Calreticulin and heat shock protein 70 expression, immune infiltration, and prognostic stratification in endometrial cancer: a retrospective cohort study

Abstract

Background: Accessible biomarkers are needed to complement conventional clinicopathological risk assessment in endometrial cancer. Calreticulin (CALR) and heat shock protein 70 (HSP70) are stress-related proteins involved in immunogenic cell death and tumor–immune interactions, but their clinicopathological and prognostic relevance remains unclear. Methods: This retrospective cohort included 286 surgically treated patients who underwent surgery between January 2016 and December 2023, with administrative follow-up through December 31, 2025. CALR and HSP70 expression was assessed by immunohistochemistry and classified as low or high using prespecified semiquantitative thresholds. CD8+ and CD68+ cell densities were evaluated as immune infiltration markers. Associations with adverse pathological features were examined using logistic regression with false-discovery rate correction. Recurrence-free survival (RFS), defined as recurrence or death, and overall survival (OS) were analyzed using Kaplan-Meier curves and Cox regression. Clinical and integrated models were compared for 3-year recurrence prediction with bootstrap validation. Results: High CALR and high HSP70 expression were independently associated with grade 3 disease, deep myometrial invasion, lymphovascular space invasion, lymph node metastasis, advanced clinicopathological stage, and a secondary adverse pathology summary endpoint after false-discovery rate correction. High expression of either marker was associated with lower CD8+ and higher CD68+ density. In multivariable RFS models including adjuvant treatment, HSP70-high expression remained associated with poorer RFS (adjusted HR 1.85, 95% CI 1.23–2.79; P = 0.003), whereas CALR-high expression did not. The CALR-high/HSP70-high phenotype was also associated with poorer RFS (adjusted HR 2.30, 95% CI 1.36–3.88; P = 0.002). OS models were exploratory because of limited deaths, although associations for HSP70-high expression and the double-high phenotype were directionally consistent. For 3-year recurrence prediction, the integrated model showed a small apparent AUC increase over the clinical model (0.779 vs. 0.763; DeLong P = 0.350), which was not retained after bootstrap optimism correction (0.717 vs. 0.720). Conclusion: CALR and HSP70 expression were associated with aggressive pathology and an immune profile characterized by reduced CD8+ and increased CD68+ infiltration. HSP70 and the CALR-high/HSP70-high phenotype showed the most consistent RFS associations, whereas OS and prediction-model findings require cautious interpretation.