Spatial/molecular heterogeneity and treatment response in HER2+ early breast cancer: Translational analyses from the DAPHNe trial

Chen, E.; Schlam, I.; Shimada, K.; Li, T.; Ogayo, E. R.; Patel, A.; Davis, J.; Lin, J.-R.; Schultz, A.; Townsend, M. G.; Zheng, K. F.; Wanderley, C. W.; Pastorello, R.; New, F.; Metzger, E.; Sinclair, N.; Spring, L. M.; Faggen, M.; Constantine, M.; Desai, N.; Tung, N.; Tayob, N.; Krop, I. E.; Tolaney, S. M.; Winer, E. P.; Guerriero, J. L.; Mittendorf, E. A.; Waks, A. G.

doi:10.64898/2026.09.17.752407

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bioRxiv · 10.64898/2026.09.17.752407

Spatial/molecular heterogeneity and treatment response in HER2+ early breast cancer: Translational analyses from the DAPHNe trial

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Abstract

Given the heterogeneity of HER2-positive breast cancer, reliable biomarkers to guide treatment are needed. We conducted multi-platform biomarker analyses of baseline tumor tissue from the DAPHNe trial of neoadjuvant paclitaxel/trastuzumab/pertuzumab (THP) for HER2-positive early breast cancer to characterize inter- and intra-tumor heterogeneity and to identify molecular predictors of response. A total of 98 patients with stage II-III HER2-positive breast cancer received neoadjuvant THP followed by surgery. Gene expression profiling, spatial protein profiling, and single-cell imaging (cyclic immunofluorescence) were performed on pre-treatment biopsies and a subset of residual disease specimens. Pathologic response was assessed using the residual cancer burden (RCB) score. Among HER2-positive patients included in the biomarker analysis, 34% of patients had node-positive breast cancer, and 66% had hormone receptor (HR)-positive tumors. HR-positive and HR-negative tumors differed significantly across gene expression, protein expression, and single-cell profiling. High ERBB2 gene signature, p53 gene signature, and HER2 protein expression predicted favorable response (RCB 0/1), while ESR1/PGR gene, estrogen receptor (ER) signaling gene signature, and ER alpha protein expression were associated with unfavorable response (RCB 2/3). Single-cell spatial analysis revealed that cancer cells clustered by shared HER2, ER, and PR expression, suggesting local expansion of phenotypically distinct subpopulations. ER-positive cancer cells were associated with lower HLA-A and PD-L1 expression, suggesting a less immunologically active tumor cell state. Baseline HER2 and ER expression are key predictors of response to neoadjuvant HER2-targeted therapy. Single-cell spatial profiling highlights intra-tumoral heterogeneity and suggests ER-driven immune exclusion as a potential mechanism of resistance.

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Chen, E., Schlam, I., Shimada, K., Li, T., Ogayo, E. R., Patel, A., Davis, J., Lin, J.-R., Schultz, A., Townsend, M. G., Zheng, K. F., Wanderley, C. W., Pastorello, R., New, F., Metzger, E., Sinclair, N., Spring, L. M., Faggen, M., Constantine, M., Desai, N., Tung, N., Tayob, N., Krop, I. E., Tolaney, S. M., Winer, E. P., Guerriero, J. L., Mittendorf, E. A., Waks, A. G.. 2026-09-18. Spatial/molecular heterogeneity and treatment response in HER2+ early breast cancer: Translational analyses from the DAPHNe trial. https://doi.org/10.64898/2026.09.17.752407

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