Abstract Background: HER2-targeted therapies are guided by HER2 protein overexpression or ERBB2 amplification. However, these biomarkers may imperfectly reflect functional dependence on HER2 signaling. We performed an integrative multi-omic analysis to quantify the discordance between clinical HER2 status and its corresponding functional biology, assessing both downstream pathway activation in HER2-positive (HER2+) tumors and inactivity in HER2-negative (HER2−) cases. Methods: Analysis was performed on the TCGA breast cancer dataset. HER2+ (n=106) and HER2− (n=595) cohorts were defined using clinical HER2 status. Transcriptomic and proteomic over-/under-expression states were derived for clinically relevant driver genes by thresholding RNA/protein expression values. For each tumor, we assessed activation of HER2-associated pathways (e.g., direct HER2 targets, PI3K-AKT-mTOR, MAPK, RTK crosstalk) by comparing expression states against the expected direction of activation. A pathway was considered 'validated' when at least half of the tested states aligned with expected activation patterns. Samples with no available data were excluded from the analysis, reducing the evaluable cases to 85 and 457 for HER2+ and HER2−, respectively. Results: Among HER2+ tumors, proximal HER2 signaling showed strong concordance with clinical status, with direct targets being active in 80.0% of cases. In contrast, canonical downstream pathways were often inactive, with PI3K-AKT-mTOR and MAPK activity validated in only 29.4% and 15.3% of tumors, respectively. This heterogeneity suggests that many HER2+ tumors may rely less on canonical HER2-driven signaling than expected. In HER2− tumors, proximal HER2 signaling and MAPK pathway activity were appropriately low as validated in 96% cases. However, the PI3K-AKT-mTOR pathway was active in 132/457 (28.9%) HER2− tumors, revealing a sizable subset with HER2-independent PI3K activation. Conclusions: Our findings highlight biological mechanisms that may underlie therapeutic resistance and reveal alternative targets for expanding precision treatment strategies across breast cancer subtypes. The limited canonical signaling in most HER2+ tumors suggests pathway-independent drivers, feedback inhibition, or gaps in transcriptomic/proteomic measurements that miss critical phosphorylation events. In contrast, activation signatures in a subset of HER2− tumors indicate therapeutically relevant HER2-low biology. The results support complementing standard testing with biology-aware signatures to (i) flag HER2+ tumors unlikely to respond to combination therapies and (ii) stratify HER2− patients into actionable groups, including those who may benefit from PI3K/mTOR inhibitors. Future work will incorporate phospho-proteomic data and link pathway signatures to treatment response to refine patient selection. Citation Format: Salim Arslan, Julian Schmidt, Cher Bass, Foivos Ntelemis, J Carl Barrett, Oscar Maiques, Jakob Nikolas Kather, Pahini Pandya. Functional pathway analysis reveals discordance between clinical HER2 status and downstream effector activation in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 57.
Arslan et al. (Fri,) studied this question.