Circulating Hsp70: a tumor biomarker for lymph node metastases and early relapse in thoracic cancer

Abstract

Background: Heat shock protein 70 (Hsp70) which is frequently overexpressed in many different cancer types is also present on the plasma membrane of tumor but not normal cells. The intensity of membrane-expressed Hsp70 (mHsp70) is associated with disease progression and treatment resistance. It has also been shown that Hsp70 can be actively released into the circulation by mHsp70 positive, viable tumor cells in the form of extracellular lipid microvesicles expressing mHsp70, the levels of which might therefore act as a potential biomarker for tumor aggressiveness in lung malignancies.

Methods: Extracellular Hsp70 (eHsp70) was measured in the plasma of patients with non-small cell lung cancer (n = 178, NSCLC) and lung metastases of extrathoracic tumors (n = 35) prior to surgery using the Hsp70-exo ELISA which detects microvesicle-associated eHsp70 and the patient`s immunophenotype was determined by flow cytometric analysis of the corresponding peripheral blood lymphocytes.

Results: eHsp70 values were significantly higher in patients with NSCLC than in healthy individuals, with no differences between adeno and squamous cell carcinomas. Levels of circulating eHsp70 which are associated with the Programmed cell death protein 1 (PD-L1) status, gradually increased from early stage to metastatic disease, and patients with lymph node metastases in surgically treatable NSCLC had significantly higher eHsp70 levels than nodal negative patients. In all tumor stages, total lymphocyte counts were significantly reduced and immunoregulatory T (Treg) cell counts were increased compared to healthy controls. Lower CD4 + T helper cell and higher CD3-/CD56+/CD94+/CD69+/NKp30+/NKp46 + NK cell ratios were only found in patients with thoracic metastases of other primary tumors. An early relapse after complete resection with curative intent correlated with significantly elevated eHsp70 levels which were measured prior to surgery, in all thoracic cancer patients.

Conclusions: In summary, we propose circulating eHsp70 levels before any treatment as a predictive biomarker for the presence of lymph node metastases and early therapy failure in patients with thoracic malignancies.

Keywords: Biomarker; Extracellular Hsp70; Immune-phenotype; Liquid biopsy; NSCLC.

Fig. 1

Flow diagram summarizing characteristics (tumor stage, histology, lymph node status, early relapse) of patients with non-small cell lung cancer (NSCLC) in different stages and lung metastases of extrathoracic primary tumors included in this study

Fig. 2

Free and vesicular eHsp70 (ng/mL) levels measured in the plasma of healthy individuals (n = 108, Healthy) and NSCLC patients (n = 178, NSCLC) with adeno (n = 61, adeno) and squamous cell (n = 59, Squamous) carcinoma subtypes. Statistically significant differences ***p < 0.001

Fig. 3

A Free and vesicular eHsp70 (ng/mL) levels measured in the plasma of healthy individuals (n = 108, Healthy), and patients with early (n = 36, Early), locally advanced (n = 89, Locally advanced) and advanced (n = 53, Advanced) NSCLC. Statistically significant differences *p < 0.05, **p < 0.01, ***p < 0.001. B ROC analysis illustrating the sensitivity and specificity of eHsp70 values in patients with NSCLC in locally advanced and advanced stages III and IV compared to healthy donors: AUC value: 0.83, sensitivity: 0.73, specificity: 0.78 at an optimal threshold value of 49.48 ng/mL (“closest top left method”)

Fig. 4

A Free and vesicular eHsp70 (ng/mL) levels measured in the plasma of NSCLC patients without (n = 32, N0) and with (n = 15, N+) pathologically proven lymph node metastases. Statistically significant differences ***p < 0.001. B ROC analysis illustrating the sensitivity and specificity of eHsp70 values in NSCLC patients with and without pathological proven lymph node metastases. AUC value: 0.82, sensitivity: 0.8, specificity: 0.71 at an optimal threshold value of 40.45 ng/mL (“closest top left method”). C Free and vesicular eHsp70 (ng/mL) levels measured in the plasma of healthy individuals (n = 108, Healthy), stage IV NSCLC patients (n = 53, NSCLC IV), and patients with lung metastases (n = 35, Thoracic metastases) of extrathoracic primary tumors. Statistically significant differences *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Proportions of lymphocyte subsets in the peripheral blood of healthy donors (n = 32), NSCLC patients with early (n = 26), locally advanced (n = 26), advanced (n = 15) tumors, and patients with lung metastases (Thoracic metastases) of extrathoracic primary tumors (n = 25). ACD45 + lymphocyte counts, B CD3-/CD19 + B cells, C CD3 + T cells, CD3+/CD4 + T helper cells, CD3+/CD8 + cytotoxic T cells, DCD3+/CD4+/FoxP3 + Treg cells, CD3+/CD8+/FoxP3 + Treg cells, E CD3+/CD56 + NKT cells, CD3+/CD56+/CD94 + NKT cells, CD3+/CD56+/CD69 + NKT cells, CD3+/CD56+/NKG2D + NKT cells, F CD3-/CD56 + NK cells, CD3-/CD56+/CD94 + NK cells, CD3-/CD56+/CD69 + NK cells, CD3-/CD56+/NKG2D + NK cells, CD3-/CD56+/NKp30 + NK cells, CD3-/CD56+/NKp46 + NK cells. Statistically significant differences *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001

Fig. 5

Proportions of lymphocyte subsets in the peripheral blood of healthy donors (n = 32), NSCLC patients with early (n = 26), locally advanced (n = 26), advanced (n = 15) tumors, and patients with lung metastases (Thoracic metastases) of extrathoracic primary tumors (n = 25). ACD45 + lymphocyte counts, B CD3-/CD19 + B cells, C CD3 + T cells, CD3+/CD4 + T helper cells, CD3+/CD8 + cytotoxic T cells, DCD3+/CD4+/FoxP3 + Treg cells, CD3+/CD8+/FoxP3 + Treg cells, E CD3+/CD56 + NKT cells, CD3+/CD56+/CD94 + NKT cells, CD3+/CD56+/CD69 + NKT cells, CD3+/CD56+/NKG2D + NKT cells, F CD3-/CD56 + NK cells, CD3-/CD56+/CD94 + NK cells, CD3-/CD56+/CD69 + NK cells, CD3-/CD56+/NKG2D + NK cells, CD3-/CD56+/NKp30 + NK cells, CD3-/CD56+/NKp46 + NK cells. Statistically significant differences *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001

Fig. 6

Free and vesicular eHsp70 (ng/mL) levels measured in the plasma of A NSCLC patients (NSCLC) and B patients with lung metastases (Thoracic metastases) of extrathoracic primary tumors without (No relapse) (n = 23, NSCLC; n = 10, Thoracic metastases) and with (Relapse) (n = 4, NSCLC 6 months; n = 7, Thoracic metastases 12 months) early relapse. Statistically significant differences *p < 0.05