Purpose The recent approval of antibody drug conjugates (ADCs) targeting HER2 (like Trastuzumab Deruxtecan or T-DXd) have led to challenges for the immunohistochemical (IHC) companion diagnostic test since the test was optimized for gene amplified levels of HER2. Here we develop and validate an objective test for low level HER2 expression toward more accurate selection of patients for T-DXd.

Experimental Design We validated the high-sensitivity HER2 assay using a mix of the requirements for an IHC assay and that of a ligand binding assay. Then we prospectively tested it on 316 core biopsy specimens received by Yale Pathology Labs from August 2022 to August 2023

Results Using a 40-case breast cancer tissue validation set, we find very high accuracy and precision with a coefficient of variation below 10% and define a reportable range for the assay in amol/mm2. These prospective cases show the dynamic range of HER2 expression, but also the discordance of pathologist scores with quantitative measurements, especially in the low range of HER2. We find that 6% of the cohort was below the limit of detection of this more sensitive assay while 71% of the IHC=0 cases were above the limit of quantification. Efforts are underway to determine a possible threshold expression level required for T-DXd response.

Conclusion This assay validation study provides a method for accurate, objective measurement of HER2 and has the potential to improve selection of patients for T-DXd or similarly targeted ADC therapies in the future.

Translational Relevance Antibody Drug Conjugates (ADCs) represent a novel class of targeted therapy, but in some ADC approvals the target is not assessed due to the lack of an objective and reproducible method for measurement of protein on slides. This paper describes, validates and tests a new fully quantitative method for ADC target assessment on histology slides.

Dr. Rimm has served as a Consultant/Advisor to Astra Zeneca, Cell Signaling Technology, Cepheid, Danaher, Halda Therapeutics, Incendia, NextCure, Nucleai, PAIGE.AI, Regeneron, and Sanofi. Astra Zeneca, Cepheid, NavigateBP, NextCure, Danaher/Leica, Regeneron and Konica/Minolta currently fund, or have previously funded, research in his lab. Patricia Gaule is now an employee of BMS Dr. Liebler is an employee and shareholder of Inotiv, Inc. Regan Fulton is an owner of Array Science, LLC, and is a paid advisor to Leica Biosystems, AbbVie, and Personalis, Inc

This work was funded by Yale Pathology Labs, a grant from the Breast Cancer Research Foundation (BCRF23-138) to DLR, and grants from the NIH including P30CA016359(DLR) and F30CA287869 (CJR).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

All tissue samples were collected under the approval of Yale Human Investigation Committee protocol #9505008219. Written informed consent, or waiver of consent, was obtained from all patients with approval of the Yale Human Investigation Committee. This work was conducted in accordance with the Declaration of Helsinki adopted by the world medical association

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

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Yes

Disclosures:

Dr. Rimm has served as a Consultant/Advisor to Astra Zeneca, Cell Signaling Technology, Cepheid, Danaher, Halda Therapeutics, Incendia, NextCure, Nucleai, PAIGE.AI, Regeneron, and Sanofi. Astra Zeneca, Cepheid, NavigateBP, NextCure, Danaher/Leica, Regeneron and Konica/Minolta currently fund, or have previously funded, research in his lab.

Patricia Gaule is now an employee of BMS

Dr. Liebler is an employee and shareholder of Inotiv, Inc.

Regan Fulton is an owner of Array Science, LLC, and is a paid advisor to Leica Biosystems, AbbVie, and Personalis, Inc

All data produced in the present study are available upon reasonable request to the authors