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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.


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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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