Abstract Background: In early-stage estrogen receptor (ER) negative breast cancer (BC), whether HER2-positive (HER2+) or triple negative (TNBC), neoadjuvant therapy is frequently employed, making diagnostic turnaround time a critical factor in treatment planning. The ERBB2 mRNA score, developed and validated to predict HER2 status according to ASCO/CAP criteria, has demonstrated high accuracy (AUC = 0.97; EBioMedicine 2022). HER2DX and TNBCDX assays, both of which incorporate this score, are performed using the same RNA-based platform, enabling rapid and integrated molecular profiling. However, whether this transcriptomic approach can replace conventional HER2 immunohistochemistry/ in situ hybridization (IHC/ISH) testing in ER-negative tumors has not yet been prospectively evaluated. Methods: In this prospective pilot study, consecutive patients with ER-negative early breast cancer (ER10% by IHC) at the Breast Cancer Unit at the Clínic Barcelona Comprehensive Cancer Center were enrolled. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue and analyzed using Reveal Genomics’ RNA platform. Based on ERBB2 mRNA expression levels, tumors classified as ERBB2-low received the TNBCDX Research Use Only (RUO) report, whereas those with medium or high ERBB2 levels received the HER2DX report. All cases underwent parallel HER2 IHC and ISH testing according to ASCO/CAP guidelines. The primary endpoint was the concordance between the HER2DX ERBB2 mRNA score and final HER2 status by IHC/ISH. Additionally, we explored the ERBB2 score in 257 ER-negative tumors (93 HER2+ and 164 TNBC) from 4 cohorts (i.e, BIONHER, DAPHNe, ATEMPT and a combined TNBC Spanish cohort from Clínic Barcelona Comprehensive Cancer Center, and Hospital Universitario 12 de Octubre). Results: As of July 1st 2025, 30 patients with newly diagnosed ER-negative BC (8 HER2+ and 22 TNBC) have been enrolled. Concordance between the HER2DX ERBB2 mRNA score and HER2 IHC/ISH status was 100%. Eight cases (26.6%) were classified as ERBB2 medium/high and received the HER2DX report, while 22 cases (73.4%) were ERBB2 low and received the TNBCDX report. The proportion of HER2+ tumors (i.e., IHC 3+ or IHC 2+ with ISH amplification) was consistent with the ERBB2 medium/high group (26.6%), whereas the ERBB2-low group was TNBC (i.e., IHC 0, 1+, 2+ without ISH amplification). Four patients had IHC 2+ tumors and underwent reflex ISH testing. Two showed no ISH amplification, consistent with the ERBB2-low classification, while the other two had confirmed amplification, aligning with the ERBB2-medium/high group. Finally, in the retrospective combined ER-negative cohort of 257 samples, the ERBB2 score accurately predicted HER2 IHC/ISH status, with 92.6% concordance and an AUC of 0.98. Among the ERBB2 medium/high group, 17 patients (15.7%) were classified as HER2-negative; whereas only 2 out of 149 (1.3%) in the ERBB2 low group were categorized as positive. Conclusions: This ongoing pilot study demonstrates the feasibility of using an upfront ERBB2 mRNA-based score to guide selection of HER2DX or TNBCDX testing in patients with newly diagnosed ER-negative breast cancer. Preliminary results show perfect concordance with standard HER2 IHC/ISH testing and support the potential of this approach as a practical and timely tool in the neoadjuvant setting. An updated analysis will be presented at the conference. Citation Format: M. Gonzalez-Rodriguez, E. Sanfeliu, M. Alva, T. Pascual, S. Pernas, A. Waks, M. Vidal, P. Tarantino, E. Ciruelos, S. Tolaney, M. Marín-Aguilera, L. Paré, W. Buckingham, F. Brasó-Maristany, A. Prat. Prospective evaluation of a mRNA ERBB2 score as a tool to guide HER2 status and accelerate neoadjuvant decision-making in ER-negative breast cancer 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 PS2-10-15.
Gonzalez-Rodriguez et al. (Tue,) studied this question.