Abstract PS3-09-14: Caught in the Middle: Discrepancies Between Immunohistochemistry and Oncotype DX in Hormone Receptor Assessment Leading to Triple Negative Reclassification—Tumour Heterogeneity or Just Low ER Expression?

Abstract Background Discrepancies in the assessment of ER, PR, and HER2 status by immunohistochemistry (IHC) and Oncotype DX (ODX) can substantially impact treatment decisions. Variations in assay results that alter tumour molecular subtype may lead to changes in recommended adjuvant therapy. We present a series of such cases and explore whether these findings reflect tumour heterogeneity or misclassification of ER expression. We show how reclassification influenced systemic treatment choices and eligibility for genetic testing, underscoring the practical implications of such discrepancies for the personalisation of breast cancer care. Methods Retrospective analysis was conducted of 682 patients who underwent ODX testing between 2020-2025 at Barts Health NHS Trusts hospitals. Data extracted from the ODX reports included the recurrence score and the qualitative and quantitative assessments of mRNA levels of ESR1, PGR, and ERBB2 receptor genes. These results were compared to ER, PR, and HER2 receptor protein status determined by standard IHC staining and scoring on the diagnostic biopsy specimens1. The standard ER IHC scoring categorises ER status as ER negative (score 0,2) ; ER low positive (score 3,4) and ER positive (score 5-8)1. All ER biopsy slides were reviewed blind by a breast pathologist (MW). Receptor IHC was repeated on the resection specimen tumour block used for ODX testing and the results compared to the ER IHC score on the diagnostic biopsy. Results were compared with 5 control cases in which the ODX as well as ER IHC on biopsy was positive. Results From a total of 682 ODX tests performed, 12 patients (1.7%; 12/682) demonstrated discordance, with a negative ESR1 gene expression score on ODX and ER IHC positive score (5-8). Upon repeat histological review, all 12 cases (100%) were confirmed to be either ER low-positive (75%; 9/12) or ER negative (25%; 3/12) on ER IHC on the resected tissue. On review of the ER IHC biopsy slides, in 7/12 cases (58.3%) the tumour displayed heterogeneous ER IHC staining on biopsy, with predominantly ER negative tumour being present on the resection block. 3/12 biopsies (25%) originally reported as ER positive on the basis of highest-staining hotspots were reclassified as overall ER low-positive on review. 2/3 (66.7%) of these cases also exhibited heterogeneity of ER IHC staining. 1/12 cases had multifocal breast cancer with one ER positive (score 8) and one ER low positive tumour (score 3) with the Oncotype test performed on the ER low positive tumour. 1/12 (8.3%) of cases was an external referral case and could not be reviewed. All 5 control cases with ODX/biopsy IHC positive were also positive by ER IHC on the resection block. All discordant tumours were grade 3 invasive ductal carcinomas presenting in women aged 30-77 years. 3 patients (25%) were BRCA1 mutation carriers. Regarding adjuvant chemotherapy, 4 patients (33.3%) were treated with ER+ regimens, while 7 patients (58.3%) received triple-negative regimens. Conclusions Discordance between IHC and ODX ER status is rare (1.7%). Most discrepancies were attributable to intratumoural heterogeneity of ER protein expression in biopsies with predominantly ER negative sub-clones in resection samples. Repeat IHC confirmed these resected cases as ER low-positive or negative, in concordance with ODX, confirming a higher-risk subgroup. Our findings as well as data from several breast receptor RT-PCR studies 2,3,4 also support the potential for subtype reclassification initially classified as ER low positive (score 3,4), with low or negative ODX ESR1 gene scores as similar in biological behaviour and chemotherapy response to ER negative tumours. This could help guide personalisation of adjuvant treatment strategies and prompt germline mutation testing. Citation Format: V. Angelis, A. Grobbelaar, A. Zamani, P. Hall, M. Phillips, P. Schmid, K. Hawkesford, M. Ullah, L. Begolli, L. Johnson, S. Ledwidge, M. Thorat, F. Irakleidis, K. Sidlauskas, D. Ryan, M. Warren. Caught in the Middle: Discrepancies Between Immunohistochemistry and Oncotype DX in Hormone Receptor Assessment Leading to Triple Negative Reclassification—Tumour Heterogeneity or Just Low ER Expression? [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-09-14.