Divergent intratumoral associations of EGFR, ERBB3 (HER3) and TACSTD2 (TROP2) transcripts with partial epithelial–mesenchymal transition in head and neck squamous cell carcinoma

Background: EGFR, HER3 and TROP2 are targets of antibody–drug conjugate (ADC) development in head and neck squamous cell carcinoma (HNSCC). We investigated how their transcripts relate to partial epithelial–mesenchymal transition (pEMT) and epithelial differentiation to define the cell-state context of therapeutic-target heterogeneity. Methods: We reanalyzed four public single-cell cohorts, with 26 patients eligible for within-patient malignant-cell analyses, focusing on tumors classified as human papillomavirus-negative or p16-negative. Using target-excluded state scores, we directly compared target-specific expression differences between upper and lower within-patient pEMT thirds. Patient-level contrasts were evaluated using sign-flip tests and Benjamini–Hochberg correction. Spatial transcriptomics, paired RNA–protein measurements, bulk transcriptomics and synthetic pseudobulks provided complementary analyses. Results: EGFR showed a more positive intratumoral pEMT association than ERBB3 (HER3) and TACSTD2 (TROP2). In discovery tumors, paired differences in high-to-low log₂ expression ratios were 1.58 (95% CI, 0.87–2.42; n = 9) for EGFR−ERBB3 and 1.30 (0.75–1.91; n = 10) for EGFR−TACSTD2 (both q = 0.006). Both contrasts replicated in a separate cohort (both q = 0.047) and were directionally concordant in two additional cohorts. Epithelial differentiation showed the opposite relative pattern, with differentiation adjustment attenuating the EGFR−TACSTD2 contrast. In Visium tumor regions from 10 patients, adjusted pEMT correlations were higher for EGFR than for either partner (both q = 0.004). Paired GeoMx profiling of 22 patients characterized tumor buds as pEMT-high, less differentiated regions. In CPTAC-HNSCC, RNA–protein correlations were 0.862 for EGFR and 0.764 for TROP2 (n = 108), and 0.404 for HER3 (n = 84). EGFR was positively associated with bulk pEMT, and HER3/TROP2 with differentiation, at both molecular levels. An EGFR-relative transcript-balance index correlated with bulk pEMT in 273 TCGA-HNSC tumors after purity adjustment (partial ρ = 0.453). Synthetic mixtures showed that both cell state and composition can produce this pattern. Conclusions: Malignant-cell state is an important dimension of therapeutic-target heterogeneity in HNSCC. The reproducible transcript-level contrasts support integrating quantitative target-protein measurements with independent cell-state markers in prospective biomarker studies of EGFR-, HER3- and TROP2-directed ADCs.