HER2 overexpression/amplification in colorectal cancer: spatial intratumoral heterogeneity as a key determinant of detection discordance and therapy response.

HER2-targeted therapy is increasingly used in colorectal cancer (CRC). However, discrepancies between conventional detection methods (immunohistochemistry [IHC]/fluorescence in situ hybridization [FISH]) and next-generation sequencing (NGS) remain to be elucidated. Furthermore, the impact of spatial intratumoral heterogeneity (SIH) in HER2 overexpression on both the methodological concordance and therapeutic efficacy is still poorly understood. Two consecutive retrospective cohorts from Peking Union Medical College Hospital (2018-2024) were analyzed. In the NGS cohort (n = 1,343), HER2-amplified cases were identified based on NGS-derived gene copy number (GCN), and their clinicopathological and molecular features were characterized using stage-matched HER2-non-amplified controls. In the IHC cohort (n = 3,585), spatial HER2 expression heterogeneity was comprehensively assessed. Finally, treatment outcomes were reviewed in four HER2-amplified patients who received anti-HER2 therapy. HER2 amplification was detected in 2.53% (34/1,343) of tumors and defined a distinct molecular subset characterized by fewer RAS/KRAS mutations and more frequent CDK12 and TP53 alterations. Concordance between NGS and IHC was higher when applying ToGA criteria (κ = 0.85) compared to the HERACLES criteria, particularly in cases with high-level amplifications (GCN ≥ 10). Notably, intrablock heterogeneity accounted for 82% of discordant cases, underscoring a major biological source of testing variability. In the IHC cohort, HER-2 positivity was defined as ≥ 10% 3 + staining or 2 + in IHC with amplification confirmed by FISH. Overall, 1.62% of CRCs were HER2-positive, of which 24.14% exhibited intrablock heterogeneity, and 31.25% demonstrated SIH across tumor blocks or between primary and metastatic sites. Among four patients treated with anti-HER2 therapy, two with relatively limited HER2 expression heterogeneity showed sustained CEA decline and stable disease (SD), whereas the two with marked heterogeneity demonstrated fluctuating CEA levels and disease progression, even in the presence of high-level amplification. HER2-amplified/overexpressed CRC constitutes a biologically distinct entity frequently characterized by SIH. This heterogeneity represents a key determinant of inter-method discordance and may influence response to HER2-targeted therapy. These findings support integrating spatial assessment into HER2 testing algorithms and warrant prospective validation of its predictive value.