Circulating Hsp70 Reflects Tumor Burden and Stage-Dependent Disease Progression Across Multiple Solid Tumor Entities

Background: Liquid biopsy-based biomarkers provide valuable insights into tumor biology, dynamics, burden, relapse prediction and therapeutic responsiveness. The stress-inducible heat shock protein 70 (Hsp70), which is frequently overexpressed in highly aggressive solid tumors and is presented on the cell membrane of tumors but not normal cells, is found in the circulation either as a free protein originating from dying cells or in the context of extracellular vesicles (EVs) that are actively released by viable tumor cells. This study demonstrates the potential value of circulating Hsp70 (eHsp70) levels across multiple solid tumor entities as an entity- and stage-dependent diagnostic biomarker reflecting tumor burden and disease stage. Methods: Circulating eHsp70 levels, as determined using the Hsp70-exo ELISA which detects free and EV-associated Hsp70, in plasma samples collected from patients with different tumor entities (n = 389) prior to the initiation of any oncological therapy and healthy controls (n = 108) between 2021 and 2025, were analyzed retrospectively. Tumor stages were categorized as early, locally advanced, or metastatic. The Kruskal-Wallis test was used for group comparisons and the Receiver Operating Characteristic (ROC) curve was used to evaluate the diagnostic performance of eHsp70 levels. DeLong's test was used to calculate differences between AUC values. Results: In tumor patients (n = 389), circulating eHsp70 levels were significantly higher than those in healthy controls (n = 108) (Kruskal-Wallis, p < 0.001). eHsp70 levels progressively increased from early-stage to locally advanced and metastatic disease in a stage-dependent manner. Although ROC analysis demonstrated the limited discriminatory performance of eHsp70 levels in early-stage disease (AUC 0.569), increased discrimination was apparent in locally advanced disease (AUC 0.751), metastatic tumors (AUC 0.784) and combined advanced tumor diseases (AUC 0.765; significant by DeLong's Test comparing early-stage to locally advanced and metastatic tumors), irrespective of the tumor entity with the highest AUC values in metastatic breast cancer (AUC 0.872), sarcoma (AUC 0.861) and non-small cell lung cancer (NSCLC) (AUC 0.835). Apart from minor entity-specific differences, the correlation of eHsp70 levels with the tumor stage remained consistent across all measured tumor entities. Conclusions: Circulating eHsp70 levels are markedly elevated in patients with highly malignant solid tumors and show a consistent, stage-dependent increase across multiple tumor types. These findings suggest that circulating eHsp70, as an indicator of tumor-associated cellular stress and overall tumor burden, represents a valuable biomarker for assessing disease stage, monitoring disease progression, and evaluating therapeutic responses.