Integrated molecular and histopathological analysis reveals genotype–phenotype correlations and diagnostic challenges in endometrial stromal sarcoma

Endometrial stromal sarcomas (ESSs) comprise a heterogeneous group of uterine mesenchymal tumors characterized by diverse morphological features and recurrent molecular alterations. Although molecular classification has substantially improved ESS diagnosis, the relationship between specific genetic alterations and morphological phenotypes remains incompletely understood. We performed an integrated molecular and histopathological analysis of ESS to investigate genotype-phenotype correlations and refine tumor classification. A total of 61 patients with histologically suspected ESSs were initially enrolled, and 51 successfully underwent targeted RNA sequencing, followed by comprehensive morphological reassessment, immunohistochemical evaluation, molecular validation, and clinicopathological correlation. Molecularly confirmed ESSs demonstrated distinct genotype-associated morphological patterns. PHF1-rearranged low-grade ESSs showed a strong association with hypocellular growth and prominent collagenized stroma. Quantitative cellularity analysis further demonstrated significantly lower tumor cellularity in PHF1-rearranged low-grade ESSs compared with JAZF1::SUZ12-rearranged tumors (p < 0.001). High-grade ESSs with YWHAE::NUTM2 and BCOR::ZC3H7B alterations showed higher mitotic activity than low-grade ESSs and demonstrated tongue-like infiltration patterns in both primary and metastatic lesions, indicating that this growth pattern, traditionally emphasized in low-grade ESSs, may represent a broader morphological feature of ESSs. Molecular reassessment led to the reclassification of 10 cases (19.6%, 10/51), identifying tumors with alternative molecular alterations that were initially diagnosed as ESSs based on morphology alone. Among these cases, a uterine sarcoma harboring a novel GATA6::SRF fusion exhibited a low-grade ESS-like morphological phenotype but lacked the characteristic molecular alterations of conventional ESSs, suggesting a potential molecularly defined uterine sarcoma subtype requiring further investigation. Our findings demonstrate that integrated molecular and histopathological analysis extends beyond diagnostic refinement by revealing genotype-phenotype relationships, expanding the molecular spectrum of ESSs and ESS-like uterine sarcomas, and providing new insights into the biological heterogeneity of these rare tumors.