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

Circulating Hsp70 - a tumor biomarker for lymph node metastases and early relapse in thoracic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Circulating Hsp70 - a tumor biomarker for lymph node metastases and early relapse in thoracic cancer Dominik Lobinger, Nicholas Taylor, Verena Messner, Sophie Seier, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4707407/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version 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 Hsp70 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 Hsp70 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 Treg 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 lung cancer patients. Conclusions: In summary, we propose circulating eHsp70 levels as a predictive biomarker for the presence of lymph node metastases and early therapy failure in patients with thoracic malignancies. NSCLC extracellular Hsp70 biomarker liquid biopsy immune-phenotype Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases, continues to be the cancer with the highest mortality rate worldwide (1). With 22,590 female and 34,100 male cases registered by the Robert Koch Institute in Germany in 2020, lung cancer remains among the most frequently newly diagnosed types of cancer in the last decade (2). Upon initial presentation, about two thirds of patients are diagnosed with metastatic NSCLC (3, 4). Survival rates are heavily influenced by the tumor stage, which besides tumor size, is determined by the presence and extent of lymphatic and distant metastases (5, 6). In early-stage NSCLC, complete resection with curative intent with systematic lymph node dissection is the standard treatment, whereas in locally advanced and metastatic NSCLC, multimodal therapies are tailored in multidisciplinary tumor boards (7-9). Large international randomized trials have demonstrated a reduction of lung cancer mortality by low-dose computed tomography (LDCT) screening, probably by increasing the proportion of cases diagnosed in earlier tumor stages (10, 11). Although LDCT has a high sensitivity for the detection of pulmonary lesions, the specificity of LDCT in differentiating a pulmonary lesion in malignant or benign disease is limited. A national lung cancer screening program for patients with specific risk factors is presently planned in Germany (12). The better classification of uncertain findings from LDCT screening, which increases the number of patients entering curative stages, requires the identification of a tumor marker which is able to determine tumor aggressiveness, especially given the rapidly growing numbers of neoadjuvant and perioperative treatment options (8, 9). With this in mind, we investigated the potential of circulating extracellular heat shock protein 70 (eHsp70), together with the immunophenotype of peripheral blood lymphocytes, as a potential tumor biomarker approach in thoracic oncology. Hsp70 (HSPA1A) plays an important role in maintaining protein homeostasis by supporting the folding and unfolding of nascent polypeptides, protein transport and preventing protein aggregation in all nucleated cells (13-15). In malignant cell types, Hsp70 is often highly overexpressed and presented on the plasma membrane in a tumor-specific manner (16). Furthermore, membrane Hsp70 (mHsp70) positive tumor cells actively release Hsp70 in the context of extracellular lipid microvesicles (17). Depending on the localization of Hsp70 in tumor cells, its role can be contradictory: whereas elevated intracellular levels are associated with an increased resistance to chemo- or radiotherapy (18), membrane-bound and circulating extracellular Hsp70 (eHsp70) can mediate anti-tumor and immunomodulatory effects. Previous studies have only explored free circulating Hsp70 as a tumor biomarker (19, 20), whereas we have developed the Hsp70-exo sandwich ELISA which is able to measure not only free Hsp70 derived from dying cells, but also Hsp70 that is actively released from living tumor cells in lipid microvesicles (21-24). The aim of this study was to investigate the potential value of eHsp70 levels, as measured using the Hsp70-exo ELISA, and the peripheral blood immunophenotype, as determined by multiparameter flow cytometry, as biomarkers for estimating the likelihood of a lung lesion being malignant and assessing disease aggressiveness in thoracic malignancies. Methods Patient cohort The patient cohort consisted of a total of 178 patients diagnosed with NSCLC (n = 178) and 35 patients with lung metastases (n = 35) of extrathoracic primary tumors (Fig. 1 ). Among the NSCLC patients, 53 (Supplementary Table 1) had undergone surgical resection with curative intent between 2020 and 2023 at the University Hospital of the Technical University of Munich (TUM) and at the München Klinik Bogenhausen, an academic teaching hospital of TUM. The male to female ratio of the patients was 1.3 to 1. Samples from 108 age-matched healthy donors were additionally included in the analyses. Patient characteristics are summarized in the flow diagram (Fig. 1 ). All EDTA blood samples for analyzing eHsp70 levels were obtained prior to surgery. The study was approved by the local ethics committee of the TUM School of Medicine and Health (TUM 2403/09 and 2428/09), and written informed consent was obtained from all patients and healthy donors before start of the study. Blood sampling was conducted in accordance to the guidelines of the Declaration of Helsinki. Hsp70-exo sandwich ELISA Plasma derived from EDTA anti-coagulated blood (S-Monovette, Sarstedt, Nümbrecht, Germany) was prepared by a centrifugation at 1500xg for 15 minutes at room temperature, followed by aliquoting (300 µL) and storage of the plasma samples at − 80°C. For the Hsp70-exo sandwich ELISA, 96-well MaxiSorp Nunc-Immuno plates (Thermo, Rochester, NY, USA) were incubated overnight with the cmHsp70.2 coating monoclonal antibody (mAb, 1 µg/mL; multimmune GmbH Munich, Germany) in sodium carbonate buffer (0.1 M sodium carbonate, 0.1 M sodium hydrogen carbonate, pH 9.6; Sigma-Aldrich (Merck, Darmstadt, Germany)) at room temperature. After a washing step in phosphate-buffered saline (PBS) containing 0.05% v/v Tween-20 (Life Technologies, Darmstadt, Germany; Calbiochem, Merck, Darmstadt, Germany) and blocking with liquid plate sealer (Candor Bioscience GmbH, Wangen i. Allgäu, Germany) plasma samples (100 µL) were diluted in StabilZyme Select (1:5; Diarect GmbH, Freiburg i. Breisgau, Germany). An 8-point Hsp70 protein standard (1-100 ng/mL) was also pre-diluted in StabilZyme Select (1:5). After an incubation for 30 minutes at room temperature and another washing, plates were incubated for 30 minutes with biotinylated cmHsp70.1 detection mAb (200 ng/mL; multimmune GmbH, Munich, Germany) dissolved in HRP-Protector™ (Candor Bioscience GmbH, Wangen i. Allgäu, Germany). Subsequently, plates were incubated for another 30 minutes with Streptavidin (57 ng/mL; Senova GmbH, Weimar, Germany) in HRP-Protector™ (Candor Bioscience GmbH, Wangern i. Allgäu, Germany). Colorimetric analysis was performed after adding the substrate reagent (100 µL; BioFX TMB Super Sensitive One Component HRP Microwell Substrate, Surmodics, Inc., Eden Prairie, MN, USA) for 15 minutes. After quenching the reaction by adding 2N H 2 SO 4 (50 µL), the absorbance was read at 450 nm using a Microplate Reader (VICTOR X4 Multilabel Plate Reader, PerkinElmer, Waltham, MA, USA), after a correction of the reference absorbance at 570 nm. Multiparameter flow cytometry of the immunophenoptype in EDTA blood Freshly taken anti-coagulated EDTA blood (100 µL) was immunophenotyped using a BD FACSCalibur™ flow cytometer (BD Biosciences, Heidelberg, Germany) and the following fluorescence antibody combinations. Combining four different fluorescence-labeled antibodies (FITC/PE/PerCP/APC) allowed the following lymphocyte subpopulations to be determined: CD45 + lymphocytes, CD3-/CD19 + B cells, CD3+/CD45 + T cells, CD3+/CD4 + helper T cells, CD3+/CD8 + cytotoxic T cells, CD3+/CD69 + activated T cells, CD3+/CD4+/CD25+/FoxP3 + and CD3+/CD8+/CD25+/FoxP3 + regulatory T cells (Treg), CD3+/CD56 + NK-like T cells (NKT), CD3+/CD94 + NKT cells, CD3+/CD69 + NKT, CD3+/NKG2D + NKT, CD3-/CD56 + NK cells, CD3-/CD94 + NK cells, CD3-/CD69 + activated NK cells, CD3-/NKG2D + NK cells, CD3-/NKp30 + NK cells, CD3-/NKp46 + NK cells. The antibody combinations and fluorescence dyes used in this study have been described previously ( 25 ). Isotype-matched control antibodies were used as negative controls. The respective percentage of a certain lymphocyte subpopulation is defined as the proportion of cells stained positively for a specific antibody within a defined viable lymphocyte gate. Statistical analysis Comparisons among different groups were conducted using ANOVA and post-hoc Tukey tests, and non-parametric data were analyzed using the Kruskal Wallis test, as appropriate. Normal distribution was tested by the Shapiro Wilk normality test. Statistical analysis was performed using the programming language R, R studio version 2024.04.2 + 764. Differences are considered to be statistically significant as follows: not significant, *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.00001. Results Patient characteristics NSCLC patients (n = 178) were classified into the following disease groups: early (n = 36, stages I and II), locally advanced (n = 89, stage III) and advanced (n = 53, stage IV) (Fig. 1). The flow diagram (Fig. 1) shows that 25 of the 178 NSCLC patients were diagnosed with stage I disease (n = 25, stage I; n = 16, stage IA; n = 9, stage IB), 11 with stage II disease (n = 11, stage II), 89 with stage III disease (n = 89, stage III; n = 49, stage IIIA; n = 40, stage IIIB), and 53 with stage IV disease (n = 53, stage IV). Sixty-one NSCLC patients had an adeno (n = 61, adeno) and 59 a squamous cell carcinoma (n = 59, squamous) (Fig. 1). Of the patients with surgically treated NSCLC (n = 53), 32 had no lymph node metastases (n = 32, N0) and 15 had multiple lymph node metastases (n = 15, N+). An early relapse was determined in 4 NSCLC patients (n = 23, no early relapse) after 6 months, and in 7 patients with lung metastases of extrathoracic primary tumors (n = 10, no early relapse) after 12 months (Fig. 1). Circulating eHsp70 concentrations in patients with adeno and squamous cell NSCLC compared to healthy controls A comparison of circulating Hsp70 levels measured with the Hsp70-exo ELISA in all NSCLC patients across all four tumor stages prior to the start of any treatment (n = 178, median 125.4 ng/mL; ***p < 0.001 ) demonstrated significantly higher plasma levels of Hsp70 in the tumor patients compared to the healthy control group (n = 108, median 16.4 ng/mL), as illustrated in Fig. 2. No difference in eHsp70 levels was found between patients with adeno (n = 61, adeno) or squamous cell carcinoma (n = 59, squamous). Patients with adeno and squamous cell carcinoma showed significantly higher eHsp70 values in the circulation ( ***p < 0.001 ) than the control group (Fig. 2). With respect to the peripheral blood immunophenotype, no significant differences were observed in the lymphocyte subpopulations of adeno and squamous cell carcinoma (data not shown). Patients with carcinoma precursors such as lepidic carcinoma (n = 2, median 15.5 ng/mL) and low-grade neuroendocrine tumors such as typical carcinoids (n = 2, median 7.7 ng/mL) exhibited considerably lower eHsp70 values than patients with advanced NSCLC. Circulating eHsp70 concentrations in patients with early, locally advanced and advanced NSCLC Circulating eHsp70 levels increased steadily from early (n = 36, stage I and II, median 37.35 ng/mL) through locally advanced (n = 89, stage III, median 137.2 ng/mL) to advanced stage with distant metastases (n = 53, stage IV, median 199.5 ng/mL), as shown in Fig. 3A. Significant different levels were found between the healthy control group (n = 108, median 16.4 ng/mL) and patients with locally advanced tumors and advanced tumors (***p = 0.001, each ), as well as between early stages and locally advanced (*p = 0.05) and advanced NSCLC cases (**p = 0.01 ). A ROC analysis of circulating eHsp70 levels in locally advanced (stage III) and advanced (stage IV) NSCLC patients compared to healthy donors yielded an AUC value of 0.83, a sensitivity of 0.73 and a specificity of 0.78 at an optimal threshold value of 49.48 ng/mL (“closest top left method”) (Fig. 3B). A comparison of the Hsp70 values in the tumor stages I (n = 25, median 27 ng/mL), II (n = 11, median 287.4 ng/mL), III (n = 89, median 137.2 ng/mL) and IV (n = 53, median 199.5 ng/mL) revealed significant differences between the control group (median 16.4 ng/mL) and grade III and IV tumors ( ***p < 0.001, each ), as well as grade I tumors and grade III and IV tumors ( **p < 0.01, each ) (Supplementary Fig. 1A). A more detailed breakdown confirms the trend described before, with significant differences between the healthy donors and stage IIIA (n = 49, median 165.3 ng/mL), IIIB (n = 40, median 88.1 ng/mL) and IV (n = 53, median 199.5 ng/mL, ***p < 0.001, each ) NSCLC. A noticeable distinction was observed between stage IA (n = 16, median 26.4 ng/mL) and stages IIIA and IV disease (each *p < 0.05 ) (Supplementary Fig. 1B). A correlation of the eHsp70 values with the largest pathologically measured NSCLC diameter after surgery showed no obvious correlation (n = 47, R 2 = 0.0616, mean diameter 3.26 cm) (Supplementary Fig. 2). Circulating eHsp70 concentrations in NSCLC patients with and without lymph node metastases In NSCLC patients that underwent surgery with curative intent, significantly higher eHsp70 values ( ***p < 0.001) were present in the subgroup of patients with lymph node metastases (n = 15, median 297.2 ng/mL), compared to those with tumor free lymph nodes (n = 32, median 23.34 ng/mL) (Fig. 4A). The ROC analysis of those cases with and without pathological lymph node involvement obtained an AUC value of 0.82 with an achieved sensitivity of 0.8 and a specificity of 0.71 for the prediction of lymph node involvement at an optimal threshold value of 40.45 ng/mL (“closest top left method”), (Fig. 4B). In patients with lymph node metastases, increased eHsp70 levels (n = 15, median 297.2 ng/mL) were predominantly found in higher tumor grades: (G3 or micropapillary/solid in 40%; G2 or acinar/papillary in 33%; G1 or lepidic in 0%; other in 26.67%), whereas in patients without lymphatic involvement low eHsp70 levels (n = 32, median 23.81 ng/mL) were predominantly found in lower tumor grades (G3 or micropapillary/solid in 29%; G2 or acinar/papillary in 41.9%; G1 or lepidic in 19.4%; other in 9.7%). PD-L1 expression, as determined by immunohistochemistry, were associated with lower eHsp70 levels. In patients with PD-L1 negative tumors (n = 17), the median eHsp70 level was 277.7 ng/mL and in those with PD-L1 positive tumors (n = 12), the median Hsp70 level was 21.21 ng/mL ( p < 0.05 ). These data indicate that eHsp70 levels might not be related to the PD-L1 expression. Circulating eHsp70 concentrations and peripheral blood immunophenotype in lung cancer patients Similar to patients with stage IV NSCLC (n = 53, median 199.5 ng/mL), patients with lung metastases of different extrathoracic primary tumors (n = 35, median 28.6 ng/mL; *p < 0.05 ) (Supplementary Table 2) also had significantly higher eHsp70 levels in the circulation than healthy controls (Fig. 4C). Immunophenotyping of the peripheral blood lymphocytes by multiparameter flow cytometry revealed significantly lower lymphocyte counts (Fig. 5A) and significantly increased immunoregulatory T (Treg) cell ratios in all tumor stages (Fig. 5D). Ratios of CD4 + T helper cells (Fig. 5C) was decreased and that of CD3-/CD56+/CD94+/NKp30+/NKp46 + NK cells (Fig. 5F) was increased in patients with thoracic metastases of other primary tumors, as compared to healthy controls (Fig. 5A-F). Circulating eHsp70 levels in thoracic cancer patients with and without early relapse A subgroup of NSCLC patients from whom the results of the first follow-ups were available revealed significantly higher preoperative eHsp70 values in patients with early recurrence in the first 6 months post-surgery (n = 4, median 626.3 ng/mL) compared to those with no evidence of recurrence in the same period (n = 23, median 47.5 ng/mL; *p < 0.05) (Fig. 6A). A similar observation was made in a cohort of patients with primary extrathoracic tumors following pulmonary metastasectomy with curative intent. In this group, patients with a relapse within the first postoperative year displayed significantly higher preoperatively determined eHsp70 values (n = 7, median 420.8 ng/mL) compared to patients without tumor relapse (n = 10, median 23.3 ng/mL; *p < 0.05 ) (Fig. 6B). Discussion In recent years, the therapeutic landscape for NSCLC has undergone rapid transformation due to a large number of new, innovative and well-tolerated approaches, particularly in the field of immunotherapy and targeted therapies ( 8 , 26 , 27 ). These achievements have not only influenced the therapy regime for patients with advanced tumors, but are also used as adjuvant and neoadjuvant approaches in early stages with favorable chances of a long-term cure ( 28 – 30 ). Efforts have been ongoing to enhance the rate of early-stage initial diagnoses, gain insight into the tumor biology aggressiveness, and identify individuals who may benefit from a more intensive therapy regime or closer follow-up care. These endeavors have led to repeated attempts to discover suitable liquid biopsy-based biomarkers having diagnostic and prognostic significance in patients with NSCLC ( 31 , 32 ). The circulating proteins CYFRA (cytokeratin fragment) and CEA (carcinoembryonic antigen, glycoprotein), which are among the earliest biomarker candidates for NSCLC, have been examined in several prior studies with the outcome that elevated plasma levels of these circulating proteins are showing a trend towards a poorer prognosis ( 33 – 38 ). Furthermore, the presence of circulating tumor cells (CTCs), which play a role in the formation and development of metastases, has been associated with poorer prognosis in patients with NSCLC ( 39 – 41 ). Most recently, a large study has delivered the first promising results regarding the potential of small RNAs in liquid biopsies for tumor detection, albeit with a low sensitivity in early stages ( 42 ). Despite these encouraging findings regarding suitable biomarker candidates, none of the candidates have yet been established as an integral part of the diagnostic or therapeutic process in the existing guidelines for the treatment of thoracic cancers. In the context of the upcoming introduction of lung cancer screening in Germany using low-dose CT, there is a significant clinical need for more specific biomarkers that could facilitate the interpretation of inconclusive results and better inform clinical decision-making. In our study, we investigated the ability of circulating eHsp70 to predict disease stage, aggressiveness and prognosis of patients with lung cancer across different stages of disease. With the established Hsp70-exo sandwich ELISA which uses the mHsp70-specific monoclonal antibodies cmHsp70.1 (aa 451–461) and cmHsp70.2 (aa 614–623), it is possible to quantify both free Hsp70 (released predominantly by dying cells) and Hsp70 bound to lipid microvesicles (primarily derived from viable cells) in the blood ( 21 ). The results of previous studies demonstrating significantly higher eHsp70 levels in the circulation of cancer patients compared to healthy controls ( 21 , 25 , 43 ) are consistent with our present findings in patients with NSCLC across all stages and patients with lung metastases of different extrathoracic primary tumors. As a molecular chaperone, cytosolic Hsp70 maintains protein homeostasis by supporting protein folding, unfolding and transport in all nucleated cell types under physiological conditions ( 44 ). In tumor cells, Hsp70 is frequently overexpressed and can contribute to a more aggressive and therapy-resistant tumor phenotype by interfering with apoptosis and proliferation pathways ( 45 – 47 ). Many studies have shown that apart from its cytosolic localization, Hsp70 can be presented on the plasma membrane of tumor cells via a mechanism which is enabled by a tumor-specific lipid composition ( 48 ). Furthermore, an elevated mHsp70 expression is associated with higher tumor stages and a more aggressive behavior in various female cancers, prostate cancer and brain tumors ( 25 , 49 , 50 ). Tumor cells expressing mHsp70 actively release eHsp70 into the extracellular space ( 17 ). Significantly increased eHsp70 levels have also been shown in patients with NSCLC compared to healthy donors ( 43 , 51 ). Herein, we found a progressive increase in circulating eHsp70 levels starting from healthy donors, to low-grade NSCLC through locally advanced and metastatic NSCLC. The ROC analysis generated an AUC value of 0.83, with a specificity of 0.78 and a sensitivity of 0.73 at an eHsp70 cut-off value of 49.48 ng/mL for the presence of locally advanced or metastatic NSCLC compared to the healthy cohort. These findings further support the crucial function of eHsp70 in mediating tumor aggressiveness in lung cancer and the significant expression of Hsp70 in advanced tumors. A second indication of Hsp70’s role as a marker for aggressiveness emerged from the detailed examination of the surgically treated cases and their histopathological results. Although some studies suggest an association between circulating eHsp70 levels and the tumor volume ( 52 , 53 ), we found no significant association of circulating eHsp70 concentrations and the largest pathologically measured NSCLC diameter after surgical resection. However, our results revealed a clear difference ( ***p = 0.001) in the eHsp70 concentration between patients with pathologically proven lymph node metastases and those without. In the guidelines for the preoperative diagnosis of possible lymph node involvement and mediastinal staging in patients with NSCLC, PET-CT plays a decisive role as a non-invasive method. The reported literature states that the sensitivity of PET-CT for the detection of lymph node metastases is between 0.74 to 0.85, with a specificity ranging from 0.85 to 0.92, but with low accuracy in the detection of micrometastases in affected lymph nodes ( 54 – 58 ). Invasive methods such as mediastinoscopy, EBUS examination or surgical lymph node dissection can provide representative histological results ( 59 ). To our knowledge, currently no established liquid biomarker exists for the detection of lymph node metastases in the pretherapeutic setting. The ROC analysis of preoperative eHsp70 in patients undergoing surgery with curative intent yielded an AUC value of 0.82 for the presence of pathological lymph node metastases with a sensitivity of 0.8 and a specificity of 0.78 at an eHsp70 value of 40.45 ng/ml. Preoperative PET-CT results of patients with histologically confirmed lymph node metastases showed a correct clear positive finding in only 6 of 15 cases and no evidence for the presence of lymph node metastasis in 5 cases. In addition, the group with affected lymph nodes and elevated eHsp70 values exhibited a higher percentage of tumors with pathologically higher and thus more dedifferentiated grading, further suggesting a potential role of Hp70 as a marker of aggressiveness. Moreover, we could show that patients with carcinoma precursors had lower eHsp70 levels than patients with malignant tumors. In summary, we assume eHsp70 measured in the blood before start of any treatment could make a significant contribution to more precise preoperative mediastinal staging, especially in unclear cases and in combination with unclear results in PET-CT. In addition to NSCLC eHsp70 levels were also measured in patients with lung metastases derived from a different primary tumor than NSCLC. Like patients with locally advanced, metastatic NSCLC, these patients also exhibited significantly higher eHsp70 values compared to healthy controls, thereby indicating that eHsp70 could provide a universal tumor biomarker not only for NSCLC, but also for other tumor entities. The third interesting evidence for the potential of eHsp70 as a tumor biomarker of aggressiveness in thoracic oncology is provided by our follow-up data. Previous studies have already shown a link between elevated eHsp70 levels and a tendency to early tumor relapse or treatment failure ( 25 , 49 , 60 ). In our study, we could clearly demonstrate that patients who had an early NSCLC relapse in the first 6 months showed significantly higher preoperative eHsp70 values compared to patients without evidence of an early tumor relapse. Moreover, the eHsp70 values of 2 patients which were additionally determined at the time of the first follow-up after 3 months showed, that already at this early time point, the patient with recurrence showed a significantly higher value than the patient without recurrence. These observations align with our recently published study, wherein eHsp70 levels rose within three months post-surgery in early-relapse patients, whereas they remained stable in relapse-free patients ( 51 ). The predictive value of eHsp70 for the occurrence of tumor relapse could also be found in patients with thoracic metastases derived form an extrathoracic primary tumor within the first 12 months following pulmonary metastasectomy. Measuring eHsp70 levels at multiple time points during follow-up and during adjuvant therapy could further confirm the results of this study and provide interesting and dynamic clues for possible recurrence and treatment response. The immunophenotyping of the peripheral blood lymphocytes of patients with lung cancer did not provide conclusive results as a predictive tumor biomarker for tumor aggressiveness. Irrespective of the stage and tumor aggressiveness all patients suffered from a lymphopenia and the number of immunosuppressive regulatory T cells were found to be significantly increased compared to healthy controls. An increase in CD3-/CD56+/CD94+/CD69+/NKp30+/NKp46 + NK cell subsets which correlated with a significant drop in CD4 + T helper cells, appeared to be restricted to patients with lung metastases of an extrathoracic primary tumor. Since reduced CD4 + T cell counts are associated with decreased levels of pro-inflammatory cytokines in the circulation [43], we assume that despite an increase in some NK cell subsets, these effector cells are unable to mediate protective anti-tumor immunity in advanced tumors. Since we found no obvious association between circulation eHsp70 levels and tumor size, we hypothesize that the tumor aggressiveness (lymph node metastases, early relapse), rather than the size of the tumor, is responsible for the elevated circulating eHsp70 levels. Considering the increased detection of incidental pulmonary nodules by lung cancer screening, our easily measurable biomarker in liquid biopsies could contribute to the management of such unclear cases. In instances where elevated eHsp70 values are associated with ambiguous results, early histological confirmation or closer follow-up could be considered. On the other hand, measuring eHsp70 not only at the time of diagnosis, but also during the treatment could help to identify patients experiencing treatment failure and thus guide the selection of alternative treatment strategies or more intensive approaches. This is especially relevant given the increasing complexity and diversity of available modalities. Conclusions With the imminent launch of a national lung cancer screening initiative in Germany and the high number of incidental pulmonary nodules on low-dose CT, there is an urgent need for reliable biomarkers to guide subsequent diagnostic algorithms. Our data support eHsp70 as an indicator of disease aggressiveness in thoracic oncology. In particular, we observed a consistent and significant increase in eHsp70 levels with advancing tumor stages. Patients with lymph node metastases and de-differentiated tumors had significantly higher eHsp70 levels compared to patients without such disease characteristics. In addition, the association between elevated preoperative eHsp70 levels and early tumor recurrence underscores its prognostic value. Further studies are warranted to validate our findings. Declarations Ethics approval and consent to participate The study was approved by the local ethics committee of the TUM School of Medicine and Health (TUM 2403/09 and 2428/09), and written informed consent was obtained from all patients and healthy donors before start of the study. Blood sampling was conducted in accordance to the Declaration of Helsinki. Consent for publication “Not applicable” Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests AGP is CEO and GM is CSO of multimmune GmbH. A granted US patent protects the Hsp70-exo ELISA for determining free and vesicular eHsp70 in liquid biopsies. Funding The research was funded by grants of BMBF 02NUK064A, Bayerische Forschungsstiftung BAYCELLator AZ-1568-22. Authors' contributions GM and SS designed and supervised the study, NT, VM, SS, ABD, ER performed the experiments, DL, NT and VM analyzed the data, DL and NT designed the Figures and drafted the manuscript, SS, DL and JB provided the clinical samples and clinical data, GM conceptualized the study, AGP proofread the manuscript, and all authors provided feedback and approved the final version of the manuscript. Acknowledgements The authors thank Hannah Herf and Anett Lange for technical and editorial support. References (WHO) WHO. Lung cancer June 2023 [Available from: https://www.who.int/news-room/fact-sheets/detail/lung-cancer. Robert Koch Institute (ed) and the Association of Population-based Cancer Registries in Germany (ed) Berlin. Cancer in Germany 2019/2020 14th edition. Kocher F, Hilbe W, Seeber A, Pircher A, Schmid T, Greil R, et al. 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Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring. Mol Cancer. 2022;21(1):25. Rolfo C, Castiglia M, Hong D, Alessandro R, Mertens I, Baggerman G, et al. Liquid biopsies in lung cancer: the new ambrosia of researchers. Biochim Biophys Acta. 2014;1846(2):539-46. Blankenburg F, Hatz R, Nagel D, Ankerst D, Reinmiedl J, Gruber C, et al. Preoperative CYFRA 21-1 and CEA as prognostic factors in patients with stage I non-small cell lung cancer: external validation of a prognostic score. Tumour Biol. 2008;29(4):272-7. Grunnet M, Sorensen JB. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer. 2012;76(2):138-43. Kato T, Ishikawa K, Aragaki M, Sato M, Okamoto K, Ishibashi T, et al. Optimal predictive value of preoperative serum carcinoembryonic antigen for surgical outcomes in stage I non-small cell lung cancer: differences according to histology and smoking status. J Surg Oncol. 2013;107(6):619-24. Matsuoka K, Sumitomo S, Nakashima N, Nakajima D, Misaki N. Prognostic value of carcinoembryonic antigen and CYFRA21-1 in patients with pathological stage I non-small cell lung cancer. Eur J Cardiothorac Surg. 2007;32(3):435-9. Hotta K, Segawa Y, Takigawa N, Kishino D, Saeki H, Nakata M, et al. Evaluation of the relationship between serum carcinoembryonic antigen level and treatment outcome in surgically resected clinical-stage I patients with non-small-cell lung cancer. Anticancer Res. 2000;20(3B):2177-80. Hsu WH, Huang CS, Hsu HS, Huang WJ, Lee HC, Huang BS, et al. Preoperative serum carcinoembryonic antigen level is a prognostic factor in women with early non-small-cell lung cancer. Ann Thorac Surg. 2007;83(2):419-24. Ma XL, Xiao ZL, Liu L, Liu XX, Nie W, Li P, et al. Meta-analysis of circulating tumor cells as a prognostic marker in lung cancer. Asian Pac J Cancer Prev. 2012;13(4):1137-44. Hofman V, Ilie MI, Long E, Selva E, Bonnetaud C, Molina T, et al. Detection of circulating tumor cells as a prognostic factor in patients undergoing radical surgery for non-small-cell lung carcinoma: comparison of the efficacy of the CellSearch Assay and the isolation by size of epithelial tumor cell method. Int J Cancer. 2011;129(7):1651-60. Hofman V, Bonnetaud C, Ilie MI, Vielh P, Vignaud JM, Flejou JF, et al. Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker. Clin Cancer Res. 2011;17(4):827-35. Sikosek T, Horos R, Trudzinski F, Jehn J, Frank M, Rajakumar T, et al. Early Detection of Lung Cancer Using Small RNAs. J Thorac Oncol. 2023;18(11):1504-23. Seier S, Bashiri Dezfouli A, Lennartz P, Pockley AG, Klein H, Multhoff G. Elevated Levels of Circulating Hsp70 and an Increased Prevalence of CD94+/CD69+ NK Cells Is Predictive for Advanced Stage Non-Small Cell Lung Cancer. Cancers (Basel). 2022;14(22). Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci. 2005;62(6):670-84. Albakova Z, Armeev GA, Kanevskiy LM, Kovalenko EI, Sapozhnikov AM. HSP70 Multi-Functionality in Cancer. Cells. 2020;9(3). Murphy ME. The HSP70 family and cancer. Carcinogenesis. 2013;34(6):1181-8. Fuqua SA, Oesterreich S, Hilsenbeck SG, Von Hoff DD, Eckardt J, Osborne CK. Heat shock proteins and drug resistance. Breast Cancer Res Treat. 1994;32(1):67-71. Gehrmann M, Liebisch G, Schmitz G, Anderson R, Steinem C, De Maio A, et al. Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS One. 2008;3(4):e1925. Xanthopoulos A, Samt AK, Guder C, Taylor N, Roberts E, Herf H, et al. Hsp70-A Universal Biomarker for Predicting Therapeutic Failure in Human Female Cancers and a Target for CTC Isolation in Advanced Cancers. Biomedicines. 2023;11(8). Baek JA, Choi JH, Gu MJ. The loss of CD44 and HSP70 overexpression is related to aggressive clinicopathologic factors in prostate cancer. Int J Clin Exp Pathol. 2018;11(11):5466-72. Safi S, Messner L, Kliebisch M, Eggert L, Ceylangil C, Lennartz P, et al. Circulating Hsp70 Levels and the Immunophenotype of Peripheral Blood Lymphocytes as Potential Biomarkers for Advanced Lung Cancer and Therapy Failure after Surgery. Biomolecules. 2023;13(5). Gunther S, Ostheimer C, Stangl S, Specht HM, Mozes P, Jesinghaus M, et al. Correlation of Hsp70 Serum Levels with Gross Tumor Volume and Composition of Lymphocyte Subpopulations in Patients with Squamous Cell and Adeno Non-Small Cell Lung Cancer. Front Immunol. 2015;6:556. Gehrmann M, Specht HM, Bayer C, Brandstetter M, Chizzali B, Duma M, et al. Hsp70--a biomarker for tumor detection and monitoring of outcome of radiation therapy in patients with squamous cell carcinoma of the head and neck. Radiat Oncol. 2014;9:131. Silvestri GA, Gould MK, Margolis ML, Tanoue LT, McCrory D, Toloza E, et al. Noninvasive staging of non-small cell lung cancer: ACCP evidenced-based clinical practice guidelines (2nd edition). Chest. 2007;132(3 Suppl):178S-201S. Gould MK, Kuschner WG, Rydzak CE, Maclean CC, Demas AN, Shigemitsu H, et al. Test performance of positron emission tomography and computed tomography for mediastinal staging in patients with non-small-cell lung cancer: a meta-analysis. Ann Intern Med. 2003;139(11):879-92. Hellwig D, Baum RP, Kirsch C. FDG-PET, PET/CT and conventional nuclear medicine procedures in the evaluation of lung cancer: a systematic review. Nuklearmedizin. 2009;48(2):59-69, quiz N8-9. Silvestri GA, Gonzalez AV, Jantz MA, Margolis ML, Gould MK, Tanoue LT, et al. Methods for staging non-small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2013;143(5 Suppl):e211S-e50S. Wang J, Welch K, Wang L, Kong FM. Negative predictive value of positron emission tomography and computed tomography for stage T1-2N0 non-small-cell lung cancer: a meta-analysis. Clin Lung Cancer. 2012;13(2):81-9. Leiro-Fernandez V, Fernandez-Villar A. Mediastinal staging for non-small cell lung cancer. Transl Lung Cancer Res. 2021;10(1):496-505. Rothammer A, Sage EK, Werner C, Combs SE, Multhoff G. Increased heat shock protein 70 (Hsp70) serum levels and low NK cell counts after radiotherapy - potential markers for predicting breast cancer recurrence? Radiat Oncol. 2019;14(1):78. Additional Declarations Competing interest reported. AGP is CEO and GM is CSO of multimmune GmbH. A granted US patent protects the Hsp70-exo ELISA for determining free and vesicular eHsp70 in liquid biopsies. Supplementary Files SupplementaryTablesFigures.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Jul, 2024 Editor assigned by journal 09 Jul, 2024 Submission checks completed at journal 09 Jul, 2024 First submitted to journal 08 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4707407","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":325722694,"identity":"0b1108d7-9c70-4a32-8560-63a71b6bec1a","order_by":0,"name":"Dominik Lobinger","email":"","orcid":"","institution":"München Klinik Bogenhausen","correspondingAuthor":false,"prefix":"","firstName":"Dominik","middleName":"","lastName":"Lobinger","suffix":""},{"id":325722696,"identity":"2f11c077-c550-4217-9b75-784225ee3f46","order_by":1,"name":"Nicholas Taylor","email":"","orcid":"","institution":"Technische Universität München 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Multhoff","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYFACHgaGBAYLEIvxAGMDAz97ewOIw0xIiwSYeeBgA4Nkz5nDRGhhQNFyIxm/Fnn3s8c+PKiQkGMQO2Nw+OMOGwkeyffHpG4wWMvh0mJ4Ji95RsIZCWMG6RyDAwfPpEnwSCezSecwpBvj1NKQY8yQ2CaR2ADW0na4zh6i5XBiAy4t/W+AWv5J1MO0AB12GKylHpcWeQmQLQ0SCQxwLRLMYC0JuBxmIPEumSHhmIRhm3RawYGzbUC/8CQbW+cYpBvitKU/9zDjjxobeX7p5I0PKtuAIcZ+8OHtnApreZy2HIAy2NDEcWkA2oLL+lEwCkbBKBgFcAAAtxtTid8JgHcAAAAASUVORK5CYII=","orcid":"","institution":"Technische Universität München (TUM)","correspondingAuthor":true,"prefix":"","firstName":"Gabriele","middleName":"","lastName":"Multhoff","suffix":""}],"badges":[],"createdAt":"2024-07-08 18:24:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4707407/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4707407/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62158192,"identity":"c6bda253-1c60-4a7d-98fa-5933313e4c21","added_by":"auto","created_at":"2024-08-09 21:29:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55867,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram summarizing characteristics (tumor stage, histology, lymph node status and 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.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/1c8ce699c6fce79a980d9e0f.png"},{"id":62159063,"identity":"514db248-fdfc-4d39-974a-1c8337cac282","added_by":"auto","created_at":"2024-08-09 21:37:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34694,"visible":true,"origin":"","legend":"\u003cp\u003eFree 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\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/edf51051f2a25d89ceae876d.png"},{"id":62158196,"identity":"5a56f185-1ace-4473-859f-2cce14119844","added_by":"auto","created_at":"2024-08-09 21:29:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56285,"visible":true,"origin":"","legend":"\u003cp\u003e(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=8, Locally advanced) and advanced (n=53, Advanced) NSCLC. Statistically significant differences *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;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”).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/1b64ed21cf9398f6c7058efc.png"},{"id":62159064,"identity":"e677aed6-8b13-4fef-b182-a4567daae7b1","added_by":"auto","created_at":"2024-08-09 21:37:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66436,"visible":true,"origin":"","legend":"\u003cp\u003e(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\u0026lt;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\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/24d340ac7d7efa96f3e37a14.png"},{"id":62159066,"identity":"39909024-2174-41ca-a6d4-8d351dde4da1","added_by":"auto","created_at":"2024-08-09 21:37:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220816,"visible":true,"origin":"","legend":"\u003cp\u003eProportions 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). (A) CD45+ lymphocyte counts, (B) CD3-/CD19+ B lymphocytes, (C) CD3+ T cells, CD3+/CD4+ T helper cells, CD3+/CD8+ cytotoxic T cells, (D) CD3+/CD4+/FoxP3+ and CD3+/CD8+/FoxP3+ Treg cells, (E) CD3+/CD56+/CD94+/CD69+/NKG2D+ NKT cells, (F) CD3-/CD56+/CD94+/CD69+/NKG2D+/NKp30+/NKp46+ NK cells. Statistically significant differences *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.00001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/e6342bb9d57d8c6714e1e2ae.png"},{"id":62158194,"identity":"9a6b25f3-3015-4ae7-90d4-b872868c7108","added_by":"auto","created_at":"2024-08-09 21:29:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":44561,"visible":true,"origin":"","legend":"\u003cp\u003eFree and vesicular eHsp70 (ng/mL) levels measured in the plasma of (A) NSCLC patients and (B) patients with lung (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 after 6 months and 12 months respectively first 6 months. Statistically significant differences *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/e592361f05ba0c5ee8f2c73c.png"},{"id":62159640,"identity":"9f1996d0-2323-498e-ac88-a35633e52560","added_by":"auto","created_at":"2024-08-09 21:53:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":951707,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/ef4062f4-1fd8-45a4-b38c-c161913bb2da.pdf"},{"id":62159349,"identity":"4f1156d1-143b-4380-b123-741ad8602ae1","added_by":"auto","created_at":"2024-08-09 21:45:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":597889,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4707407/v1/90a4865ed5189139ac66bc1d.docx"}],"financialInterests":"Competing interest reported. AGP is CEO and GM is CSO of multimmune GmbH. A granted US patent protects the Hsp70-exo ELISA for determining free and vesicular eHsp70 in liquid biopsies.","formattedTitle":"Circulating Hsp70 - a tumor biomarker for lymph node metastases and early relapse in thoracic cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases, continues to be the cancer with the highest mortality rate worldwide\u0026nbsp;(1).\u0026nbsp;\u0026nbsp;With 22,590 female and 34,100 male cases registered by the Robert Koch Institute in Germany in 2020, lung cancer remains among the most frequently newly diagnosed types of cancer in the last decade\u0026nbsp;(2).\u0026nbsp;Upon initial presentation, about two thirds of patients are diagnosed with metastatic NSCLC\u0026nbsp;(3, 4).\u0026nbsp;Survival rates are heavily influenced by the tumor stage, which besides tumor size, is determined by the presence and extent of lymphatic and distant metastases\u0026nbsp;(5, 6). In early-stage NSCLC, complete resection with curative intent with systematic lymph node dissection is the standard treatment, whereas in locally advanced and metastatic NSCLC, multimodal therapies are tailored in multidisciplinary tumor boards\u0026nbsp;(7-9).\u003c/p\u003e\n\u003cp\u003eLarge international randomized trials have demonstrated a reduction of lung cancer mortality by low-dose computed tomography (LDCT) screening, probably by increasing the proportion of cases diagnosed in earlier tumor stages\u0026nbsp;(10, 11). Although LDCT has a high sensitivity for the detection of pulmonary lesions, the specificity of LDCT in differentiating a pulmonary lesion in malignant or benign disease is limited. A national lung cancer screening program for patients with specific risk factors is presently planned in Germany\u0026nbsp;(12).\u0026nbsp;The better classification of uncertain findings from LDCT screening, which increases the number of patients entering curative stages, requires the identification of a tumor marker which is able to determine tumor aggressiveness, especially given the rapidly growing numbers of neoadjuvant and perioperative treatment options\u0026nbsp;(8, 9). With this in mind, we investigated the potential of circulating extracellular heat shock protein 70 (eHsp70), together with the immunophenotype of peripheral blood lymphocytes, as a potential tumor biomarker approach in thoracic oncology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHsp70 (HSPA1A) plays an important role in maintaining protein homeostasis by supporting the folding and unfolding of nascent polypeptides, protein transport and preventing protein aggregation in all nucleated cells\u0026nbsp;(13-15). In malignant cell types,\u0026nbsp;Hsp70 is often highly overexpressed and presented on the plasma membrane in a tumor-specific manner\u0026nbsp;(16).\u0026nbsp;Furthermore, membrane\u0026nbsp;Hsp70 (mHsp70) positive tumor cells actively release Hsp70 in the context of extracellular lipid microvesicles\u0026nbsp;(17).\u0026nbsp;Depending on the localization of Hsp70 in tumor cells, its role can be contradictory: whereas elevated intracellular levels are associated with an increased resistance to chemo- or radiotherapy\u0026nbsp;(18), membrane-bound and circulating extracellular Hsp70 (eHsp70) can mediate anti-tumor and immunomodulatory effects. Previous\u0026nbsp;studies have only explored free circulating Hsp70 as a tumor biomarker\u0026nbsp;(19, 20),\u0026nbsp;whereas we have developed the Hsp70-exo sandwich ELISA which is able to measure not only free Hsp70 derived from dying cells, but also Hsp70 that is actively released from living tumor cells in lipid microvesicles\u0026nbsp;(21-24).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to investigate the potential value of eHsp70 levels, as measured using the Hsp70-exo ELISA, and the peripheral blood immunophenotype, as determined by multiparameter flow cytometry, as biomarkers for estimating the likelihood of a lung lesion being malignant and assessing disease aggressiveness in thoracic malignancies.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\n\u003ch3\u003ePatient cohort\u003c/h3\u003e\n\u003cp\u003eThe patient cohort consisted of a total of 178 patients diagnosed with NSCLC (n\u0026thinsp;=\u0026thinsp;178) and 35 patients with lung metastases (n\u0026thinsp;=\u0026thinsp;35) of extrathoracic primary tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the NSCLC patients, 53 (Supplementary Table\u0026nbsp;1) had undergone surgical resection with curative intent between 2020 and 2023 at the University Hospital of the Technical University of Munich (TUM) and at the M\u0026uuml;nchen Klinik Bogenhausen, an academic teaching hospital of TUM. The male to female ratio of the patients was 1.3 to 1. Samples from 108 age-matched healthy donors were additionally included in the analyses. Patient characteristics are summarized in the flow diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All EDTA blood samples for analyzing eHsp70 levels were obtained prior to surgery. The study was approved by the local ethics committee of the TUM School of Medicine and Health (TUM 2403/09 and 2428/09), and written informed consent was obtained from all patients and healthy donors before start of the study. Blood sampling was conducted in accordance to the guidelines of the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHsp70-exo sandwich ELISA\u003c/h3\u003e\n\u003cp\u003ePlasma derived from EDTA anti-coagulated blood (S-Monovette, Sarstedt, N\u0026uuml;mbrecht, Germany) was prepared by a centrifugation at 1500xg for 15 minutes at room temperature, followed by aliquoting (300 \u0026micro;L) and storage of the plasma samples at \u0026minus;\u0026thinsp;80\u0026deg;C. For the Hsp70-exo sandwich ELISA, 96-well MaxiSorp Nunc-Immuno plates (Thermo, Rochester, NY, USA) were incubated overnight with the cmHsp70.2 coating monoclonal antibody (mAb, 1 \u0026micro;g/mL; multimmune GmbH Munich, Germany) in sodium carbonate buffer (0.1 M sodium carbonate, 0.1 M sodium hydrogen carbonate, pH 9.6; Sigma-Aldrich (Merck, Darmstadt, Germany)) at room temperature. After a washing step in phosphate-buffered saline (PBS) containing 0.05% v/v Tween-20 (Life Technologies, Darmstadt, Germany; Calbiochem, Merck, Darmstadt, Germany) and blocking with liquid plate sealer (Candor Bioscience GmbH, Wangen i. Allg\u0026auml;u, Germany) plasma samples (100 \u0026micro;L) were diluted in StabilZyme Select (1:5; Diarect GmbH, Freiburg i. Breisgau, Germany). An 8-point Hsp70 protein standard (1-100 ng/mL) was also pre-diluted in StabilZyme Select (1:5). After an incubation for 30 minutes at room temperature and another washing, plates were incubated for 30 minutes with biotinylated cmHsp70.1 detection mAb (200 ng/mL; multimmune GmbH, Munich, Germany) dissolved in HRP-Protector\u0026trade; (Candor Bioscience GmbH, Wangen i. Allg\u0026auml;u, Germany). Subsequently, plates were incubated for another 30 minutes with Streptavidin (57 ng/mL; Senova GmbH, Weimar, Germany) in HRP-Protector\u0026trade; (Candor Bioscience GmbH, Wangern i. Allg\u0026auml;u, Germany). Colorimetric analysis was performed after adding the substrate reagent (100 \u0026micro;L; BioFX TMB Super Sensitive One Component HRP Microwell Substrate, Surmodics, Inc., Eden Prairie, MN, USA) for 15 minutes. After quenching the reaction by adding 2N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (50 \u0026micro;L), the absorbance was read at 450 nm using a Microplate Reader (VICTOR X4 Multilabel Plate Reader, PerkinElmer, Waltham, MA, USA), after a correction of the reference absorbance at 570 nm.\u003c/p\u003e\n\u003ch3\u003eMultiparameter flow cytometry of the immunophenoptype in EDTA blood\u003c/h3\u003e\n\u003cp\u003eFreshly taken anti-coagulated EDTA blood (100 \u0026micro;L) was immunophenotyped using a BD FACSCalibur\u0026trade; flow cytometer (BD Biosciences, Heidelberg, Germany) and the following fluorescence antibody combinations. Combining four different fluorescence-labeled antibodies (FITC/PE/PerCP/APC) allowed the following lymphocyte subpopulations to be determined: CD45\u0026thinsp;+\u0026thinsp;lymphocytes, CD3-/CD19\u0026thinsp;+\u0026thinsp;B cells, CD3+/CD45\u0026thinsp;+\u0026thinsp;T cells, CD3+/CD4\u0026thinsp;+\u0026thinsp;helper T cells, CD3+/CD8\u0026thinsp;+\u0026thinsp;cytotoxic T cells, CD3+/CD69\u0026thinsp;+\u0026thinsp;activated T cells, CD3+/CD4+/CD25+/FoxP3\u0026thinsp;+\u0026thinsp;and CD3+/CD8+/CD25+/FoxP3\u0026thinsp;+\u0026thinsp;regulatory T cells (Treg), CD3+/CD56\u0026thinsp;+\u0026thinsp;NK-like T cells (NKT), CD3+/CD94\u0026thinsp;+\u0026thinsp;NKT cells, CD3+/CD69\u0026thinsp;+\u0026thinsp;NKT, CD3+/NKG2D\u0026thinsp;+\u0026thinsp;NKT, CD3-/CD56\u0026thinsp;+\u0026thinsp;NK cells, CD3-/CD94\u0026thinsp;+\u0026thinsp;NK cells, CD3-/CD69\u0026thinsp;+\u0026thinsp;activated NK cells, CD3-/NKG2D\u0026thinsp;+\u0026thinsp;NK cells, CD3-/NKp30\u0026thinsp;+\u0026thinsp;NK cells, CD3-/NKp46\u0026thinsp;+\u0026thinsp;NK cells. The antibody combinations and fluorescence dyes used in this study have been described previously (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Isotype-matched control antibodies were used as negative controls. The respective percentage of a certain lymphocyte subpopulation is defined as the proportion of cells stained positively for a specific antibody within a defined viable lymphocyte gate.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eComparisons among different groups were conducted using ANOVA and post-hoc Tukey tests, and non-parametric data were analyzed using the Kruskal Wallis test, as appropriate. Normal distribution was tested by the Shapiro Wilk normality test. Statistical analysis was performed using the programming language R, R studio version 2024.04.2\u0026thinsp;+\u0026thinsp;764. Differences are considered to be statistically significant as follows: not significant, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003ePatient characteristics\u003c/h2\u003e\n \u003cp\u003eNSCLC patients (n = 178) were classified into the following disease groups: early (n = 36, stages I and II), locally advanced (n = 89, stage III) and advanced (n = 53, stage IV) (Fig.\u0026nbsp;1). The flow diagram (Fig.\u0026nbsp;1) shows that 25 of the 178 NSCLC patients were diagnosed with stage I disease (n = 25, stage I; n = 16, stage IA; n = 9, stage IB), 11 with stage II disease (n = 11, stage II), 89 with stage III disease (n = 89, stage III; n = 49, stage IIIA; n = 40, stage IIIB), and 53 with stage IV disease (n = 53, stage IV). Sixty-one NSCLC patients had an adeno (n = 61, adeno) and 59 a squamous cell carcinoma (n = 59, squamous) (Fig.\u0026nbsp;1). Of the patients with surgically treated NSCLC (n = 53), 32 had no lymph node metastases (n = 32, N0) and 15 had multiple lymph node metastases (n = 15, N+). An early relapse was determined in 4 NSCLC patients (n = 23, no early relapse) after 6 months, and in 7 patients with lung metastases of extrathoracic primary tumors (n = 10, no early relapse) after 12 months (Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eCirculating eHsp70 concentrations in patients with adeno and squamous cell NSCLC compared to healthy controls\u003c/h2\u003e\n \u003cp\u003eA comparison of circulating Hsp70 levels measured with the Hsp70-exo ELISA in all NSCLC patients across all four tumor stages prior to the start of any treatment (n = 178, median 125.4 ng/mL; \u003cem\u003e***p \u0026lt; 0.001\u003c/em\u003e) demonstrated significantly higher plasma levels of Hsp70 in the tumor patients compared to the healthy control group (n = 108, median 16.4 ng/mL), as illustrated in Fig.\u0026nbsp;2. No difference in eHsp70 levels was found between patients with adeno (n = 61, adeno) or squamous cell carcinoma (n = 59, squamous). Patients with adeno and squamous cell carcinoma showed significantly higher eHsp70 values in the circulation (\u003cem\u003e***p \u0026lt; 0.001\u003c/em\u003e) than the control group (Fig.\u0026nbsp;2). With respect to the peripheral blood immunophenotype, no significant differences were observed in the lymphocyte subpopulations of adeno and squamous cell carcinoma (data not shown). Patients with carcinoma precursors such as lepidic carcinoma (n = 2, median 15.5 ng/mL) and low-grade neuroendocrine tumors such as typical carcinoids (n = 2, median 7.7 ng/mL) exhibited considerably lower eHsp70 values than patients with advanced NSCLC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eCirculating eHsp70 concentrations in patients with early, locally advanced and advanced NSCLC\u003c/h2\u003e\n \u003cp\u003eCirculating eHsp70 levels increased steadily from early (n = 36, stage I and II, median 37.35 ng/mL) through locally advanced (n = 89, stage III, median 137.2 ng/mL) to advanced stage with distant metastases (n = 53, stage IV, median 199.5 ng/mL), as shown in Fig.\u0026nbsp;3A. Significant different levels were found between the healthy control group (n = 108, median 16.4 ng/mL) and patients with locally advanced tumors and advanced tumors \u003cem\u003e(***p = 0.001, each\u003c/em\u003e), as well as between early stages and locally advanced \u003cem\u003e(*p = 0.05)\u003c/em\u003e and advanced NSCLC cases \u003cem\u003e(**p = 0.01\u003c/em\u003e). A ROC analysis of circulating eHsp70 levels in locally advanced (stage III) and advanced (stage IV) NSCLC patients compared to healthy donors yielded an AUC value of 0.83, a sensitivity of 0.73 and a specificity of 0.78 at an optimal threshold value of 49.48 ng/mL (“closest top left method”) (Fig.\u0026nbsp;3B).\u003c/p\u003e\n \u003cp\u003eA comparison of the Hsp70 values in the tumor stages I (n = 25, median 27 ng/mL), II (n = 11, median 287.4 ng/mL), III (n = 89, median 137.2 ng/mL) and IV (n = 53, median 199.5 ng/mL) revealed significant differences between the control group (median 16.4 ng/mL) and grade III and IV tumors (\u003cem\u003e***p \u0026lt; 0.001, each\u003c/em\u003e), as well as grade I tumors and grade III and IV tumors (\u003cem\u003e**p \u0026lt; 0.01, each\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;1A). A more detailed breakdown confirms the trend described before, with significant differences between the healthy donors and stage IIIA (n = 49, median 165.3 ng/mL), IIIB (n = 40, median 88.1 ng/mL) and IV (n = 53, median 199.5 ng/mL, \u003cem\u003e***p \u0026lt; 0.001, each\u003c/em\u003e) NSCLC. A noticeable distinction was observed between stage IA (n = 16, median 26.4 ng/mL) and stages IIIA and IV disease (each \u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;1B).\u003c/p\u003e\n \u003cp\u003eA correlation of the eHsp70 values with the largest pathologically measured NSCLC diameter after surgery showed no obvious correlation (n = 47, R\u003csup\u003e2\u003c/sup\u003e = 0.0616, mean diameter 3.26 cm) (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eCirculating eHsp70 concentrations in NSCLC patients with and without lymph node metastases\u003c/h2\u003e\n \u003cp\u003eIn NSCLC patients that underwent surgery with curative intent, significantly higher eHsp70 values (\u003cem\u003e***p \u0026lt; 0.001)\u003c/em\u003e were present in the subgroup of patients with lymph node metastases (n = 15, median 297.2 ng/mL), compared to those with tumor free lymph nodes (n = 32, median 23.34 ng/mL) (Fig.\u0026nbsp;4A). The ROC analysis of those cases with and without pathological lymph node involvement obtained an AUC value of 0.82 with an achieved sensitivity of 0.8 and a specificity of 0.71 for the prediction of lymph node involvement at an optimal threshold value of 40.45 ng/mL (“closest top left method”), (Fig.\u0026nbsp;4B). In patients with lymph node metastases, increased eHsp70 levels (n = 15, median 297.2 ng/mL) were predominantly found in higher tumor grades: (G3 or micropapillary/solid in 40%; G2 or acinar/papillary in 33%; G1 or lepidic in 0%; other in 26.67%), whereas in patients without lymphatic involvement low eHsp70 levels (n = 32, median 23.81 ng/mL) were predominantly found in lower tumor grades (G3 or micropapillary/solid in 29%; G2 or acinar/papillary in 41.9%; G1 or lepidic in 19.4%; other in 9.7%). PD-L1 expression, as determined by immunohistochemistry, were associated with lower eHsp70 levels. In patients with PD-L1 negative tumors (n = 17), the median eHsp70 level was 277.7 ng/mL and in those with PD-L1 positive tumors (n = 12), the median Hsp70 level was 21.21 ng/mL (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e). These data indicate that eHsp70 levels might not be related to the PD-L1 expression.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eCirculating eHsp70 concentrations and peripheral blood immunophenotype in lung cancer patients\u003c/h2\u003e\n \u003cp\u003eSimilar to patients with stage IV NSCLC (n = 53, median 199.5 ng/mL), patients with lung metastases of different extrathoracic primary tumors (n = 35, median 28.6 ng/mL; \u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e) (Supplementary Table\u0026nbsp;2) also had significantly higher eHsp70 levels in the circulation than healthy controls (Fig.\u0026nbsp;4C). Immunophenotyping of the peripheral blood lymphocytes by multiparameter flow cytometry revealed significantly lower lymphocyte counts (Fig.\u0026nbsp;5A) and significantly increased immunoregulatory T (Treg) cell ratios in all tumor stages (Fig.\u0026nbsp;5D). Ratios of CD4 + T helper cells (Fig.\u0026nbsp;5C) was decreased and that of CD3-/CD56+/CD94+/NKp30+/NKp46 + NK cells (Fig.\u0026nbsp;5F) was increased in patients with thoracic metastases of other primary tumors, as compared to healthy controls (Fig.\u0026nbsp;5A-F).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eCirculating eHsp70 levels in thoracic cancer patients with and without early relapse\u003c/h2\u003e\n \u003cp\u003eA subgroup of NSCLC patients from whom the results of the first follow-ups were available revealed significantly higher preoperative eHsp70 values in patients with early recurrence in the first 6 months post-surgery (n = 4, median 626.3 ng/mL) compared to those with no evidence of recurrence in the same period (n = 23, median 47.5 ng/mL; \u003cem\u003e*p \u0026lt; 0.05)\u003c/em\u003e (Fig.\u0026nbsp;6A). A similar observation was made in a cohort of patients with primary extrathoracic tumors following pulmonary metastasectomy with curative intent. In this group, patients with a relapse within the first postoperative year displayed significantly higher preoperatively determined eHsp70 values (n = 7, median 420.8 ng/mL) compared to patients without tumor relapse (n = 10, median 23.3 ng/mL; \u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e) (Fig.\u0026nbsp;6B).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, the therapeutic landscape for NSCLC has undergone rapid transformation due to a large number of new, innovative and well-tolerated approaches, particularly in the field of immunotherapy and targeted therapies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). These achievements have not only influenced the therapy regime for patients with advanced tumors, but are also used as adjuvant and neoadjuvant approaches in early stages with favorable chances of a long-term cure (\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Efforts have been ongoing to enhance the rate of early-stage initial diagnoses, gain insight into the tumor biology aggressiveness, and identify individuals who may benefit from a more intensive therapy regime or closer follow-up care. These endeavors have led to repeated attempts to discover suitable liquid biopsy-based biomarkers having diagnostic and prognostic significance in patients with NSCLC (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe circulating proteins CYFRA (cytokeratin fragment) and CEA (carcinoembryonic antigen, glycoprotein), which are among the earliest biomarker candidates for NSCLC, have been examined in several prior studies with the outcome that elevated plasma levels of these circulating proteins are showing a trend towards a poorer prognosis (\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Furthermore, the presence of circulating tumor cells (CTCs), which play a role in the formation and development of metastases, has been associated with poorer prognosis in patients with NSCLC (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Most recently, a large study has delivered the first promising results regarding the potential of small RNAs in liquid biopsies for tumor detection, albeit with a low sensitivity in early stages (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Despite these encouraging findings regarding suitable biomarker candidates, none of the candidates have yet been established as an integral part of the diagnostic or therapeutic process in the existing guidelines for the treatment of thoracic cancers. In the context of the upcoming introduction of lung cancer screening in Germany using low-dose CT, there is a significant clinical need for more specific biomarkers that could facilitate the interpretation of inconclusive results and better inform clinical decision-making.\u003c/p\u003e \u003cp\u003eIn our study, we investigated the ability of circulating eHsp70 to predict disease stage, aggressiveness and prognosis of patients with lung cancer across different stages of disease. With the established Hsp70-exo sandwich ELISA which uses the mHsp70-specific monoclonal antibodies cmHsp70.1 (aa 451\u0026ndash;461) and cmHsp70.2 (aa 614\u0026ndash;623), it is possible to quantify both free Hsp70 (released predominantly by dying cells) and Hsp70 bound to lipid microvesicles (primarily derived from viable cells) in the blood (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The results of previous studies demonstrating significantly higher eHsp70 levels in the circulation of cancer patients compared to healthy controls (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) are consistent with our present findings in patients with NSCLC across all stages and patients with lung metastases of different extrathoracic primary tumors.\u003c/p\u003e \u003cp\u003eAs a molecular chaperone, cytosolic Hsp70 maintains protein homeostasis by supporting protein folding, unfolding and transport in all nucleated cell types under physiological conditions (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In tumor cells, Hsp70 is frequently overexpressed and can contribute to a more aggressive and therapy-resistant tumor phenotype by interfering with apoptosis and proliferation pathways (\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Many studies have shown that apart from its cytosolic localization, Hsp70 can be presented on the plasma membrane of tumor cells via a mechanism which is enabled by a tumor-specific lipid composition (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Furthermore, an elevated mHsp70 expression is associated with higher tumor stages and a more aggressive behavior in various female cancers, prostate cancer and brain tumors (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Tumor cells expressing mHsp70 actively release eHsp70 into the extracellular space (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Significantly increased eHsp70 levels have also been shown in patients with NSCLC compared to healthy donors (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Herein, we found a progressive increase in circulating eHsp70 levels starting from healthy donors, to low-grade NSCLC through locally advanced and metastatic NSCLC. The ROC analysis generated an AUC value of 0.83, with a specificity of 0.78 and a sensitivity of 0.73 at an eHsp70 cut-off value of 49.48 ng/mL for the presence of locally advanced or metastatic NSCLC compared to the healthy cohort. These findings further support the crucial function of eHsp70 in mediating tumor aggressiveness in lung cancer and the significant expression of Hsp70 in advanced tumors.\u003c/p\u003e \u003cp\u003eA second indication of Hsp70\u0026rsquo;s role as a marker for aggressiveness emerged from the detailed examination of the surgically treated cases and their histopathological results. Although some studies suggest an association between circulating eHsp70 levels and the tumor volume (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), we found no significant association of circulating eHsp70 concentrations and the largest pathologically measured NSCLC diameter after surgical resection. However, our results revealed a clear difference (\u003cem\u003e***p\u0026thinsp;=\u0026thinsp;0.001)\u003c/em\u003e in the eHsp70 concentration between patients with pathologically proven lymph node metastases and those without. In the guidelines for the preoperative diagnosis of possible lymph node involvement and mediastinal staging in patients with NSCLC, PET-CT plays a decisive role as a non-invasive method. The reported literature states that the sensitivity of PET-CT for the detection of lymph node metastases is between 0.74 to 0.85, with a specificity ranging from 0.85 to 0.92, but with low accuracy in the detection of micrometastases in affected lymph nodes (\u003cspan additionalcitationids=\"CR55 CR56 CR57\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Invasive methods such as mediastinoscopy, EBUS examination or surgical lymph node dissection can provide representative histological results (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). To our knowledge, currently no established liquid biomarker exists for the detection of lymph node metastases in the pretherapeutic setting. The ROC analysis of preoperative eHsp70 in patients undergoing surgery with curative intent yielded an AUC value of 0.82 for the presence of pathological lymph node metastases with a sensitivity of 0.8 and a specificity of 0.78 at an eHsp70 value of 40.45 ng/ml. Preoperative PET-CT results of patients with histologically confirmed lymph node metastases showed a correct clear positive finding in only 6 of 15 cases and no evidence for the presence of lymph node metastasis in 5 cases. In addition, the group with affected lymph nodes and elevated eHsp70 values exhibited a higher percentage of tumors with pathologically higher and thus more dedifferentiated grading, further suggesting a potential role of Hp70 as a marker of aggressiveness. Moreover, we could show that patients with carcinoma precursors had lower eHsp70 levels than patients with malignant tumors. In summary, we assume eHsp70 measured in the blood before start of any treatment could make a significant contribution to more precise preoperative mediastinal staging, especially in unclear cases and in combination with unclear results in PET-CT.\u003c/p\u003e \u003cp\u003eIn addition to NSCLC eHsp70 levels were also measured in patients with lung metastases derived from a different primary tumor than NSCLC. Like patients with locally advanced, metastatic NSCLC, these patients also exhibited significantly higher eHsp70 values compared to healthy controls, thereby indicating that eHsp70 could provide a universal tumor biomarker not only for NSCLC, but also for other tumor entities.\u003c/p\u003e \u003cp\u003eThe third interesting evidence for the potential of eHsp70 as a tumor biomarker of aggressiveness in thoracic oncology is provided by our follow-up data. Previous studies have already shown a link between elevated eHsp70 levels and a tendency to early tumor relapse or treatment failure (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). In our study, we could clearly demonstrate that patients who had an early NSCLC relapse in the first 6 months showed significantly higher preoperative eHsp70 values compared to patients without evidence of an early tumor relapse. Moreover, the eHsp70 values of 2 patients which were additionally determined at the time of the first follow-up after 3 months showed, that already at this early time point, the patient with recurrence showed a significantly higher value than the patient without recurrence. These observations align with our recently published study, wherein eHsp70 levels rose within three months post-surgery in early-relapse patients, whereas they remained stable in relapse-free patients (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe predictive value of eHsp70 for the occurrence of tumor relapse could also be found in patients with thoracic metastases derived form an extrathoracic primary tumor within the first 12 months following pulmonary metastasectomy. Measuring eHsp70 levels at multiple time points during follow-up and during adjuvant therapy could further confirm the results of this study and provide interesting and dynamic clues for possible recurrence and treatment response. The immunophenotyping of the peripheral blood lymphocytes of patients with lung cancer did not provide conclusive results as a predictive tumor biomarker for tumor aggressiveness. Irrespective of the stage and tumor aggressiveness all patients suffered from a lymphopenia and the number of immunosuppressive regulatory T cells were found to be significantly increased compared to healthy controls. An increase in CD3-/CD56+/CD94+/CD69+/NKp30+/NKp46\u0026thinsp;+\u0026thinsp;NK cell subsets which correlated with a significant drop in CD4\u0026thinsp;+\u0026thinsp;T helper cells, appeared to be restricted to patients with lung metastases of an extrathoracic primary tumor. Since reduced CD4\u0026thinsp;+\u0026thinsp;T cell counts are associated with decreased levels of pro-inflammatory cytokines in the circulation [43], we assume that despite an increase in some NK cell subsets, these effector cells are unable to mediate protective anti-tumor immunity in advanced tumors.\u003c/p\u003e \u003cp\u003eSince we found no obvious association between circulation eHsp70 levels and tumor size, we hypothesize that the tumor aggressiveness (lymph node metastases, early relapse), rather than the size of the tumor, is responsible for the elevated circulating eHsp70 levels. Considering the increased detection of incidental pulmonary nodules by lung cancer screening, our easily measurable biomarker in liquid biopsies could contribute to the management of such unclear cases. In instances where elevated eHsp70 values are associated with ambiguous results, early histological confirmation or closer follow-up could be considered. On the other hand, measuring eHsp70 not only at the time of diagnosis, but also during the treatment could help to identify patients experiencing treatment failure and thus guide the selection of alternative treatment strategies or more intensive approaches. This is especially relevant given the increasing complexity and diversity of available modalities.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWith the imminent launch of a national lung cancer screening initiative in Germany and the high number of incidental pulmonary nodules on low-dose CT, there is an urgent need for reliable biomarkers to guide subsequent diagnostic algorithms. Our data support eHsp70 as an indicator of disease aggressiveness in thoracic oncology. In particular, we observed a consistent and significant increase in eHsp70 levels with advancing tumor stages. Patients with lymph node metastases and de-differentiated tumors had significantly higher eHsp70 levels compared to patients without such disease characteristics. In addition, the association between elevated preoperative eHsp70 levels and early tumor recurrence underscores its prognostic value. Further studies are warranted to validate our findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the local ethics committee of the TUM School of Medicine and Health (TUM 2403/09 and 2428/09), and written informed consent was obtained from all patients and healthy donors before start of the study. Blood sampling was conducted in accordance to the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e“Not applicable”\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAGP is CEO and GM is CSO of multimmune GmbH. A granted US patent protects the Hsp70-exo ELISA for determining free and vesicular eHsp70 in liquid biopsies.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by grants of BMBF 02NUK064A, Bayerische Forschungsstiftung BAYCELLator AZ-1568-22.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGM and SS designed and supervised the study, NT, VM, SS, ABD, ER performed the experiments, DL, NT and VM analyzed the data, DL and NT designed the Figures and drafted the manuscript, SS, DL and JB provided the clinical samples and clinical data, GM conceptualized the study, AGP proofread the manuscript, and all authors provided feedback and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors thank Hannah Herf and Anett Lange for technical and editorial support.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e(WHO) WHO. 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Radiat Oncol. 2019;14(1):78.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NSCLC, extracellular Hsp70, biomarker, liquid biopsy, immune-phenotype","lastPublishedDoi":"10.21203/rs.3.rs-4707407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4707407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 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 Hsp70 and the patient`s immunophenotype was determined by flow cytometric analysis of the corresponding peripheral blood lymphocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 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 Hsp70 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 Treg 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 lung cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e In summary, we propose circulating eHsp70 levels as a predictive biomarker for the presence of lymph node metastases and early therapy failure in patients with thoracic malignancies.\u003c/p\u003e","manuscriptTitle":"Circulating Hsp70 - a tumor biomarker for lymph node metastases and early relapse in thoracic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 21:29:54","doi":"10.21203/rs.3.rs-4707407/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-11T14:33:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-09T08:31:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-09T08:29:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-07-08T18:23:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a9c5c1b-0634-44c9-af18-de72c7234991","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T10:38:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 21:29:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4707407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4707407","identity":"rs-4707407","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}