Circulating Hsp70 Levels and the Immunophenotype of Peripheral Blood Lymphocytes as Potential Biomarkers for Advanced Lung Cancer and Therapy Failure after Surgery

Seyer Safi, Luis Messner, Merten Kliebisch, Linn Eggert, Ceyra Ceylangil, Philipp Lennartz, Benedict Jefferies, Henriette Klein, Moritz Schirren, Michael Dommasch, Dominik Lobinger, Gabriele Multhoff, Seyer Safi, Luis Messner, Merten Kliebisch, Linn Eggert, Ceyra Ceylangil, Philipp Lennartz, Benedict Jefferies, Henriette Klein, Moritz Schirren, Michael Dommasch, Dominik Lobinger, Gabriele Multhoff

Abstract

Lung cancer remains a devastating disease with a poor clinical outcome. A biomarker signature which could distinguish lung cancer from metastatic disease and detect therapeutic failure would significantly improve patient management and allow for individualized, risk-adjusted therapeutic decisions. In this study, circulating Hsp70 levels were measured using ELISA, and the immunophenotype of the peripheral blood lymphocytes were measured using multiparameter flow cytometry, to identify a predictive biomarker signature for lung cancer patients pre- and post-operatively, in patients with lung metastases and in patients with COPD as an inflammatory lung disease. The lowest Hsp70 concentrations were found in the healthy controls followed by the patients with advanced COPD. Hsp70 levels sequentially increased with an advancing tumor stage and metastatic disease. In the early-recurrence patients, Hsp70 levels started to increase within the first three months after surgery, but remained unaltered in the recurrence-free patients. An early recurrence was associated with a significant drop in B cells and an increase in Tregs, whereas the recurrence-free patients had elevated T and NK cell levels. We conclude that circulating Hsp70 concentrations might have the potential to distinguish lung cancer from metastatic disease, and might be able to predict an advanced tumor stage and early recurrence in lung cancer patients. Further studies with larger patient cohorts and longer follow-up periods are needed to validate Hsp70 and immunophenotypic profiles as predictive biomarker signatures.

Keywords: advanced COPD; biomarker; circulating Hsp70; early recurrence; immunophenotypic profile; lung cancer; surgery.

Conflict of interest statement

The authors declare there is no conflict of interest. G.M. is the founder and Chief Scientific Officer of multimmune GmbH, Munich, Germany. The funders had no role in the design of the study, in the collection, analyses, or interpretation of the data, in the writing of the manuscript or in the decision to publish the results.

Figures

Figure 1
Figure 1
Patient flow diagram of the healthy controls, lung tumor (LT) and advanced-COPD patients. Blood samples were taken from patients with adeno carcinoma (ADENO-CA), squamous cell carcinoma of the lung (SCC), other lung cancers (OTLC) and lung metastases (MET) before (Pre) and three months (3m Post) post-operatively. Patients were divided into a recurrence-free (RFC) and early recurrent (RC) cohort. The numbers in parentheses indicate the number of blood samples which were available for Hsp70 measurements using ELISA and for immunophenotyping using multiparameter flow cytometry.
Figure 2
Figure 2
Circulating Hsp70 levels in (a) healthy individuals (Healthy, n = 108), patients with adeno carcinoma of the lung (ADENO-CA, n = 17), squamous cell carcinoma (SCC, n = 7) of the lung, other types of lung cancers (OTLC, n = 8), lung metastases (MET, n = 17) and advanced COPD (n = 18). (b) Hsp70 levels in the healthy control cohort (n = 108) and primary lung tumor patients in stage I (n = 7), II (n = 5), III (n = 11) and IV (n = 9). * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 3
Figure 3
Measurement of Hsp70 in the peripheral blood of healthy individuals (n = 108), patients with primary lung tumors in stages I-III (n = 6), stage IV (n = 3) and lung metastases (MET, n = 7), before (Pre) and three months (Post) post-operatively, * p < 0.05.
Figure 4
Figure 4
Measurement of Hsp70 levels in the peripheral blood of healthy human volunteers (Healthy, n = 108) and lung cancer patients before (Pre) and three months after (Post) surgery in a recurrence-free (RFC, n = 10) and early recurrence (RC, n = 6) cohort, * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Representative example of a gating strategy to identify lymphocyte subpopulations (a) such as CD3−/CD19+ B cells (c), CD3−/CD56+ NK cells (d), CD3+/CD45+ leukocytes (e), CD3+/CD4+ T helper (e), CD3+/CD8+ cytotoxic T cells (e), CD4+ and CD8+ CD3+/CD25+/FoxP3+ Treg cells (e). (b) negative control gating, red circle, lymphocyte gate; green box, gated cell population for subpopulation analysis.
Figure 6
Figure 6
Composition of lymphocytes and lymphocyte subpopulations in the peripheral blood of the healthy volunteers (Healthy, n = 16) and in a cohort of recurrence-free (RFC, n = 8) and early recurrence (RC, n = 6) lung cancer patients before (Pre) and after (Post) surgery, as determined by using multiparameter flow cytometry. (a) The proportion of CD45+ lymphocytes in the healthy volunteers, pre-surgery recurrence-free (Pre RFC), post-surgery recurrence-free (Post RFC), pre-surgery early recurrence (Pre RC) and post-surgery early recurrence (Post RC) lung cancer patients. (b) The proportion of CD19+ B cells in all the indicated groups. (c) The proportion of CD3+ T cells, CD4+ T helper cells and CD8+ cytotoxic T cells in all the indicated groups. (d) The proportion of CD4+ and CD8+ CD3+/CD25+/FoxP3+ Treg cells in all the indicated groups. (e) The proportion of different NK cell subpopulations including CD3−/CD16+ (CD16), CD3−/NKG2D+ (NKG2D), CD3−/CD56+ (CD56), CD3−/NKp30+ (NKp30), CD3−/NKp46+ (NKp46), CD3−/CD69+ (CD69) and CD3−/CD94+ (CD94) in all the indicated groups (* p < 0.05, ** p < 0.01, **** p < 0.0001).

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