Circulating levels of High Mobility Group Box-1 and Nucleophosmin/B23 proteins and clinical significance in debut Non-Small Cell Lung Cancer patients

Circulating levels of High Mobility Group Box-1 and Nucleophosmin/B23 proteins and clinical significance in debut Non-Small Cell Lung Cancer patients | 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 Article Circulating levels of High Mobility Group Box-1 and Nucleophosmin/B23 proteins and clinical significance in debut Non-Small Cell Lung Cancer patients Tan Hui, Liu Lu, Yi Ying, Carmen Valenzuela, Zhao Qiang, Zhang Zhiwei, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7021384/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Alarmins are endogenous molecules that alert the immune system to circumvent the invading antigens in the host. Here, we investigated the serum levels of the high mobility group box 1 (HMGB-1) and nucleophosmin alarmins in a cohort of 162 newly diagnosed patients with non-small cell lung cancer and 60 age-matched healthy individuals. Interestingly, patients exhibited lower median values of HMGB-1 than healthy individuals, but higher levels of HMGB-1 were associated with a tumor size of > 2.0 cm. In addition, nucleophosmin levels were significantly increased in patients compared with the healthy individuals and those with tumors of > 2 cm in diameter also displayed higher circulating nucleophosmin. Furthermore, a strong association was reported between HMGB-1 and nucleophosmin levels in patients (r = 0.679; P = 0.0001) compared with healthy individuals. Interestingly, the strongest association between both alarmins was obtained in patients with Spread Through Airspaces pattern and tumors of ≤2 cm in size (r = 0.900; P = 0.0001). Co-expression of both alarmins was also observed in tumor tissues, although to a lesser extent. Altogether, our data indicate that high levels of each alarmin were individually associated with larger tumor size, however the strongest associations between both alarmins in NSCLC seemed to be primarily linked to early disease progression parameters. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Oncology High Mobility Group Box-1 HMGB-1 Nucleophosmin NPM/B23 Alarmins Non-Small Cell Cancer NSCLC Figures Figure 1 Introduction Lung cancer remains one of the leading causes of cancer-related deaths each year worldwide, largely due to its late clinical presentation and the limited availability of biomarkers capable of predicting early diagnosis, disease progression, clinical response and overall survival. However, currently, the majority of the identified biomarkers for non-small cell lung cancer (NSCLC) are actionable gene mutations, which promote personalized molecular targeted therapies. The above therapies yield a successful objective clinical response in ~30% of patients 1 . Therefore, identifying new biomarkers reflecting the natural course of NSCLC from its onset, is of great importance for public health worldwide. Ideally, such biomarkers should be easily measurable and preferably detectable by non-invasive methods. Alarmins, which can promote, recruit, activate, and mature dendritic cells 2 , could potentially serve as soluble biomarkers for the diagnosis, progression, and/or treatment of NSCLC. More particularly, the present study primarily focused on the clinical significance of circulating levels of high mobility group box 1 (HMGB-1), which is a widely studied alarmin not only in cancer but also in autoimmunity and diabetes 3 . Additionally, the coexistence of circulating HMGB-1 with nucleophosmin/B23 (NPM/B23), a molecule that has not been studied as alarmin in cancer, was also investigated. On the other hand, a dual role, both pro- and anti-tumor, has been suggested for alarmin by other authors 4 . For example, a study indicated that intracellular HMGB-1, a non-histone chromatin-associated protein, could play a crucial role in tumor growth, affect response to therapy and promote both cell death and survival 5 . However, it was also reported that this alarmin could be involved in different cancer progression-related pathways and could enhance the efficacy of immune checkpoint blockade in patients with cancer 5 . As a putative biomarker in cancer, the role of HMGB-1 in cancer has been supported by several studies. For example, a study demonstrated that the expression levels of HMGB-1 were increased in tumor tissues and serum of patients with urothelial carcinoma of the bladder 6 . Additionally, in advanced lung cancer, small cell lung carcinoma (SCLC) and mesothelioma, high HMGB1 levels were associated with shorter overall survival in patients with NSCLC 7 . In pancreatic ductal adenocarcinoma (PDAC), HMGB1 displayed a better diagnostic value compared with the traditional biomarkers, such as carbohydrate antigen 19-9 or carcinoembryonic antigen 8 . Furthermore, other studies revealed that HMGB-1 could predict gastric cancer progression 9 and response to chemotherapy in breast cancer 10 . Intracellular NPM/B23, also known as NPM1, B23, No38 or numatrin, is a phosphoprotein mainly localized in the nucleolus 11 . It is involved in several biological processes, including mRNA transport, chromatin remodeling, genome stability and apoptosis. NPM/B23 is commonly overexpressed in solid tumors and it is therefore mainly associated with increased mitotic activity and metastasis 12 . However, there is limited data on the presence of circulating NPM/B23 in the serum of patients with cancer and/or its potential role as an alarmin. To the best our knowledge, no prior studies have investigated the clinical significance of the coexistence of circulating HMGB-1 and NPM/B23 in the serum of patients with cancer. Therefore, the present study aimed to investigate whether these two proteins were associated with each other and/or with clinicopathological parameters in newly diagnosed patients with NSCLC. Results Demographic and clinicopathological characteristics. The mean age of patients in the NSCLC group was 61.2 ± 8 years, while that in the control group was 62.4 ± 11.8 years. Among the total of 232 patients with NSCLC enrolled in the present study, 98 (42.2%) and 134 (57.8%) were females and males, respectively. The control group composed of 30 male and 30 female subjects. Among all patients with NSCLC, 162 (69.8%) were diagnosed with LUAD and 70 (30,2%) with LUSC. Clinical stages were categorized into two major cohorts: Stage I and stages II, III and IV. The mean tumor size, according to the maximum diameter, was 2.3 ± 1.3 cm. In addition, other clinical conditions considered in the current study included the presence or absence of lymphatic metastasis (23 vs. 191 patients), pleural invasion (23 vs. 191 patients) and STAS (79 vs. 135 patients). Tumor differentiation was classified as low (65 patients), mid (125 patients), mid-low (5 patients), high (23 patients) and high-mid (1 patient; Table I). Patients with missing clinical data or inadequate samples were excluded from the correlation analysis. Data on clinical response to standard anticancer therapy and overall survival were collected. Levels of circulating alarmins in patients with NSCLC. As shown in Fig. 1 A, the median HMGB-1 levels were significantly lower in patients with NSCLC (30.04 ng/ml) compared with healthy individuals (37.30 ng/ml; P = 0.02). Interestingly, patients with LUAD, but not those with LUSC, displayed markedly reduced HMGB-1 levels compared with the control group (P < 0.01). However, HMGB-1 levels were not significantly different between patients with LUAD and LUSC. By contrast, the median NPM/B23 levels were notably enhanced in patients with NSCLC (812 pg/ml) compared with healthy individuals (551 pg/ml; P = 0.001; Fig. 1 B). When both histological subtypes were analyzed separately, circulating NPM/B23 levels were also significantly higher in both patients with LUAD and LUSC compared with the control group (P < 0.001; Fig. 1 B). No significant difference in circulating NPM/B23 levels was observed between patients with LUAD and LUSC. Association between circulating alarmin levels and clinicopathological features of patients with NSCLC . After assessing the serum levels of both alarmins in patients with NSCLC, the present study then investigated whether these levels were associated with any particular clinicopathological feature. Interestingly, the univariate analysis shown in Table I revealed a strong association between tumor size and HMGB-1 serum levels in patients with NSCLC. More particularly, significant higher HMGB-1 levels were positively associated with tumors of > 2.0 cm in maximum diameter (P = 0.004), regardless of tumor histology. However, no other associations were recorded between circulating HMGB-1 levels and other clinicopathological features. When generalized linear models were applied using HMGB-1 as a dependent variable and incorporating all available clinical features, it was verified that only tumor size was associated with HMGB-1 levels (Table SI). Subsequently, potential associations between circulating NPM/B23 levels and particular clinicopathological features in patients with NSCLC were evaluated. As shown in Table II, the presence of STAS pattern (P = 0.043) and a tumor size of > 2 cm (P = 0.0001) were associated with higher serum levels of NPM/B23 in patients with NSCLC. However, data from the multivariate analysis revealed that only a tumor size of > 2 cm was significantly associated with circulating NPM/B23 levels (Table SII). Correlation between circulating HMGB-1 and NPM/B23 serum levels in patients with NSCLC. To verify whether circulating HMGB-1 and NPM/B23 levels were associated with each other, the presence of both alarmins in the serum of healthy individuals and patients with NSCLC were analyzed. Therefore, as shown in Table III, the serum levels of both HMGB-1 and NPM/B23 were associated with each other in both cohorts. However, their association was more significant in patients with NSCLC compared with healthy individuals (r = 0.679 vs. r = 0.278). As tumor size and the presence of STAS pattern were associated with the serum levels of circulating alarmins in the NSCLC group, the present study aimed to further investigate the association of the above two clinicopathological features with HMGB-1 and NPM/B23 levels. Interestingly, the analysis showed that the levels of both alarmins were significantly associated with a tumor size of > 2 cm (r = 0.70), regardless the presence or not of STAS (Table IV). However, the strongest association between both circulating alarmins was observed in patients with NSCLC with tumors of ≤ 2 cm in diameter and the presence of STAS (r = 0.900; P = 0.0001; Table IV). The presence of STAS pattern indicates the spread of tumor cells, as micropapillary structures, solid nests, and/or single cells, within the airspaces of the lung parenchyma beyond the edge of the main tumor 13 . It is also associated with a higher risk of recurrence in small lung adenocarcinomas, where limited resection can be insufficient for patients exhibiting STAS 13 . Therefore, the strong association between HMGB-1 and NPM/B23 levels in the presence of STAS and tumors of ≤ 2 cm in diameter could suggest either early disease progression or a more aggressive form of NSCLC. Expression of HMGB-1 and NPM/B23 in tumor and adjacent healthy lung tissues. Finally, the current study aimed to explore the intratumoral expression of both HMGB-1 and NPM/B23 alarmins in patients with NSCLC by immunohistochemistry, using tumor biopsies and adjacent healthy lung tissues, located 5 cm from tumor, as internal control. Representative immunohistochemistry images of intracellular HMGB-1 expression in both tumor and adjacent healthy tissues are illustrated in Fig. S1 . HMGB-1 was predominantly expressed in the nuclear compartment, not only in alveolar epithelial cells, but also in phagocytes in the alveolar space, fiber cells in the alveolar septum and fibrocytes. To semi-quantify the immunohistochemistry results, the HMGB-1 positive expression rate was calculated as the sum of patients exhibiting low (+), moderate (++) and high (+++) intracellular expression. As shown in Table V, among 167 patients with NSCLC, the positive expression rate of HMGB-1 in tumor tissues was 78.44% compared with 86.83% in adjacent healthy lung tissues (P = 0.043). Notably, a distinct dichotomous expression pattern of HMGB-1 was observed based on NSCLC histological subtypes. Therefore, in patients with LUAD the positive expression rate of HMGB-1 was significantly lower compared with that in adjacent healthy tissues (P = 0.0001). By contrast, in patients diagnosed with LUSC the positive expression rate was notably higher in tumor tissues compared with adjacent healthy ones (P = 0.001). Interestingly, more than 50% of adjacent healthy tissues also displayed positive HMGB-1 expression. Subsequently, whether a potential association existed between intracellular HMGB-1 levels in tumors and adjacent healthy tissues was assessed. Therefore, a statistically significant, but weak, positive association was found between intracellular HMGB-1 expression in tumors and their corresponding adjacent normal tissues counterpart (P = 0.0001; r = 0.348; Table SIII). However, no significant association was observed between circulating serum HMGB-1 levels and tumor or normal tissues (Table SIII). By contrast, the immunohistochemistry analysis revealed a nuclear or nuclear/nucleolar expression pattern for the NPM/B23 alarmin (Fig. S2 ). Unlike HMGB-1, the overall positive NPM/B23 expression rate in tumor tissues was notably higher compared with that observed in adjacent healthy tissues (92.22 vs. 85.03%; P = 0.0387; Table VI). More particularly, the positive NPM/B23 expression rate in tumor tissues from patients with LUAD was 91.54% compared with 83.08% recorded in adjacent healthy tissues. Interestingly, in patients with LUSC the positive expression rate of NPM/B23 was not significantly different between tumor and adjacent healthy tissues (94.60 vs. 91.89%; P = 0.6433; Table VI). Nonetheless, NPM/B23 showed a significantly increased expression rate in patients with NSCLC. Similarly, with HMGB-1, a significant but weak positive association was also observed between intracellular NPM/B23 expression in tumor and adjacent healthy tissues (P = 0.0001; r = 0.394; Table SIV). Consistently, no association was obtained between circulating NPM/B23 levels in serum and tissue expression in either tumor or normal tissues (Table SIV). Given the increased positive expression rate of both HMGB-1 and NPM/B23 in adjacent healthy tissues, the expression of Ki-67, a cell proliferation biomarker that is not an alarmin, was determined to verify specificity. As expected, Ki-67 expression was very weak in adjacent healthy tissues compared with NSCLC tissues (2.2 vs 56.7%; P < 0.001). Finally, to assess whether HMGB-1 and NPM/B23 alarmins were associated at the tissue level, a Spearman coefficient analysis was performed in both malignant and adjacent normal tissues. Unlike the findings in serum, only a weak correlation between the two alarmins was observed in tumor tissues (P = 0.02; r = 0.223; Table SV), but not in adjacent healthy tissues (Table SVI). Discussion Early detection of novel disease biomarkers in NSCLC can optimize clinical decision-making for therapy, but can also complement computed tomography (CT) scan and chest radiography, thus helping to mitigate cancer-related mortality and improve overall prognosis in lung cancer. As mentioned above, alarmins have been proposed as biomarkers for the diagnosis, progression, and/or treatment of NSCLC. In particular, previous meta-analyses suggested that the HMGB-1 alarmin was upregulated in both tumors and serum of patients with NSCLC compared with healthy individuals 14 , 15 , supporting its potential as a biomarker for predicting disease progression and survival. However, other studies demonstrated that HMGB1 was downregulated in NSCLC tumor tissues compared with matched normal lung tissues 16 . The present study investigated the relevance of pretherapeutic circulating and tissue levels of HMGB-1, as well as those of MPN/B23, which has been suggested as a secreted alarmin in sepsis and found to be elevated in patients with NSCLC 17 , 18 . More particularly, herein, the clinical significance of the co-expression of both proteins in relation to key clinicopathological parameters in newly diagnosed patients with NSCLC was assessed. In addition, the association between HMGB-1 and NPM/B23 at both systemic and tissue levels was investigated in tumor samples. The results demonstrated that the median global circulating HMGB-1 levels in the serum of patients with NSCLC were lower compared with those observed in healthy individuals, and more particularly in patients diagnosed with LUAD (Fig. 1 A). It is currently known that different oxidation states of HMGB-1 can lead in three mutually exclusive functions, acting as an alarmin 19 , and promoting chemoattraction 20 or tolerance 21 . Therefore, it would be of great interest to determine whether a specific redox status of HMGB-1 occurs in patients with LUAD and whether this status could be associated with clinical outcome, disease progression, or survival. Nonetheless, the results showed that elevated individual HMGB-1 levels were positively associated with a tumor size of > 2 cm in diameter (P = 0.004; Table I), regardless of NSCLC histological subtype. This finding could be associated with the clinical staging of patients and could serve as an indicator of tumor progression in both LUAD and LUSC. These results were consistent with previous ones showing positive association between circulating HMGB-1 levels and tumor size, among other clinicopathological features 14 . Although a correlation between HMGB-1 with other clinicopathological parameters were not found in patients with NSCLC, the current study was the first to report a 2-cm tumor size threshold as clinically significant in relation with HMGB-1 expression. Notably, none of the correlations observed between alarmins and clinicopathological features were associated with the NSCLC histological subtype, which could be due to the sample size imbalance in the cohort included in the study (LUAD, n = 124 vs. LUSC, n = 38). NPM/B23, a multifunctional protein involved in cancer, exerts chaperone activity and plays a key role in ribosomal biogenesis. Previous studies demonstrated that NPM/B23 was upregulated in patients with cancer 11 , 12 , 22 . Although only a few studies have investigated the extracellular role of NPM/B23 as an alarmin, experimental evidences have suggested that beyond being a potential biomarker, it can also serve as a therapeutic target. For instance, a previous study indicated that NPM/B23 was physiologically secreted by endothelial cells under stress, thus exerting proangiogenic activity both in vitro and in vivo through the secretion of vascular endothelial growth factor-A, hepatocyte growth factor, stromal derived factor-1, fibroblast growth factor-2, platelet derived growth factor-B, and matrix metallopeptidase 9 23 . Consistently, recombinant NPM/B23 could induce the release of the proinflammatory cytokines, tumor necrosis factor-, IL-6 and monocyte chemoattractant protein-1 from RAW264.7 cells and increase the expression levels of intercellular adhesion molecule 1 in human umbilical vein endothelial cells, thus supporting its additional role in systemic inflammation 17 . Interestingly, the toll-like receptor 4/myeloid differentiation factor 2 complex has been identified as a receptor which is required for the extracellular function of NPM/B23 24 . Consistently, other studies reported elevated circulating levels of NPM/B23 in patients with NSCLC compared with healthy individuals, a positive association with the presence of nodules, and markedly higher levels of NPM/B23 in patients with LUAD compared with those with LUSC 18 . In agreement with the aforementioned findings, the results of the present study revealed significantly higher levels of NPN/B23 in patients with NSCLC across both histological subtypes (Fig. 1 B). Furthermore, both the univariate and multivariate analyses identified a notable association between elevated NPM/B23 levels and tumor size of > 2 cm (P = 0.0001; Table II), regardless of histological subtype. Additionally, higher circulating levels of NPM/B23 were modestly associated with the presence of STAS pattern (P = 0.043). Since the presence of STAS pattern in NSCLC indicated early spread of tumor cells through airspaces adjacent to the primary tumor 25 , NPM/B23 could serve as a valuable prognostic biomarker for assessing the risk of disease progression. However, the univariate and multivariate analyses did not reveal any significant associations between NPM/B23 levels and other clinicopathological features. The above findings indicated that both HMGB-1 and NPM/B23 were individually associated with clinical indicators of disease progression, such as tumor size and STAS pattern, independent of NSCLC histological subtype. In the case of B23/NPM, the results could provide further evidence supporting the role of NPM/B23 as an alarmin in newly diagnosed patients with NSCLC. After evaluating the clinical relevance of HMGB-1 and NPM/B23, the present study aimed to analyze the association between both proteins in serum and tissue samples from patients with NSCLC. Importantly, the results revealed a positive association between both proteins in the serum of newly diagnosed patients with NSCLC (Table III), reinforcing the hypothesis that NPM/B23 could also act as an alarmin in this clinical setting. Considering that the individual analysis of circulating HMGB-1 and NPM/B23 levels was associated with clinical parameters involved in disease progression, such as tumor size and the presence of STAS, whether this correlation was maintained across the abovementioned clinicopathological parameters was subsequently investigated. A significant positive correlation was observed between HMGB-1 and NPM/B23 levels when tumor diameters were 2 cm, a weak but significant association was obtained between both alarmins, regardless of the presence of STAS pattern. To the best of our knowledge, this study was first to report a direct positive association between circulating HGMB-1 and NPM/B23 levels in patients with cancer, as well as their association with specific tumor size thresholds and early tumor cell spreading. Although elevated individual levels of circulating HMGB1 and NPM/B23 could reflect changes associated with disease progression, the strong correlation observed between both proteins in smaller tumors accompanied with STAS could be useful for the early detection of the disease and/or for predicting the risk of NSCLC progression, particularly when combined with non-invasive imaging procedures, such as CT scan and Magnetic Resonance Imaging. In addition, early detection could assist oncologist practitioners in developing more effective therapeutic strategies. Therefore, the results of the current study warrant validation in larger patient cohorts. Considering that alarmins can be released from tumor cells during the tumor-host immune interactions and the necrotic processes inherent to solid tumors, subsequently, the expression levels of HMGB-1 and NPM/B23 were detected in tumor and normal adjacent lung tissues. The results indicated that, although the positivity rate or HMGB-1 expression in tumor tissues was > 50%, it was lower than that observed in matched adjacent healthy tissues, especially among patients with LUAD (Table V). By contrast, in patients with LUSC, this expression pattern was rather reversed, possibly due to the function of HMGB-1 as an alarmin. Therefore, further studies are needed to elucidate the significance of the differential expression of HMGB-1 in tumor tissues compared with adjacent healthy lung tissues in patients with LUAD (Table VI). On the other hand, the positive expression rate of NPM/B23 was > 80% in both tumor and healthy tissues, which was a novel observation in this study. By contrast, Ki-67, a common biomarker of cell proliferation which does not act as an alarmin, showed a very low positive expression rate (2.2%) in healthy tumor tissues of patients with NSCLC. To evaluate whether the co-expression of HMGB-1 and NPM/B23 in tumor tissues behaved similarly to that observed in the circulating levels, a correlation analysis was carried out. Interestingly, a weak correlation (P = 0.02; r = 0.223) was obtained, thus suggesting that future studies with a greater number of patients with NSCLC are needed to strengthen the above finding. Nonetheless, the weak correlation might be influenced by the semiquantitative output of immunohistochemistry compared with ELISA that is fully quantitative. No relevant associations between the intracellular expression levels of both proteins and individual clinicopathological parameters were identified. In addition, other potential biomarkers linked to tumor cell proliferation and lung cancer like Ki-67, AKT, phospho-AKT, Protein Kinase CK2 (CK2A1, CK2A2, CK2B), CDC37, and PDL-1 were investigated by immunohistochemistry in tumor biopsies but they did not exhibit the same correlation patterns with clinical parameters (unpublished data). Therefore, circulating alarmin-based biomarkers could be more useful over such biomarkers to support disease progression of NSCLC. In summary, the present study investigated the individual and correlative features of HMGB-1 and NPM/B23 alarmins in the serum and tissues of patients with NSCLC, who had not received any anticancer therapy. This study was the first to describe the correlations between these two proteins in the serum of patients with NSCLC and their clinical characteristics at the onset of the disease. However, future directions of the research must be focused on the validation of this potential alarmin-based biomarker of disease progression in larger NSCLC patient cohorts. Methods Subjects : A total of 167 NSCLC patients and 60 age-matched healthy individuals were enrolled in this prospective study. The study was approved by the Ethics Committee of the First Affiliated Hospital of University of South China, Hunan Province and reviewed lot number No.2021LL0115001. Also, the study was conducted in accordance with ethical principles from Helsinki declaration (2013), the regulations on Biomedical Ethics Review involving human (2016), the regulations on Clinical Research Ethics review in traditional Chinese medicine and the International Ethical Guidelines for Biomedical Research. In addition, a consent was obtained by all the subjects included in this study. Although patient identification records are not published in this work, a consent for publication of the general data in this work was also obtained. Enrollment criteria for NSCLC patients and sample collection : Only debut patients without previous chemoradiotherapy, targeted therapy or immunotherapy before surgery, were included. Also, patients with paraffin histology diagnosis after radical resection of lung cancer with LUAD or LUSC were eligible for the study. Those patients with poor quality of serum, other histology types or previous lung cancer surgery were excluded. All the patients’ samples originated pairs of wax blocks (cancer and its corresponding normal lung tissue taken 5 cm away from tumor). Five milliliters of blood from patients and healthy individuals were obtained and serum was collected and kept at -80 o C until used for measuring HMGB-1 and NPM/B23 by ELISA. Measurement of HMGB 1 and NPM/B23 in serum. HMGB-1 in serum was quantified used a commercially available Human High-Mobility Protein 1 (HMGB 1) ELISA Kit (ml026429), Shanghai mlbio Technology Co., LTD. On the other hand, NPM/B23 was determined by using a Human Nucleolar Phosphoryl Protein (NPM) ELISA Kit (ml058225), Shanghai mlbio Technology Co., LTD. Immunohistochemistry and scoring criteria in tissue samples from NSCLC patients. In total, 167 paraffin-embedded NSCLC and healthy tissue samples were submitted to immunohistochemistry analysis by using a Ready to Use Immunohistochemical Hypersensitivity UltraSensitiveTM SP kit (rabbit / rat) (KIT-9729), Fuzhou Maixin Biotechnology Development Co., LTD. Serial sections of tumor specimens were cut and stained with antibodies specific for both alarmins. Recombinant Anti-HMGB1 Antibody [EPR3507] (ab79823), and Recombinant Anti-Nucleophosmin Antibody [EP1848Y] (ab52644), Abcam (Shanghai) Trading Co., Ltd, were used as primary antibodies for detecting HMGB-1 and NPM/B23 respectively. Samples were scored independently by two observers. Briefly, the slides were scored based on intensity (negative, 0; weak, 1+; weak positive, 2+; positive, 3+; and strongly positive, 4+), and percentage of positively stained tumor cells 75% (4+). Data were expressed as positive expression rate which correspond to the number of positive sections respect total sections × 100. Statistical Analysis: The quantitative variables are reported as mean, standard deviation (SD), median, interquartile range, and range. The qualitative are reported as absolute and relative frequencies. The expression of NPM/B23 and HMGB-1 in patients and healthy subjects was compared using the Mann-Whitney U test. A generalized linear model was used to explore the association between NPM/B23 and HMGB-1 (each one) regarding clinicopathological parameters. Spearman’s rank correlation was used to assess relationships among NPM/B23 and HMGB-1 globally and stratified by tumoral size and Spread Through Airspaces (STAS). Statistical analyses were done using SPSS version 26. The level of significance chosen was α = 0.05. Declarations Competing interest: The authors have nothing to declare Funding. This work was supported by MOST "National key R&D program of China (2021YFE0192100)" and “The Science and Technology Innovation Program of Hunan Province”, China, (2024RC4027)”. Author Contribution YP and SEP conceived the original research idea for the project. YP is the principal investigator of the project. SEP wrote the manuscript. WL contributed to the conceptualization and overall supervision. CV performed and conducted data analysis. TH and LL conducted the experimentation in this work. YY carried out technical assistance and data collection. Both QZ and ZZ co-supervised the work in the project and helped with technical training, data analysis and interpretation of data. All authors have read and approved final version of the manuscript. Acknowledgement The authors would like to thank to Ke Yang and Changyuan Tan for administrative and logistic supporting of this work. Data Availability Data is provided within the manuscript or supplementary information files References Arbour, K. C. & Riely, G. J. Systemic Therapy for Locally Advanced and Metastatic Non-Small Cell Lung Cancer: A Review. JAMA 322 , 764–774 (2019). Nie, Y., Yang, D. & Oppenheim, J. J. Alarmins and Antitumor Immunity. Clin. Ther. 38 (5), 1042–1053 (2016). Zhang, J. et al. Extracellular HMGB1 exacerbates autoimmune progression and recurrence of type 1 diabetes by impairing regulatory T cell stability. Diabetologia 63 , 987–1001 (2020). Bertheloot, D. & Latz, E. HMGB1, IL-1α, IL-33 and S100 proteins: dual-function alarmins. Cell. Mol. Immunol. 14 , 43–64 (2017). Idoudi, S. et al. Role of HMGB1 and its associated signaling pathways in human malignancies. Cell. Signal. 112 , 110904 (2023). Singh, A. et al. 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Rep. 22 , 535–539 (2009). Nawa, Y. et al. Nucleophosmin may act as an alarmin: implications for severe sepsis. J. Leukoc. Biol. 86 , 645–653 (2009). Akın, G., Esbah, O. & Eröz, R. Could nucleolin and nucleophosmin levels be prognostic indicators in non-small cell lung cancer? Acta fac. medic Naissensis . 39 , 433–442 (2022). Yang, H. et al. Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1). Mol. Med. 27 , 58 (2021). Venereau, E. et al. Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release. J. Exp. Med. 209 , 1519–1528 (2012). Kazama, H. et al. Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity 29 , 21–32 (2008). Lindström, M. S. NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling. Biochem. Res. Int. 2011 , 195209 (2011). DiCarlo, A. et al. The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo. Int. J. Mol. Sci. 22 , 3672 (2021). Nakatomi, K. et al. TLR4/MD–2 is a receptor for extracellular nucleophosmin 1. Biomedical Rep. 14 , 21 (2021). Jia, M., Yu, S., Gao, H. & Sun, P. L. Spread Through Air Spaces (STAS) in Lung Cancer: A Multiple-Perspective and Update Review. Cancer Manag Res. 12 , 2743–2752 (2020). Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx FigureS1.jpg Figure S1: Illustrative immunohistochemistry of HMGB-1 protein expression in tissues from NSCLC patients. Paraffin-embedded tumor and healthy tissue samples were sliced and submitted to immunohistochemistry analysis by using a recombinant anti-HMGB1 antibody and UltraSensitiveTM SP kit. Illustrative light microscopy images of HMGB-1 expression in tumor and healthy tissues from the same patient, are presented at 40X magnification. Healthy adjacent tissue (A) and tumor tissue (B) from one LUAD patient. Healthy adjacent tissue (C)and tumor tissue (D) from one LUSC patient. Brown staining at the nuclear area corresponds to HMGB-1 expression. FigureS2.jpg Figure S2: Illustrative immunohistochemistry of NPM/B23 protein expression in tissues from NSCLC patients. Paraffin-embedded tumor and healthy tissue samples were sliced and submitted to immunohistochemistry analysis by using a recombinant anti-NPM/B23 antibody and UltraSensitiveTM SP kit. Illustrative light microscopy images of NPM/B23 expression in tumor and healthy tissues from the same patient are presented at 40X magnification. Healthy adjacent tissue (A)and tumor tissue (B) from one LUAD patient. Healthy adjacent tissue (C)and tumor tissue (D) from one LUSC patient. Brown staining at nucleolar and nuclear area corresponds to NPM/B23 expression. SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Feb, 2026 Reviews received at journal 29 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 05 Dec, 2025 Reviews received at journal 06 Oct, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers invited by journal 10 Jul, 2025 Editor assigned by journal 10 Jul, 2025 Editor invited by journal 10 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 08 Jul, 2025 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. 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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-7021384","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":484466347,"identity":"0e9004fb-61ff-4d82-a352-2734962014a5","order_by":0,"name":"Tan Hui","email":"","orcid":"","institution":"University of South China","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"","lastName":"Hui","suffix":""},{"id":484466348,"identity":"707cf43a-e9f4-42f5-8c10-2502fbed45b3","order_by":1,"name":"Liu Lu","email":"","orcid":"","institution":"The First Affiliated Hospital of University of South China","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Lu","suffix":""},{"id":484466349,"identity":"569edd8e-d635-47af-b810-d081c67b51eb","order_by":2,"name":"Yi Ying","email":"","orcid":"","institution":"China-Cuba Biotechnology Joint Innovation Center (CCBJIC), Yongzhou Development and Construction Investment Co., Ltd. (YDCI)","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Ying","suffix":""},{"id":484466350,"identity":"ae4feda1-cb47-4d4f-b9f2-6cc4b651ca90","order_by":3,"name":"Carmen Valenzuela","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Valenzuela","suffix":""},{"id":484466351,"identity":"ff39f813-0f21-409e-8718-14ab6fcd31ef","order_by":4,"name":"Zhao Qiang","email":"","orcid":"","institution":"The First Affiliated Hospital of University of South China","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Qiang","suffix":""},{"id":484466352,"identity":"1aae3d0e-91e5-4795-890d-0a9e5aa3751b","order_by":5,"name":"Zhang Zhiwei","email":"","orcid":"","institution":"University of South China","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Zhiwei","suffix":""},{"id":484466353,"identity":"54a6c050-e572-4f56-92d7-7fcb1d8e4ec3","order_by":6,"name":"Li Wen","email":"","orcid":"","institution":"China-Cuba Biotechnology Joint Innovation Center (CCBJIC), Yongzhou Development and Construction Investment Co., Ltd. (YDCI)","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wen","suffix":""},{"id":484466354,"identity":"3618bbc9-fc29-4241-a706-9b70323f96af","order_by":7,"name":"Silvio E. Perea","email":"","orcid":"","institution":"China-Cuba Biotechnology Joint Innovation Center (CCBJIC), Yongzhou Development and Construction Investment Co., Ltd. (YDCI)","correspondingAuthor":false,"prefix":"","firstName":"Silvio","middleName":"E.","lastName":"Perea","suffix":""},{"id":484466355,"identity":"8bfc5892-4d73-4b45-b544-f9d191603887","order_by":8,"name":"Yasser Perera","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYLCDAwwVDAwGDAcbSNFyhlQtDIxtIC0EgMHt5mMfPu6xy+PnX/vwcOE8O3lzxsONHxhqbKJxarlzLHnmjGfJxZIznhscnrkt2XBnw8FmCYZjabm4nGdwI8eYmecAc+KGG8cYDvNuO5BgcOBgGwNjw2E8WvI/M/85UJ+4H6xlDlFacpiZGQ4cTtzA3wbU0kCEFsk7x4wZew4cT5xxg43hMM8xqF8S8PiF73bzY4YfB6oT+/uPMX/mqQGGmMTxhx8+1Njg1KJwA8aSSIAxDjAwJGBTCwXyM2As/gMwBi7jR8EoGAWjYKQCANttZ4nBLcc+AAAAAElFTkSuQmCC","orcid":"","institution":"China-Cuba Biotechnology Joint Innovation Center (CCBJIC), Yongzhou Development and Construction Investment Co., Ltd. (YDCI)","correspondingAuthor":true,"prefix":"","firstName":"Yasser","middleName":"","lastName":"Perera","suffix":""}],"badges":[],"createdAt":"2025-07-01 14:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7021384/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7021384/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-43471-2","type":"published","date":"2026-03-07T15:58:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86674308,"identity":"b09029c3-6f2c-489c-8f90-4ded89eef7b9","added_by":"auto","created_at":"2025-07-14 11:53:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculating levels of HMGB-1 and NPM/B23 in serum.\u003c/strong\u003e Levels of each alarmin were determined by ELISA in serum from 162 NSCLC patients and 60 healthy individuals. NSCLC patients were segregated according to LUAD and LUSC histology. Statistical Significant is represented as * (p\u0026lt;0.05); ** (p\u0026lt;0.01); *** (p\u0026lt;0.001). \u003cstrong\u003eA\u003c/strong\u003e: a significant reduction of HMGB-1 in NSCLC (*) and LUAD (**) patients compared to healthy individuals was observed. \u003cstrong\u003eB\u003c/strong\u003e: a significant increase of NPM/B23 in NSCLC (***), LUAD (***) and LUSC (***) compared to healthy individuals was observed.\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/383c3b4933cd89e24edfb834.jpeg"},{"id":104252006,"identity":"43e0de8a-c197-48d3-b6da-45b24445e0f5","added_by":"auto","created_at":"2026-03-09 16:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":758313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/9f780708-a1bc-4f23-b198-f7f119ec42a7.pdf"},{"id":86674309,"identity":"2db4d4cb-0db9-43ba-989e-e8c1324aa28a","added_by":"auto","created_at":"2025-07-14 11:53:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34676,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/01e57b602e191025e370e50b.docx"},{"id":86674314,"identity":"0dc04531-404d-4ba8-914a-19c8a9f2145b","added_by":"auto","created_at":"2025-07-14 11:53:03","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":340109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1:\u003c/strong\u003e \u003cstrong\u003eIllustrative immunohistochemistry of HMGB-1 protein expression in tissues from NSCLC patients.\u003c/strong\u003e Paraffin-embedded tumor and healthy tissue samples were sliced and submitted to immunohistochemistry analysis by using a recombinant anti-HMGB1 antibody and UltraSensitiveTM SP kit. Illustrative light microscopy images of HMGB-1 expression in tumor and healthy tissues from the same patient, are presented at 40X magnification. Healthy adjacent tissue \u003cstrong\u003e(A)\u003c/strong\u003e and tumor tissue \u003cstrong\u003e(B)\u003c/strong\u003e from one LUAD patient. Healthy adjacent tissue \u003cstrong\u003e(C)\u003c/strong\u003eand tumor tissue \u003cstrong\u003e(D)\u003c/strong\u003e from one LUSC patient. Brown staining at the nuclear area corresponds to HMGB-1 expression.\u003c/p\u003e","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/e26ea29acd8558dd73def323.jpg"},{"id":86674316,"identity":"15d54bf5-bbc3-4114-8537-ee022148252b","added_by":"auto","created_at":"2025-07-14 11:53:03","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":335006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2:\u003c/strong\u003e \u003cstrong\u003eIllustrative immunohistochemistry of NPM/B23 protein expression in tissues from NSCLC patients.\u003c/strong\u003e Paraffin-embedded tumor and healthy tissue samples were sliced and submitted to immunohistochemistry analysis by using a recombinant anti-NPM/B23 antibody and UltraSensitiveTM SP kit. Illustrative light microscopy images of NPM/B23 expression in tumor and healthy tissues from the same patient are presented at 40X magnification. Healthy adjacent tissue \u003cstrong\u003e(A)\u003c/strong\u003eand tumor tissue \u003cstrong\u003e(B)\u003c/strong\u003e from one LUAD patient. Healthy adjacent tissue \u003cstrong\u003e(C)\u003c/strong\u003eand tumor tissue \u003cstrong\u003e(D)\u003c/strong\u003e from one LUSC patient. Brown staining at nucleolar and nuclear area corresponds to NPM/B23 expression.\u003c/p\u003e","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/7e1f6b09c9b36ffd067b8d40.jpg"},{"id":86676770,"identity":"fa62f721-b167-46be-aad1-088766771f99","added_by":"auto","created_at":"2025-07-14 12:17:04","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":31961,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7021384/v1/16a47f7b6c62732a6b3a8d37.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circulating levels of High Mobility Group Box-1 and Nucleophosmin/B23 proteins and clinical significance in debut Non-Small Cell Lung Cancer patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer remains one of the leading causes of cancer-related deaths each year worldwide, largely due to its late clinical presentation and the limited availability of biomarkers capable of predicting early diagnosis, disease progression, clinical response and overall survival. However, currently, the majority of the identified biomarkers for non-small cell lung cancer (NSCLC) are actionable gene mutations, which promote personalized molecular targeted therapies. The above therapies yield a successful objective clinical response in ~30% of patients\u0026nbsp;\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e. Therefore, identifying new biomarkers reflecting the natural course of NSCLC from its onset, is of great importance for public health worldwide. Ideally, such biomarkers should be easily measurable and preferably detectable by non-invasive methods. Alarmins, which can promote, recruit, activate, and mature dendritic cells\u0026nbsp;\u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e, could potentially serve as soluble biomarkers for the diagnosis, progression, and/or treatment of NSCLC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMore particularly, the present study primarily focused on the clinical significance of circulating levels of high mobility group box 1 (HMGB-1), which is a widely studied alarmin not only in cancer but also in autoimmunity and diabetes\u0026nbsp;\u003cstrong\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e. Additionally, the coexistence of circulating HMGB-1 with nucleophosmin/B23 (NPM/B23), a molecule that has not been studied as alarmin in cancer, was also investigated.\u003c/p\u003e\n\u003cp\u003eOn the other hand, a dual role, both pro- and anti-tumor, has been suggested for alarmin by other authors\u0026nbsp;\u003cstrong\u003e\u003csup\u003e4\u003c/sup\u003e\u003c/strong\u003e. For example, a study indicated that intracellular HMGB-1, a non-histone chromatin-associated protein, could play a crucial role in tumor growth, affect response to therapy and promote both cell death and survival\u0026nbsp;\u003cstrong\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/strong\u003e. However, it was also reported that this alarmin could be involved in different cancer progression-related pathways and could enhance the efficacy of immune checkpoint blockade in patients with cancer\u0026nbsp;\u003cstrong\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/strong\u003e. As a putative biomarker in cancer, the role of HMGB-1 in cancer has been supported by several studies. For example, a study demonstrated that the expression levels of HMGB-1 were increased in tumor tissues and serum of patients with urothelial carcinoma of the bladder\u0026nbsp;\u003cstrong\u003e\u003csup\u003e6\u003c/sup\u003e\u003c/strong\u003e. Additionally, in advanced lung cancer, small cell lung carcinoma (SCLC) and mesothelioma, high HMGB1 levels were associated with shorter overall survival in patients with NSCLC\u0026nbsp;\u003cstrong\u003e\u003csup\u003e7\u003c/sup\u003e\u003c/strong\u003e. In pancreatic ductal adenocarcinoma (PDAC), HMGB1 displayed a better diagnostic value compared with the traditional biomarkers, such as carbohydrate antigen 19-9 or carcinoembryonic antigen\u0026nbsp;\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. Furthermore, other studies revealed that HMGB-1 could predict gastric cancer progression\u0026nbsp;\u003cstrong\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/strong\u003e and response to chemotherapy in breast cancer\u0026nbsp;\u003cstrong\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntracellular NPM/B23, also known as NPM1, B23, No38 or numatrin, is a phosphoprotein mainly localized in the nucleolus\u0026nbsp;\u003cstrong\u003e\u003csup\u003e11\u003c/sup\u003e\u003c/strong\u003e. It is involved in several biological processes, including mRNA transport, chromatin remodeling, genome stability and apoptosis. NPM/B23 is commonly overexpressed in solid tumors and it is therefore mainly associated with increased mitotic activity and metastasis\u0026nbsp;\u003cstrong\u003e\u003csup\u003e12\u003c/sup\u003e\u003c/strong\u003e. However, there is limited data on the presence of circulating NPM/B23 in the serum of patients with cancer and/or its potential role as an alarmin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo the best our knowledge, no prior studies have investigated the clinical significance of the coexistence of circulating HMGB-1 and NPM/B23 in the serum of patients with cancer. Therefore, the present study aimed to investigate whether these two proteins were associated with each other and/or with clinicopathological parameters in newly diagnosed patients with NSCLC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDemographic and clinicopathological characteristics.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mean age of patients in the NSCLC group was 61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8 years, while that in the control group was 62.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 years. Among the total of 232 patients with NSCLC enrolled in the present study, 98 (42.2%) and 134 (57.8%) were females and males, respectively. The control group composed of 30 male and 30 female subjects. Among all patients with NSCLC, 162 (69.8%) were diagnosed with LUAD and 70 (30,2%) with LUSC. Clinical stages were categorized into two major cohorts: Stage I and stages II, III and IV. The mean tumor size, according to the maximum diameter, was 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 cm. In addition, other clinical conditions considered in the current study included the presence or absence of lymphatic metastasis (23 \u003cem\u003evs.\u003c/em\u003e 191 patients), pleural invasion (23 \u003cem\u003evs.\u003c/em\u003e 191 patients) and STAS (79 \u003cem\u003evs.\u003c/em\u003e 135 patients). Tumor differentiation was classified as low (65 patients), mid (125 patients), mid-low (5 patients), high (23 patients) and high-mid (1 patient; Table I). Patients with missing clinical data or inadequate samples were excluded from the correlation analysis. Data on clinical response to standard anticancer therapy and overall survival were collected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLevels of circulating alarmins in patients with NSCLC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the median HMGB-1 levels were significantly lower in patients with NSCLC (30.04 ng/ml) compared with healthy individuals (37.30 ng/ml; P\u0026thinsp;=\u0026thinsp;0.02). Interestingly, patients with LUAD, but not those with LUSC, displayed markedly reduced HMGB-1 levels compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, HMGB-1 levels were not significantly different between patients with LUAD and LUSC. By contrast, the median NPM/B23 levels were notably enhanced in patients with NSCLC (812 pg/ml) compared with healthy individuals (551 pg/ml; P\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). When both histological subtypes were analyzed separately, circulating NPM/B23 levels were also significantly higher in both patients with LUAD and LUSC compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). No significant difference in circulating NPM/B23 levels was observed between patients with LUAD and LUSC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssociation between circulating alarmin levels and clinicopathological features of patients with NSCLC\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAfter assessing the serum levels of both alarmins in patients with NSCLC, the present study then investigated whether these levels were associated with any particular clinicopathological feature. Interestingly, the univariate analysis shown in Table I revealed a strong association between tumor size and HMGB-1 serum levels in patients with NSCLC. More particularly, significant higher HMGB-1 levels were positively associated with tumors of \u0026gt;\u0026thinsp;2.0 cm in maximum diameter (P\u0026thinsp;=\u0026thinsp;0.004), regardless of tumor histology. However, no other associations were recorded between circulating HMGB-1 levels and other clinicopathological features. When generalized linear models were applied using HMGB-1 as a dependent variable and incorporating all available clinical features, it was verified that only tumor size was associated with HMGB-1 levels (Table SI). Subsequently, potential associations between circulating NPM/B23 levels and particular clinicopathological features in patients with NSCLC were evaluated. As shown in Table II, the presence of STAS pattern (P\u0026thinsp;=\u0026thinsp;0.043) and a tumor size of \u0026gt;\u0026thinsp;2 cm (P\u0026thinsp;=\u0026thinsp;0.0001) were associated with higher serum levels of NPM/B23 in patients with NSCLC. However, data from the multivariate analysis revealed that only a tumor size of \u0026gt;\u0026thinsp;2 cm was significantly associated with circulating NPM/B23 levels (Table SII).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorrelation between circulating HMGB-1 and NPM/B23 serum levels in patients with NSCLC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify whether circulating HMGB-1 and NPM/B23 levels were associated with each other, the presence of both alarmins in the serum of healthy individuals and patients with NSCLC were analyzed. Therefore, as shown in Table III, the serum levels of both HMGB-1 and NPM/B23 were associated with each other in both cohorts. However, their association was more significant in patients with NSCLC compared with healthy individuals (r\u0026thinsp;=\u0026thinsp;0.679 \u003cem\u003evs.\u003c/em\u003e r\u0026thinsp;=\u0026thinsp;0.278). As tumor size and the presence of STAS pattern were associated with the serum levels of circulating alarmins in the NSCLC group, the present study aimed to further investigate the association of the above two clinicopathological features with HMGB-1 and NPM/B23 levels. Interestingly, the analysis showed that the levels of both alarmins were significantly associated with a tumor size of \u0026gt;\u0026thinsp;2 cm (r\u0026thinsp;=\u0026thinsp;0.70), regardless the presence or not of STAS (Table IV). However, the strongest association between both circulating alarmins was observed in patients with NSCLC with tumors of \u0026le;\u0026thinsp;2 cm in diameter and the presence of STAS (r\u0026thinsp;=\u0026thinsp;0.900; P\u0026thinsp;=\u0026thinsp;0.0001; Table IV). The presence of STAS pattern indicates the spread of tumor cells, as micropapillary structures, solid nests, and/or single cells, within the airspaces of the lung parenchyma beyond the edge of the main tumor \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. It is also associated with a higher risk of recurrence in small lung adenocarcinomas, where limited resection can be insufficient for patients exhibiting STAS \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, the strong association between HMGB-1 and NPM/B23 levels in the presence of STAS and tumors of \u0026le;\u0026thinsp;2 cm in diameter could suggest either early disease progression or a more aggressive form of NSCLC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of HMGB-1 and NPM/B23 in tumor and adjacent healthy lung tissues.\u003c/b\u003e Finally, the current study aimed to explore the intratumoral expression of both HMGB-1 and NPM/B23 alarmins in patients with NSCLC by immunohistochemistry, using tumor biopsies and adjacent healthy lung tissues, located 5 cm from tumor, as internal control. Representative immunohistochemistry images of intracellular HMGB-1 expression in both tumor and adjacent healthy tissues are illustrated in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. HMGB-1 was predominantly expressed in the nuclear compartment, not only in alveolar epithelial cells, but also in phagocytes in the alveolar space, fiber cells in the alveolar septum and fibrocytes. To semi-quantify the immunohistochemistry results, the HMGB-1 positive expression rate was calculated as the sum of patients exhibiting low (+), moderate (++) and high (+++) intracellular expression. As shown in Table V, among 167 patients with NSCLC, the positive expression rate of HMGB-1 in tumor tissues was 78.44% compared with 86.83% in adjacent healthy lung tissues (P\u0026thinsp;=\u0026thinsp;0.043). Notably, a distinct dichotomous expression pattern of HMGB-1 was observed based on NSCLC histological subtypes. Therefore, in patients with LUAD the positive expression rate of HMGB-1 was significantly lower compared with that in adjacent healthy tissues (P\u0026thinsp;=\u0026thinsp;0.0001). By contrast, in patients diagnosed with LUSC the positive expression rate was notably higher in tumor tissues compared with adjacent healthy ones (P\u0026thinsp;=\u0026thinsp;0.001). Interestingly, more than 50% of adjacent healthy tissues also displayed positive HMGB-1 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequently, whether a potential association existed between intracellular HMGB-1 levels in tumors and adjacent healthy tissues was assessed. Therefore, a statistically significant, but weak, positive association was found between intracellular HMGB-1 expression in tumors and their corresponding adjacent normal tissues counterpart (P\u0026thinsp;=\u0026thinsp;0.0001; r\u0026thinsp;=\u0026thinsp;0.348; Table SIII). However, no significant association was observed between circulating serum HMGB-1 levels and tumor or normal tissues (Table SIII). By contrast, the immunohistochemistry analysis revealed a nuclear or nuclear/nucleolar expression pattern for the NPM/B23 alarmin (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Unlike HMGB-1, the overall positive NPM/B23 expression rate in tumor tissues was notably higher compared with that observed in adjacent healthy tissues (92.22 \u003cem\u003evs.\u003c/em\u003e 85.03%; P\u0026thinsp;=\u0026thinsp;0.0387; Table VI). More particularly, the positive NPM/B23 expression rate in tumor tissues from patients with LUAD was 91.54% compared with 83.08% recorded in adjacent healthy tissues. Interestingly, in patients with LUSC the positive expression rate of NPM/B23 was not significantly different between tumor and adjacent healthy tissues (94.60 \u003cem\u003evs.\u003c/em\u003e 91.89%; P\u0026thinsp;=\u0026thinsp;0.6433; Table VI). Nonetheless, NPM/B23 showed a significantly increased expression rate in patients with NSCLC. Similarly, with HMGB-1, a significant but weak positive association was also observed between intracellular NPM/B23 expression in tumor and adjacent healthy tissues (P\u0026thinsp;=\u0026thinsp;0.0001; r\u0026thinsp;=\u0026thinsp;0.394; Table SIV). Consistently, no association was obtained between circulating NPM/B23 levels in serum and tissue expression in either tumor or normal tissues (Table SIV). Given the increased positive expression rate of both HMGB-1 and NPM/B23 in adjacent healthy tissues, the expression of Ki-67, a cell proliferation biomarker that is not an alarmin, was determined to verify specificity. As expected, Ki-67 expression was very weak in adjacent healthy tissues compared with NSCLC tissues (2.2 \u003cem\u003evs\u003c/em\u003e 56.7%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally, to assess whether HMGB-1 and NPM/B23 alarmins were associated at the tissue level, a Spearman coefficient analysis was performed in both malignant and adjacent normal tissues. Unlike the findings in serum, only a weak correlation between the two alarmins was observed in tumor tissues (P\u0026thinsp;=\u0026thinsp;0.02; r\u0026thinsp;=\u0026thinsp;0.223; Table SV), but not in adjacent healthy tissues (Table SVI).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEarly detection of novel disease biomarkers in NSCLC can optimize clinical decision-making for therapy, but can also complement computed tomography (CT) scan and chest radiography, thus helping to mitigate cancer-related mortality and improve overall prognosis in lung cancer. As mentioned above, alarmins have been proposed as biomarkers for the diagnosis, progression, and/or treatment of NSCLC. In particular, previous meta-analyses suggested that the HMGB-1 alarmin was upregulated in both tumors and serum of patients with NSCLC compared with healthy individuals \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, supporting its potential as a biomarker for predicting disease progression and survival. However, other studies demonstrated that HMGB1 was downregulated in NSCLC tumor tissues compared with matched normal lung tissues \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The present study investigated the relevance of pretherapeutic circulating and tissue levels of HMGB-1, as well as those of MPN/B23, which has been suggested as a secreted alarmin in sepsis and found to be elevated in patients with NSCLC \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. More particularly, herein, the clinical significance of the co-expression of both proteins in relation to key clinicopathological parameters in newly diagnosed patients with NSCLC was assessed. In addition, the association between HMGB-1 and NPM/B23 at both systemic and tissue levels was investigated in tumor samples. The results demonstrated that the median global circulating HMGB-1 levels in the serum of patients with NSCLC were lower compared with those observed in healthy individuals, and more particularly in patients diagnosed with LUAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It is currently known that different oxidation states of HMGB-1 can lead in three mutually exclusive functions, acting as an alarmin \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, and promoting chemoattraction \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e or tolerance \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, it would be of great interest to determine whether a specific redox status of HMGB-1 occurs in patients with LUAD and whether this status could be associated with clinical outcome, disease progression, or survival. Nonetheless, the results showed that elevated individual HMGB-1 levels were positively associated with a tumor size of \u0026gt; 2 cm in diameter (P = 0.004; Table I), regardless of NSCLC histological subtype. This finding could be associated with the clinical staging of patients and could serve as an indicator of tumor progression in both LUAD and LUSC. These results were consistent with previous ones showing positive association between circulating HMGB-1 levels and tumor size, among other clinicopathological features \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Although a correlation between HMGB-1 with other clinicopathological parameters were not found in patients with NSCLC, the current study was the first to report a 2-cm tumor size threshold as clinically significant in relation with HMGB-1 expression. Notably, none of the correlations observed between alarmins and clinicopathological features were associated with the NSCLC histological subtype, which could be due to the sample size imbalance in the cohort included in the study (LUAD, n = 124 \u003cem\u003evs.\u003c/em\u003e LUSC, n = 38).\u003c/p\u003e\u003cp\u003eNPM/B23, a multifunctional protein involved in cancer, exerts chaperone activity and plays a key role in ribosomal biogenesis. Previous studies demonstrated that NPM/B23 was upregulated in patients with cancer \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Although only a few studies have investigated the extracellular role of NPM/B23 as an alarmin, experimental evidences have suggested that beyond being a potential biomarker, it can also serve as a therapeutic target. For instance, a previous study indicated that NPM/B23 was physiologically secreted by endothelial cells under stress, thus exerting proangiogenic activity both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e through the secretion of vascular endothelial growth factor-A, hepatocyte growth factor, stromal derived factor-1, fibroblast growth factor-2, platelet derived growth factor-B, and matrix metallopeptidase 9 \u003csup\u003e\u003cb\u003e23\u003c/b\u003e\u003c/sup\u003e. Consistently, recombinant NPM/B23 could induce the release of the proinflammatory cytokines, tumor necrosis factor-, IL-6 and monocyte chemoattractant protein-1 from RAW264.7 cells and increase the expression levels of intercellular adhesion molecule 1 in human umbilical vein endothelial cells, thus supporting its additional role in systemic inflammation \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Interestingly, the toll-like receptor 4/myeloid differentiation factor 2 complex has been identified as a receptor which is required for the extracellular function of NPM/B23 \u003csup\u003e\u003cb\u003e24\u003c/b\u003e\u003c/sup\u003e. Consistently, other studies reported elevated circulating levels of NPM/B23 in patients with NSCLC compared with healthy individuals, a positive association with the presence of nodules, and markedly higher levels of NPM/B23 in patients with LUAD compared with those with LUSC \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In agreement with the aforementioned findings, the results of the present study revealed significantly higher levels of NPN/B23 in patients with NSCLC across both histological subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, both the univariate and multivariate analyses identified a notable association between elevated NPM/B23 levels and tumor size of \u0026gt; 2 cm (P = 0.0001; Table II), regardless of histological subtype. Additionally, higher circulating levels of NPM/B23 were modestly associated with the presence of STAS pattern (P = 0.043). Since the presence of STAS pattern in NSCLC indicated early spread of tumor cells through airspaces adjacent to the primary tumor \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, NPM/B23 could serve as a valuable prognostic biomarker for assessing the risk of disease progression. However, the univariate and multivariate analyses did not reveal any significant associations between NPM/B23 levels and other clinicopathological features. The above findings indicated that both HMGB-1 and NPM/B23 were individually associated with clinical indicators of disease progression, such as tumor size and STAS pattern, independent of NSCLC histological subtype. In the case of B23/NPM, the results could provide further evidence supporting the role of NPM/B23 as an alarmin in newly diagnosed patients with NSCLC.\u003c/p\u003e\u003cp\u003eAfter evaluating the clinical relevance of HMGB-1 and NPM/B23, the present study aimed to analyze the association between both proteins in serum and tissue samples from patients with NSCLC. Importantly, the results revealed a positive association between both proteins in the serum of newly diagnosed patients with NSCLC (Table III), reinforcing the hypothesis that NPM/B23 could also act as an alarmin in this clinical setting. Considering that the individual analysis of circulating HMGB-1 and NPM/B23 levels was associated with clinical parameters involved in disease progression, such as tumor size and the presence of STAS, whether this correlation was maintained across the abovementioned clinicopathological parameters was subsequently investigated. A significant positive correlation was observed between HMGB-1 and NPM/B23 levels when tumor diameters were \u0026lt; 2 cm and in the presence of STAS (P = 0.0001; r = 0.900; Table VI). For tumors \u0026gt; 2 cm, a weak but significant association was obtained between both alarmins, regardless of the presence of STAS pattern. To the best of our knowledge, this study was first to report a direct positive association between circulating HGMB-1 and NPM/B23 levels in patients with cancer, as well as their association with specific tumor size thresholds and early tumor cell spreading. Although elevated individual levels of circulating HMGB1 and NPM/B23 could reflect changes associated with disease progression, the strong correlation observed between both proteins in smaller tumors accompanied with STAS could be useful for the early detection of the disease and/or for predicting the risk of NSCLC progression, particularly when combined with non-invasive imaging procedures, such as CT scan and Magnetic Resonance Imaging. In addition, early detection could assist oncologist practitioners in developing more effective therapeutic strategies. Therefore, the results of the current study warrant validation in larger patient cohorts.\u003c/p\u003e\u003cp\u003eConsidering that alarmins can be released from tumor cells during the tumor-host immune interactions and the necrotic processes inherent to solid tumors, subsequently, the expression levels of HMGB-1 and NPM/B23 were detected in tumor and normal adjacent lung tissues. The results indicated that, although the positivity rate or HMGB-1 expression in tumor tissues was \u0026gt; 50%, it was lower than that observed in matched adjacent healthy tissues, especially among patients with LUAD (Table V). By contrast, in patients with LUSC, this expression pattern was rather reversed, possibly due to the function of HMGB-1 as an alarmin. Therefore, further studies are needed to elucidate the significance of the differential expression of HMGB-1 in tumor tissues compared with adjacent healthy lung tissues in patients with LUAD (Table VI). On the other hand, the positive expression rate of NPM/B23 was \u0026gt; 80% in both tumor and healthy tissues, which was a novel observation in this study. By contrast, Ki-67, a common biomarker of cell proliferation which does not act as an alarmin, showed a very low positive expression rate (2.2%) in healthy tumor tissues of patients with NSCLC. To evaluate whether the co-expression of HMGB-1 and NPM/B23 in tumor tissues behaved similarly to that observed in the circulating levels, a correlation analysis was carried out. Interestingly, a weak correlation (P = 0.02; r = 0.223) was obtained, thus suggesting that future studies with a greater number of patients with NSCLC are needed to strengthen the above finding. Nonetheless, the weak correlation might be influenced by the semiquantitative output of immunohistochemistry compared with ELISA that is fully quantitative. No relevant associations between the intracellular expression levels of both proteins and individual clinicopathological parameters were identified. In addition, other potential biomarkers linked to tumor cell proliferation and lung cancer like Ki-67, AKT, phospho-AKT, Protein Kinase CK2 (CK2A1, CK2A2, CK2B), CDC37, and PDL-1 were investigated by immunohistochemistry in tumor biopsies but they did not exhibit the same correlation patterns with clinical parameters (unpublished data). Therefore, circulating alarmin-based biomarkers could be more useful over such biomarkers to support disease progression of NSCLC.\u003c/p\u003e\u003cp\u003eIn summary, the present study investigated the individual and correlative features of HMGB-1 and NPM/B23 alarmins in the serum and tissues of patients with NSCLC, who had not received any anticancer therapy. This study was the first to describe the correlations between these two proteins in the serum of patients with NSCLC and their clinical characteristics at the onset of the disease. However, future directions of the research must be focused on the validation of this potential alarmin-based biomarker of disease progression in larger NSCLC patient cohorts.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eSubjects\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eA total of 167 NSCLC patients and 60 age-matched healthy individuals were enrolled in this prospective study. The study was approved by the Ethics Committee of the First Affiliated Hospital of University of South China, Hunan Province and reviewed lot number No.2021LL0115001. Also, the study was conducted in accordance with ethical principles from Helsinki declaration (2013), the regulations on Biomedical Ethics Review involving human (2016), the regulations on Clinical Research Ethics review in traditional Chinese medicine and the International Ethical Guidelines for Biomedical Research. In addition, a consent was obtained by all the subjects included in this study. Although patient identification records are not published in this work, a consent for publication of the general data in this work was also obtained.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnrollment criteria for NSCLC patients and sample collection\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eOnly debut patients without previous chemoradiotherapy, targeted therapy or immunotherapy before surgery, were included. Also, patients with paraffin histology diagnosis after radical resection of lung cancer with LUAD or LUSC were eligible for the study. Those patients with poor quality of serum, other histology types or previous lung cancer surgery were excluded. All the patients’ samples originated pairs of wax blocks (cancer and its corresponding normal lung tissue taken 5 cm away from tumor). Five milliliters of blood from patients and healthy individuals were obtained and serum was collected and kept at -80\u003csup\u003eo\u003c/sup\u003eC until used for measuring HMGB-1 and NPM/B23 by ELISA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of HMGB 1 and NPM/B23 in serum.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHMGB-1 in serum was quantified used a commercially available Human High-Mobility Protein 1 (HMGB 1) ELISA Kit (ml026429), Shanghai mlbio Technology Co., LTD. On the other hand, NPM/B23 was determined by using a Human Nucleolar Phosphoryl Protein (NPM) ELISA Kit (ml058225), Shanghai mlbio Technology Co., LTD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry and scoring criteria in tissue samples from NSCLC patients.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn total, 167 paraffin-embedded NSCLC and healthy tissue samples were submitted to immunohistochemistry analysis by using a Ready to Use Immunohistochemical Hypersensitivity UltraSensitiveTM SP kit (rabbit / rat) (KIT-9729), Fuzhou Maixin Biotechnology Development Co., LTD. Serial sections of tumor specimens were cut and stained with antibodies specific for both alarmins. Recombinant Anti-HMGB1 Antibody [EPR3507] (ab79823), and Recombinant Anti-Nucleophosmin Antibody [EP1848Y] (ab52644), Abcam (Shanghai) Trading Co., Ltd, were used as primary antibodies for detecting HMGB-1 and NPM/B23 respectively. Samples were scored independently by two observers. Briefly, the slides were scored based on intensity (negative, 0; weak, 1+; weak positive, 2+; positive, 3+; and strongly positive, 4+), and percentage of positively stained tumor cells \u0026lt; 5% (negative); 5–20% (1+); 21–50% (2+); 51–75% (3+) and \u0026gt; 75% (4+). Data were expressed as positive expression rate which correspond to the number of positive sections respect total sections × 100.\u003c/p\u003e\u003ch2\u003eStatistical Analysis:\u003c/h2\u003e\u003cp\u003eThe quantitative variables are reported as mean, standard deviation (SD), median, interquartile range, and range. The qualitative are reported as absolute and relative frequencies. The expression of NPM/B23 and HMGB-1 in patients and healthy subjects was compared using the Mann-Whitney U test. A generalized linear model was used to explore the association between NPM/B23 and HMGB-1 (each one) regarding clinicopathological parameters. Spearman’s rank correlation was used to assess relationships among NPM/B23 and HMGB-1 globally and stratified by tumoral size and Spread Through Airspaces (STAS). Statistical analyses were done using SPSS version 26. The level of significance chosen was α = 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interest:\u003c/h2\u003e\n\u003cp\u003eThe authors have nothing to declare\u003c/p\u003e\n\u003ch2\u003eFunding.\u003c/h2\u003e\n\u003cp\u003eThis work was supported by MOST \u0026quot;National key R\u0026amp;D program of China (2021YFE0192100)\u0026quot; and \u0026ldquo;The Science and Technology Innovation Program of Hunan Province\u0026rdquo;, China, (2024RC4027)\u0026rdquo;.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eYP and SEP conceived the original research idea for the project. YP is the principal investigator of the project. SEP wrote the manuscript. WL contributed to the conceptualization and overall supervision. CV performed and conducted data analysis. TH and LL conducted the experimentation in this work. YY carried out technical assistance and data collection. Both QZ and ZZ co-supervised the work in the project and helped with technical training, data analysis and interpretation of data. All authors have read and approved final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank to Ke Yang and Changyuan Tan for administrative and logistic supporting of this work.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArbour, K. C. \u0026amp; Riely, G. J. 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Int.\u003c/em\u003e \u003cb\u003e2011\u003c/b\u003e, 195209 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiCarlo, A. et al. The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 3672 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakatomi, K. et al. TLR4/MD\u0026ndash;2 is a receptor for extracellular nucleophosmin 1. \u003cem\u003eBiomedical Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 21 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia, M., Yu, S., Gao, H. \u0026amp; Sun, P. L. Spread Through Air Spaces (STAS) in Lung Cancer: A Multiple-Perspective and Update Review. \u003cem\u003eCancer Manag Res.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 2743\u0026ndash;2752 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"High Mobility Group Box-1, HMGB-1, Nucleophosmin, NPM/B23, Alarmins, Non-Small Cell Cancer, NSCLC","lastPublishedDoi":"10.21203/rs.3.rs-7021384/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7021384/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlarmins are endogenous molecules that alert the immune system to circumvent the invading antigens in the host. Here, we investigated the serum levels of the high mobility group box 1 (HMGB-1) and nucleophosmin alarmins in a cohort of 162 newly diagnosed patients with non-small cell lung cancer and 60 age-matched healthy individuals. Interestingly, patients exhibited lower median values of HMGB-1 than healthy individuals, but higher levels of HMGB-1 were associated with a tumor size of \u0026gt; 2.0 cm. In addition, nucleophosmin levels were significantly increased in patients compared with the healthy individuals and those with tumors of \u0026gt; 2 cm in diameter also displayed higher circulating nucleophosmin. Furthermore, a strong association was reported between HMGB-1 and nucleophosmin levels in patients (r = 0.679; P = 0.0001) compared with healthy individuals. Interestingly, the strongest association between both alarmins was obtained in patients with Spread Through Airspaces pattern and tumors of ≤2 cm in size (r = 0.900; P = 0.0001). Co-expression of both alarmins was also observed in tumor tissues, although to a lesser extent. Altogether, our data indicate that high levels of each alarmin were individually associated with larger tumor size, however the strongest associations between both alarmins in NSCLC seemed to be primarily linked to early disease progression parameters.\u003c/p\u003e","manuscriptTitle":"Circulating levels of High Mobility Group Box-1 and Nucleophosmin/B23 proteins and clinical significance in debut Non-Small Cell Lung Cancer patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:52:59","doi":"10.21203/rs.3.rs-7021384/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-06T17:42:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T16:24:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285720455610576006266703459443211548050","date":"2026-01-12T15:43:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278004968974803225610162254267795539575","date":"2025-12-05T17:33:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T13:10:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59747084691583281320954303602704529766","date":"2025-09-12T07:31:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299472727240602218117748447930181848105","date":"2025-09-10T10:48:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68225562732931368533824299414418293080","date":"2025-08-06T14:32:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81162427610554979253208805921946888320","date":"2025-07-23T14:11:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T13:52:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-10T13:34:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-10T05:59:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T03:54:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-09T03:50:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f424ea0-b72e-4c69-9a8a-195a85b83da3","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51439167,"name":"Health sciences/Biomarkers"},{"id":51439168,"name":"Biological sciences/Cancer"},{"id":51439169,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-09T16:14:36+00:00","versionOfRecord":{"articleIdentity":"rs-7021384","link":"https://doi.org/10.1038/s41598-026-43471-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-07 15:58:59","publishedOnDateReadable":"March 7th, 2026"},"versionCreatedAt":"2025-07-14 11:52:59","video":"","vorDoi":"10.1038/s41598-026-43471-2","vorDoiUrl":"https://doi.org/10.1038/s41598-026-43471-2","workflowStages":[]},"version":"v1","identity":"rs-7021384","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7021384","identity":"rs-7021384","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}