 10.1038/modpathol.2011.9. Epub 2011 Feb 11. PMID: 21317877.\u003c/li\u003e\n\u003cli\u003eBartlett JM, Campbell FM, Ibrahim M, O\u0026apos;Grady A, Kay E, Faulkes C, Collins N, Starczynski J, Morgan JM, Jasani B, Miller K. A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay. Am J Clin Pathol. 2011 Jan;135(1):157-62. doi: 10.1309/AJCPVPRKK1ENEDGQ. PMID: 21173138.\u003c/li\u003e\n\u003cli\u003eBr\u0026uuml;gmann A, Lelkaitis G, Nielsen S, Jensen KG, Jensen V. Testing HER2 in breast cancer: a comparative study on BRISH, FISH, and IHC. Appl Immunohistochem Mol Morphol. 2011 May;19(3):203-11. doi: 10.1097/PAI.0b013e3181f7118e. PMID: 21475037.\u003c/li\u003e\n\u003cli\u003eMollerup J, Henriksen U, M\u0026uuml;ller S, Sch\u0026oslash;nau A. Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization. BMC Clin Pathol. 2012 Feb 14;12:3. doi: 10.1186/1472-6890-12-3. PMID: 22333181; PMCID: PMC3305592.\u003c/li\u003e\n\u003cli\u003eLim SJ, Cantillep A, Carpenter PM. Validation and workflow optimization of human epidermal growth factor receptor 2 testing using INFORM HER2 dual-color in situ hybridization. Hum Pathol. 2013 Nov;44(11):2590-6. doi: 10.1016/j.humpath.2013.07.005. Epub 2013 Sep 25. PMID: 24075600.\u003c/li\u003e\n\u003cli\u003eJacquemier J, Spyratos F, Esterni B, Mozziconacci MJ, Antoine M, Arnould L, Lizard S, Bertheau P, Lehmann-Che J, Fournier CB, Krieger S, Bibeau F, Lamy PJ, Chenard MP, Legrain M, Guinebreti\u0026egrave;re JM, Loussouarn D, Macgrogan G, Hostein I, Mathieu MC, Lacroix L, Valent A, Robin YM, Revillion F, Triki ML, Seaume A, Salomon AV, de Cremoux P, Portefaix G, Xerri L, Vacher S, Bi\u0026egrave;che I, Penault-Llorca F. SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases. BMC Cancer. 2013 Jul 22;13:351. doi: 10.1186/1471-2407-13-351. PMID: 23875536; PMCID: PMC3729815.\u003c/li\u003e\n\u003cli\u003eGao FF, Dabbs DJ, Cooper KL, Bhargava R. Bright-field HER2 dual in situ hybridization (DISH) assay vs fluorescence in situ hybridization (FISH): focused study of immunohistochemical 2+ cases. Am J Clin Pathol. 2014 Jan;141(1):102-10. doi: 10.1309/AJCP6CXS8OSRHXIR. PMID: 24343743.\u003c/li\u003e\n\u003cli\u003eMansfield AS, Sukov WR, Eckel-Passow JE, Sakai Y, Walsh FJ, Lonzo M, Wiktor AE, Dogan A, Jenkins RB. Comparison of fluorescence in situ hybridization (FISH) and dual-ISH (DISH) in the determination of HER2 status in breast cancer. Am J Clin Pathol. 2013 Feb;139(2):144-50. doi: 10.1309/AJCP13GJAOJAYJMW. PMID: 23355198.\u003c/li\u003e\n\u003cli\u003eHorii R, Matsuura M, Iwase T, Ito Y, Akiyama F. Comparison of dual-color in-situ hybridization and fluorescence in-situ hybridization in HER2 gene amplification in breast cancer. Breast Cancer. 2014 Sep;21(5):598-604. doi: 10.1007/s12282-012-0436-0. Epub 2013 Jan 12. PMID: 23307494.\u003c/li\u003e\n\u003cli\u003eTang SM, Soong IS, Luk MY, Suen DT, Hioe F, Man EP, Tsun OK, Khoo US. Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong. Hong Kong Med J. 2016 Apr;22(2):144-51. doi: 10.12809/hkmj144458. PMID: 26823385.\u003c/li\u003e\n\u003cli\u003eLayfield LJ, Wallander ML, Tripp SR, Redpath S, Banks PM. Comparison of Dual-ISH (DISH) With Fluorescence In Situ Hybridization (FISH) and Correlation With Immunohistochemical Findings for HER2/Neu Status in Breast Carcinoma. Appl Immunohistochem Mol Morphol. 2017 Apr;25(4):231-236. doi: 10.1097/PAI.0000000000000304. PMID: 26766122.\u003c/li\u003e\n\u003cli\u003eRathi A, Sahay A, Shet TM, Patil A, Desai SB. Validation of Dual-Color Dual In Situ Hybridization for HER2/neu Gene in Breast Cancer. Arch Pathol Lab Med. 2023 Jul 25. doi: 10.5858/arpa.2022-0543-OA. Epub ahead of print. PMID: 37490416.\u003c/li\u003e\n\u003cli\u003eGajaria PK, Tambe S, Pai T, Patil A, Desai SB, Shet TM. Dual-color dual-hapten in situ hybridization (D-DISH) - Comparison with fluorescence in situ hybridization (FISH) for HER2/neu testing in breast cancer. Indian J Pathol Microbiol. 2020 Apr-Jun;63(2):194-199. doi: 10.4103/IJPM.IJPM_861_19. PMID: 32317514.\u003c/li\u003e\n\u003cli\u003eBahreini F, Soltanian AR, Mehdipour P. A meta-analysis on concordance between immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to detect HER2 gene overexpression in breast cancer. Breast Cancer. 2015 Nov;22(6):615-25. doi: 10.1007/s12282-014-0528-0. Epub 2014 Apr 10. PMID: 24718809.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HER2, Breast neoplasms, Immunohistochemistry, In situ hybridization, Concordance analysis","lastPublishedDoi":"10.21203/rs.3.rs-4830804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4830804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe human epidermal growth factor receptor 2 (HER2) gene encodes a tyrosine kinase receptor, implicated in 15\u0026ndash;20% of breast cancers. HER2 amplification is both prognostic and predictive of response to HER2-targeted therapy. IHC is the preferred initial test due to its cost-effectiveness and simplicity. While FISH is the gold standard test for HER2 amplification, it has several limitations, prompting consideration of DISH as an alternative. This study aims to evaluate the concordance between HER2 IHC and DISH in lymph node metastasized breast cancer and compares the results to existing data on HER2 IHC and FISH.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective study included 1,307 cases of pathologically confirmed lymph node metastasized invasive breast carcinoma with HER2 IHC scores of 2+ (481 cases) and 3+ (826 cases). Interpretation of HER2 IHC was performed using the 2023 ASCO/CAP guideline. HER2 DISH was conducted and evaluated by the HER2/Chromosome 17 signals ratio.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHER2 amplification was detected in 933 cases, including 92% (760) of IHC 3\u0026thinsp;+\u0026thinsp;cases and 36% (173) of IHC 2\u0026thinsp;+\u0026thinsp;cases. Comparison with the meta-analysis data of IHC and FISH showed no significant differences, indicating that DISH is a reliable alternative to FISH.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study demonstrates no significant difference in the positive rates of HER2 IHC to DISH when compared with prior data of IHC to FISH, reaffirming the use of HER2 DISH as an effective and more accessible alternative to FISH in breast cancer. The high concordant rate between HER2 IHC and DISH in the IHC 3\u0026thinsp;+\u0026thinsp;group and low positive rate in the IHC 2\u0026thinsp;+\u0026thinsp;group support the recommendation to proceed with HER2-targeted therapy in IHC 3\u0026thinsp;+\u0026thinsp;cases and to perform ISH in IHC 2\u0026thinsp;+\u0026thinsp;cases.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of HER2 Immunohistochemistry (IHC) and Brightfield Dual-Color In Situ Hybridization (DISH) in Lymph Node Metastasized Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 21:34:47","doi":"10.21203/rs.3.rs-4830804/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09be5301-0c4f-4052-b14d-1ec7be96abae","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-30T08:09:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 21:34:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4830804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4830804","identity":"rs-4830804","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}