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Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer


The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

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Fig. 1: Schematic overview of the low HER2 assay.
Fig. 2: HER2 Calibration CMA from Array Science.
Fig. 3: Validation and quality control of the antibody reagents.
Fig. 4: Regressions of the control cell lines show reproducibility and correlation at each tested antibody concentration.
Fig. 5: The distribution of breast cancer cases in a serially collected population showing many cases above the LOQ that would be considered negative by the conventional assay.
Fig. 6: An illustration of HER2 low expressing cases with membranous expression pattern.


  1. 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. Arch Pathol Lab Med 2018; 142: 1364–1382; e-pub ahead of print 2018/05/31;

  2. Pegram MD, Lipton A, Hayes DF, Weber BL, Baselga JM, Tripathy D, et al. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998; 16: 2659–2671;

  3. Howie LJ, Scher NS, Amiri-Kordestani L, Zhang L, King-Kallimanis BL, Choudhry Y, et al. FDA Approval summary: Pertuzumab for adjuvant treatment of HER2-positive early breast cancer. Clin Cancer Res 2019; 25: 2949–2955; e-pub ahead of print 2018/12/16;

  4. Ryan Q, Ibrahim A, Cohen MH, Johnson J, Ko CW, Sridhara R, et al. FDA drug approval summary: lapatinib in combination with capecitabine for previously treated metastatic breast cancer that overexpresses HER-2. Oncologist 2008 13: 1114–1119; e-pub ahead of print 2008/10/14;

  5. Koleva-Kolarova RG, Oktora MP, Robijn AL, Greuter MJW, Reyners AKL, Buskens E, et al. Increased life expectancy as a result of non-hormonal targeted therapies for HER2 or hormone receptor positive metastatic breast cancer: A systematic review and meta-analysis. Cancer Treat Rev 2017; 55: 16–25;

  6. von Minckwitz G, Huang C-S, Mano MS, Loibl S, Mamounas EP, Untch M, et al. Trastuzumab Emtansine for residual invasive HER2-positive breast cancer. New Engl J Med 2018; 380: 617–628

  7. Saura C, Oliveira M, Feng Y-H, Dai M-S, Chen S-W, Hurvitz SA, et al. Neratinib Plus Capecitabine Versus Lapatinib Plus Capecitabine in HER2-positive metastatic breast cancer previously treated with ≥ 2 HER2-directed regimens: Phase III NALA Trial. J Clin Oncol 2020; 38: 3138–3149

  8. Murthy RK, Loi S, Okines A, Paplomata E, Hamilton E, Hurvitz SA, et al. Tucatinib, Trastuzumab, and Capecitabine for HER2-positive metastatic breast cancer. New Engl J Med 2019; 382: 597–609

  9. Rugo HS, Im S-A, Cardoso F, Cortés J, Curigliano G, Musolino A, et al. Efficacy of Margetuximab vs Trastuzumab in patients with pretreated ERBB2-positive advanced breast cancer: a Phase 3 randomized clinical trial. JAMA Oncol 2021; 7: 573–584

  10. Hudis CA. Trastuzumab-mechanism of action and use in clinical practice. N Engl J Med 2007; 357: 39–51.

  11. 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: 1951–1962; e-pub ahead ofprint 2020/04/25

  12. Nakada T, Sugihara K, Jikoh T, Abe Y, Agatsuma T. The latest research and development into the antibody-drug conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 cancer therapy. Chem Pharm Bull 2019; 67: 173–185; e-pub ahead of print 2019/03/05

  13. Modi S, Park H, Murthy RK, Iwata H, Tamura K, Tsurutani J, et al. Antitumor activity and safety of Trastuzumab Deruxtecan in patients With HER2-low–expressing advanced breast cancer: results from a Phase Ib Study. J Clin Oncol 2020; 38: 1887–1896

  14. Fernandez AI, Liu M, Bellizzi A, Brock J, Fadare O, Hanley K, et al. Examination of low ERBB2 protein expression in breast cancer tissue. JAMA Oncol 2022;

  15. DeFazio-Eli L, Strommen K, Dao-Pick T, Parry G, Goodman L, Winslow J. Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action. Breast Cancer Res 2011; 13: R44

  16. Onsum MD, Geretti E, Paragas V, Kudla AJ, Moulis SP, Luus L, et al. Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients. Am J Pathol 2013; 183: 1446–1460; e-pub ahead of print 2013/09/17

  17. McCabe A, Dolled-Filhart M, Camp RL, Rimm DL. Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J Natl Cancer Inst 2005; 97: 1808–1815.

  18. Morales-Betanzos CA, Lee H, Gonzalez Ericsson PI, Balko JM, Johnson DB, Zimmerman LJ, et al. Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma. Mol Cell Proteom 2017; 16: 1705–1717; e-pub ahead of print 2017/05/27

  19. Liebler DC, Holzer TR, Haragan A, Morrison RD, O’Neill Reising L, Ackermann BL, et al. Analysis of immune checkpoint drug targets and tumor proteotypes in non-small cell lung cancer. Sci Rep 2020; 10: 9805; e-pub ahead of print 2020/06/20

  20. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 2010; 26: 966–968; e-pub ahead of print 2010/02/12

  21. Carvajal-Hausdorf DE, Schalper KA, Neumeister VM, Rimm DL. Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic. Lab Invest 2015; 95: 385–396

  22. Schrohl, A-S., Pedersen, HC., Jensen, SS., Nielsen, SL. & Brünner, N. Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies. Histopathology 59, 975–983, (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Sci Rep 2017; 7: 16878

  24. Camp, R L, Chung, G G & Rimm, D L Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med 8, 1323–1327 (2002).

    CAS  Article  Google Scholar 

  25. Subik K, Lee JF, Baxter L, Strzepek T, Costello D, Crowley P, et al. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer 2010; 4: 35–41; e-pub ahead of print 2010/08/11.

  26. Gustavo González A, Ángeles Herrador M. A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles. TrAC Trends Anal Chem 2007; 26: 227–238;

  27. Hubert P, Chiap P, Crommen J, Boulanger B, Chapuzet E, Mercier N, et al. The SFSTP guide on the validation of chromatographic methods for drug bioanalysis: from the Washington Conference to the laboratory. Analytica Chimica Acta 1999; 391: 135–148;

  28. Inczedy JnLTsUAMIUoP, Applied C. Compendium of analytical nomenclature: definitive rules 1997. Blackwell Science: Osney Mead, Oxford; Malden, MA, 1998.

  29. Huber L. Validation and Qualification in Analytical Laboratories. Interpharm Presss, East Englewood, CO, USA 1998.

  30. Moatamed NA, Nanjangud G, Pucci R, Lowe A, Shintaku IP, Shapourifar-Tehrani S, et al. Effect of ischemic time, fixation time, and fixative type on HER2/neu immunohistochemical and fluorescence in situ hybridization results in breast cancer. Am J Clin Pathol 2011; 136: 754–761

  31. Bai Y, Tolles J, Cheng H, Siddiqui S, Gopinath A, Pectasides E, et al. Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables. Lab Investig 2011; 91: 1253–1261

  32. Siddiqui S, Rimm DL. Pre-analytic variables and phospho-specific antibodies: The Achilles heel of immunohistochemistry. Breast Cancer Research (Editorial) 2010; 12;

  33. Modi, S., Saura, C., Yamashita, T., Park, Y H., Kim, S-B. & Tamura, K. et al. Trastuzumab Deruxtecan in previously treated HER2-positive breast cancer. New Engl J Med 382, 610–621, (2019).

    Article  PubMed  Google Scholar 

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We would like to thank Lori Charette, and the team at the Yale Pathology Tissue Service and Developmental Histology Facility for production of the high-quality tissue sections and TMAs. This study was supported by a sponsored research agreement with InviCRO/Konica/Minolta and the Breast Cancer Research Foundation (DLR) and the Yale Cancer Cancer (P30CA016359). Myrto Moutafi was supported by a scholarship from the Hellenic Society of Medical Oncologists (HESMO).

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Authors and Affiliations



MM: Data collection, formal analysis, investigation, visualization, methodology, writing including the original draft, and review and editing of the manuscript. CR: Data collection and curation, formal analysis, validation, and review and editing of the manuscript. VY: Data collection and curation and review and editing of the manuscript. SM-M: Data collection and curation and review and editing of the manuscript. VX: Data collection and curation and review and editing of the manuscript. YB; Data collection and curation and review and editing of the manuscript. YS: Data collection and curation and review and editing of the manuscript. PG: Data collection and curation and review and editing of the manuscript. JK: Data collection and curation, project administration and supervision, and review and editing of the manuscript. KB: Data collection and curation, project administration and supervision, and review and editing of the manuscript. SH: Data collection and curation and review and editing of the manuscript. D: Data collection and curation and review and editing of the manuscript. RF: Data collection and curation, resource provision, and review and editing of the manuscript DLR: Conceptualization, resources, formal analysis, supervision, funding acquisition, writing including the original draft, project administration, writing, review, and editing of the final draft.

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Correspondence to David L. Rimm.

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Competing interests

DLR has served as an advisor for AstraZeneca, Agendia, Amgen, BMS, Cell Signaling Technology, Cepheid, Danaher, Daiichi Sankyo, Novartis, GSK, Konica Minolta, Merck, NanoString, PAIGE.AI, Perkin Elmer, Regeneron, Roche, Sanofi, Ventana, and Ultivue. Amgen, Cepheid, Konica Minolta, NavigateBP, NextCure, and Lilly have funded research in his lab. JK and KB are employees of Invicro, a division of Konica/Minolta. DCL and SH are employees of Protypia, Inc. Regan Fulton is the majority owner of Array Science, LLC and serves as a consultant to Leica Biosystems, Personalis, Inc., and Konica/Minolta (Invicro). All other authors declare no conflict of interest.

Ethics approval and consent to participate

All tissue samples were collected with the approval from the Yale Human Investigation Committee protocol #9505008219. Written informed consent, or waiver of consent, was obtained from all patients with the approval of the Yale Human Investigation Committee.

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Moutafi, M., Robbins, C.J., Yaghoobi, V. et al. Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer. Lab Invest (2022).

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