Prognostic Significance of Programmed Death-Ligand 1 Expression in Betel Nut Chewing Patients with Oral Squamous Cell Carcinoma

Prognostic Significance of Programmed Death-Ligand 1 Expression in Betel Nut Chewing Patients with Oral Squamous Cell Carcinoma | 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 Prognostic Significance of Programmed Death-Ligand 1 Expression in Betel Nut Chewing Patients with Oral Squamous Cell Carcinoma Hui-Zhu Yang, Ling-Yu Kung, Yu-Chun Lin, Cheng-Yu Yang, Gu-Jiun Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7777087/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in BMC Oral Health → Version 1 posted 11 You are reading this latest preprint version Abstract Objective: This study aimed to investigate the prognostic significance of programmed death-ligand 1 (PD-L1) expression in Taiwanese patients with oral squamous cell carcinoma (OSCC) who underwent surgical treatment, particularly in those with a history of betel-nut chewing. Materials and Methods: We retrospectively analyzed the medical records of patients with OSCC diagnosed and treated at a single tertiary hospital between 2017 and 2023. Immunohistochemical evaluation of formalin-fixed, paraffin-embedded tumor specimens was performed using the validated anti–PD-L1 antibody clone 22C3. PD-L1 expression was evaluated using the Combined Positive Score (CPS), and CPS≥1 and ≥20 were defined as PD-L1 positivity and overexpression, respectively. An additional score was obtained by subtracting the CPS from the Tumor Proportion Score (TPS) to clarify the effect of the immune cells. The associations between PD-L1 expression and overall survival (OS), and disease-free survival were assessed using Kaplan–Meier and Cox regression analyses. Results: Of the 118 OSCCs, 56.8% and 31.4% exhibited PD-L1 positivity and overexpression, respectively. PD-L1 overexpression and TPS-CPS≥10 were significantly associated with improved OS ( p =0.022 and p =0.035, respectively). Multivariate analysis confirmed that PD-L1 overexpression and cancer stage were independent prognostic factors for OS ( p =0.010 and p =0.001, respectively). However, PD-L1 overexpression was not significantly correlated with tumor stage, primary site, or adverse pathological features. Conclusion: High PD-L1 expression is a favorable prognostic biomarker in patients with surgically treated OSCC, particularly in populations with prevalent betel nut use. These findings suggest that PD-L1 expression can guide immunotherapy decisions and risk stratification in OSCC management. Clinical relevance : Patients with OSCC exhibiting high PD-L1 expression have a better survival rate after surgery. PD-L1 testing may help in risk stratification and guiding immunotherapy decisions. Oral squamous cell carcinoma programmed death-ligand 1 immunohistochemistry prognosis betel nut immune checkpoint Combined Positive Score Figures Figure 1 Figure 2 Introduction Oral squamous cell carcinoma (OSCC), one of the most prevalent and aggressive malignancies in the head and neck region, results in high morbidity and mortality worldwide [1, 2]. Despite advancements in surgical techniques and adjuvant therapies, the prognosis of OSCC remains poor with a 5-year survival rate of approximately 60%, particularly in patients with advanced-stage disease or those with other pathologically adverse factors [3-5]. Thus, identifying reliable prognostic factors for risk stratification and informing therapeutic decision-making is necessary. Programmed death-ligand 1 (PD-L1), also known as CD274 or B7-H1, is a ligand of programmed death-protein 1 (PD-1) [6]. It is a transmembrane protein commonly expressed on the surface of tumor epithelial and immune cells [6, 7] and suppresses T-cell-mediated immune responses by binding to PD-1, allowing tumor cells to evade immune surveillance [3]. Elevated PD-L1 expression is associated with poor outcomes in several malignancies, including non-small cell lung cancer, advanced-stage melanoma, and gastrointestinal tumors [3, 8, 9]. However, its prognostic value in OSCC remains an area of active investigation, with several studies yielding conflicting results. Evaluation of PD-L1 expression may provide insights into the potential utility of immune checkpoint inhibitors (ICIs) as adjuvant or neoadjuvant strategies in surgically managed OSCC. Several scoring systems have been developed for quantifying PD-L1 expression in tumor specimens, including Tumor Proportion Score (TPS), Combined Positive Score (CPS), and Tumor Cell scoring [6, 10, 11]. TPS measures the percentage of PD-L1–positive tumor cells relative to all viable tumor cells, whereas CPS indicates the sum of PD-L1–positive tumor cells and immune cells (e.g., lymphocytes and macrophages). Conversely, the Tumor Cell score specifically evaluates PD-L1 expression in the tumor cells. These scoring systems are critical for predicting responses to ICIs and guiding cancer immunotherapy strategies [6, 12]. The choice of scoring method is often influenced by the monoclonal antibody clone used for staining, because different clones (such as 22C3 or 28-8) may result in distinct staining intensities and cellular localization patterns [6, 13]. This study aimed to elucidate the prognostic significance of PD-L1 expression in patients who underwent surgery for OSCC. By synthesizing the current evidence, we sought to clarify its role in predicting oncological outcomes and explore its implications for personalized treatment strategies. Patients and Methods Selection Criteria This retrospective observational study included 118 patients with OSCC diagnosed and managed at the Tri-Service General Hospital between January 2017 and December 2023 and followed up until death or December 2024. Patients with advanced-stage disease in whom surgery was infeasible and those with poor physical conditions, distant metastases, or incomplete data were excluded. Sample Preparation Formalin-fixed, paraffin-embedded oral cancer tissue specimens were sectioned at 4 µm thickness using a microtome and mounted on positively charged glass slides to enhance tissue adhesion. The slides were then baked at 60°C for at least 1 h to ensure proper fixation before deparaffinization and rehydration using xylene and graded ethanol, respectively. Antigen retrieval was performed using a heat-induced method in citrate or ethylenediaminetetraacetic acid buffer to unmask PD-L1 epitopes. Immunohistochemical (IHC) staining was performed using a validated PD-L1 antibody (22c3) and an automated or manual staining procedure. The slides were counterstained with hematoxylin for tissue contrast. After dehydration and application of a cover slip, the stained slides were examined by two board-certified pathologists under a light microscope. IHC Evaluation For CPS calculations, the two pathologists independently evaluated PD-L1 expression at 200× magnification. PD-L1–positive tumor cells, lymphocytes, and macrophages were counted across multiple high-power fields to estimate their proportions relative to the total number of viable tumor cells. Necrotic regions and artifacts were excluded, and disagreements between the two pathologists were resolved through consensus. CPS<1, ≥1, and ≥20 were defined as negative PD-L1 expression (Figure 1), positive PD-L1 expression, and PD-L1 overexpression, respectively (Figure 2). An additional score was obtained by subtracting CPS from TPS to determine further whether PD-L1 positivity was highly concentrated in tumor cells and whether immune cells affect the prognosis. Survival Patient survival data were obtained from the clinical records. The follow-up period was from the date of diagnosis to death or until December 2024. Overall survival (OS) was defined as the time from the date of diagnosis to death due to any cause. Disease-free survival was defined as the time from the date of curative treatment to the first instance of disease recurrence (local, regional, or distant metastasis) or death from any cause. Statistical analysis All statistical analyses were performed using the SPSS (version 20.0; IBM Corp., Armonk, NY, USA). The chi-square test was used to compare categorical variables, including age, sex, site, adverse factors for OSCC, alcohol consumption, cigarette smoking, and PD-L1 marker expression. Univariate Kaplan–Meier and multivariate Cox regression analyses were performed to determine the correlation of PD-L1 overexpression with OS, and disease-free survival after treatment. Statistical significance was set at p < 0.05. Results Clinicopathological characteristics of the patients This retrospective study included 118 patients, and the detailed clinicopathological characteristics of the study cohort are presented in Table 1. The cohort consisted predominantly of males (105 [89%]) and 13 females (11%). The median age at diagnosis was 57.9 years (range, 34–94 years), and the mean follow-up duration was 22.54 months (range, 0–76 months). According to the American Joint Committee on Cancer, 8th edition, most patients had stage IV disease (62/118, 52.5%) at diagnosis, followed by stages I (20/118, 16.9%) and III (20/118, 16.9%). The most common primary tumor subsites were the tongue (40/118, 33.9%) and buccal mucosa (37/118, 31.4%). PD-L1 expression in OSCC was stained using 22c3 IHC staining for PD-L1 was observed in the membranes, tumor cells, lymphocytes, and macrophages. Representative images of IHC staining for PD-L1 are shown in Figures 1 and 2. PD-L1 positivity and overexpression were observed in 56.8% (67/118) and 31.4% (37/118) of tumors, respectively. PD-L1 overexpression was observed in 55.2% (37/67) of all cases with PD-L1 positivity. Comparative analysis of PD-L1 expression and association with clinicopathological characteristics in OSCC. The clinicopathological characteristics of patients with OSCC, grouped according to PD-L1 expression level, are shown in Table 2. PD-L1 overexpression was not significantly associated with age, sex, primary site, adverse pathological factors, disease stage, cigarette smoking, or alcohol consumption. Survival analysis Kaplan–Meier curves revealed that OS was significantly better in patients with PD-L1-positive tumors than in those with PD-L1-negative tumors ( p=0.022 ). Univariate analysis showed that disease stage and PD-L1 overexpression were significantly correlated with OS ( p =0.001 and p =0.022, respectively). Multivariate analysis (Cox regression model) of these factors showed that disease stage and PD-L1 overexpression were significantly correlated with OS ( p =0.001 and p =0.010, respectively; Table 3). Furthermore, TPS-CPS≥10 was significantly associated with improved OS ( p =0.035). Discussion This study focused on a clinically and culturally distinct patient population. All participants were Taiwanese individuals with a history of betel nut chewing, which is a well-established risk factor for OSCC in Southeast Asia [4]. The inclusion of this relatively homogeneous cohort provides a unique opportunity to evaluate the prognostic significance of PD-L1 overexpression in high-risk groups with common environmental and behavioral exposures [14]. Considering the strong etiological association between betel nut use and oral carcinogenesis, our findings may reflect a tumor immune microenvironment specific to this subgroup, offering perspectives that differ from those of other studies based on populations without this exposure. Apart from betel nut chewing, cigarette smoking and alcohol consumption are common risk factors for OSCC [15, 16]. Some studies have reported lower PD-L1 expression in smokers due to reduced interferon-γ signaling, which is a potent PD-L1 inducer [2]. However, our results showed no significant association between cigarette smoking and OS. Generally, the prognosis of oral cancer is evaluated based on the presence of adverse pathological factors including extranodal extension, perineural invasion, lymphovascular invasion, depth of invasion, positive or close surgical margins, and metastatic lymph nodes [5, 17]. These histopathological features are associated with a higher risk of recurrence and poorer OS and are critical in guiding postoperative treatment decisions and prognosis assessment [3, 5, 17]. Additionally, advanced disease stage represents a worse prognosis, and the multivariate analysis in our study showed that staging was significantly correlated with OS. In this study, we evaluated PD-L1 expression using IHC in patients with locally advanced OSCC who underwent surgical resection. Notably, patients with PD-L1 overexpression had significantly better survival outcomes than those without. This observation contrasts with previous reports suggesting that PD-L1 overexpression correlates with poor prognosis in various malignancies, such as renal cell carcinoma, colorectal cancer, and lung cancer [14]. However, studies on patients with metastatic melanoma, non-small cell lung cancer, Merkel cell carcinoma, and laryngeal cancer have shown that PD-L1 overexpression, particularly when accompanied by a high density of tumor-infiltrating lymphocytes, is associated with prolonged survival [18, 19]. To emphasize the influence of tumor-infiltrating lymphocytes, we analyzed the differences between TPS and CPS. In this study, the survival rate was better in patients with PD-L1 overexpression than in those without. Conversely, most previous studies have reported an association between high PD-L1 expression and poor prognosis. Therefore, we examined the contribution of immune-cell staining to this outcome because the difference between TPS and CPS might reflect the influence of PD-L1 expression on immune cells more clearly [20]. This approach could help illustrate whether PD-L1 positivity within the tumor microenvironment, independent of tumor-cell staining, correlates with improved clinical outcomes. Previous studies have suggested that PD-L1 expression in tumor-infiltrating immune cells may reflect a preexisting antitumor immune response, which could partly explain the observed survival benefit. These findings highlight the importance of considering tumor-intrinsic and immune-related PD-L1 expression when evaluating its prognostic relevance in OSCC [11]. In this study, TPS-CPS≥10 was significantly correlated with OS. A key distinguishing feature of this study was the more stringent definition of PD-L1 overexpression adopted for patient stratification. Although most previous studies have used relatively low thresholds, commonly CPS>1 or 5, to define PD-L1 positivity, our analysis applied a higher cut-off value of CPS≥20 [2, 3, 6]. The higher threshold was selected to identify cases with biologically and clinically meaningful PD-L1 expression, thereby potentially improving the specificity of its prognostic value. By setting stricter criteria, our study aimed to better delineate the subset of patients who may truly benefit from ICIs and identify a distinct correlation between PD-L1 overexpression and clinical outcomes [21-23]. This methodological distinction strengthens the validity of our findings and contributes to ongoing discussions regarding the optimal CPS cutoff in head and neck cancer immuno-oncology research [23, 24]. Immune oncology, an evolving field that uses the body’s own immune system to fight cancer, has gained significant attention as a novel treatment approach, particularly for patients with recurrent or metastatic head and neck squamous cell carcinoma [25, 26]. ICIs targeting the PD-1/PD-L1 axis have emerged as important second-line therapies, especially for patients who do not respond to standard concurrent chemoradiotherapy [27]. Nonetheless, despite its growing clinical application, the prognostic value of PD-L1 expression in advanced head and neck squamous cell carcinoma remains controversial [28]. This study had some limitations. First, the relatively small sample size of 118 patients may have limited the statistical power and generalizability of the findings. Therefore, the association between PD-L1 overexpression and survival outcomes should be interpreted with caution. Second, the study population consisted exclusively of Taiwanese individuals with a habit of chewing betel nuts. Although this homogeneity reduced confounding factors and allowed for focused analysis, it introduced the risk of selection bias that may limit the applicability of the results to broader and more diverse populations. Conclusion Our findings suggest that PD-L1 overexpression, defined as CPS ≥20, may serve as a favorable prognostic marker in patients with OSCC with a habit of chewing betel nuts. This finding highlights the relevance of immune checkpoint markers for risk stratification and treatment planning. Based on the unique exposure to betel nut chewing, our results offer population-specific insights. Further prospective studies are required to validate the prognostic value of PD-L1 and explore its potential as a therapeutic guide. Abbreviation PD-L1, programmed death-ligand 1; OSCC, oral squamous cell carcinoma; CPS, Combined Positive Score; TPS, Tumor Proportion Score; OS, overall survival; ICI, immune checkpoint inhibitor; IHC, Immunohistochemical. Declarations Acknowledgements The statistical consulting company Estat provided consultation and editing services for this research. The authors also appreciated the team of Cancer Registry Group and Medical Records Room of Tri-Service General Hospital. Conflicts of interest The authors declare that they have no conflicts of interest. Data availability statement The datasets generated and analyzed during the current study are not publicly available due to institutional and ethical restrictions. Still, they are available from the corresponding author on reasonable request and with approval from the Tri-Service General Hospital Institutional Review Board. Ethics statement The study was approved by the Institutional Review Board of Tri-Service General Hospital (IRB no: C202405205), and informed consent was obtained from all participants. The study was retrospective; clinical trial number: not applicable. Disclaimer The authors affirm that no generative AI tools were used for data collection, analysis, or figure creation in this study. The research was conducted independently by the authors, and all interpretations and conclusions are solely those of the authors and do not necessarily reflect the views of the affiliated institutions. Funding This research was supported by Tri-Service General Hospital, Republic of China [Grant Nos. TSGH-D-114061]. Author Contributions Hui-Zhu Yang: Writing – Original draft preparation. Ling-Yu Kung, Yu-Chun Lin: Writing – Figure and figure legends. Cheng-Yu Yang: Data curation. Gu-Jiun Lin, Yuan-Wu Chen: Validation. Wei-Chin Chang: Supervision References Jiang, S., et al., Prognostic value of PD-1, PD-L1 and PD-L2 deserves attention in head and neck cancer. Front Immunol, 2022. 13 : p. 988416. Leporace-Jiménez, F., et al., Revisiting the Role of PD-L1 Overexpression in Prognosis and Clinicopathological Features in Patients with Oral Squamous Cell Carcinoma. Onco, 2024. 4 (3): p. 131-142. Lenouvel, D., et al., Prognostic and clinicopathological significance of PD-L1 overexpression in oral squamous cell carcinoma: A systematic review and comprehensive meta-analysis. Oral Oncol, 2020. 106 : p. 104722. Kilaru, S., et al., PD-L1 expression in head and neck squamous cell carcinoma and its clinical significance: A prospective observational study from a tertiary care centre. J Cancer Res Ther, 2024. 20 (1): p. 46-51. Caudell, J.J., et al., NCCN Guidelines® Insights: Head and Neck Cancers, Version 1.2022. J Natl Compr Canc Netw, 2022. 20 (3): p. 224-234. Paolino, G., et al., PD-L1 evaluation in head and neck squamous cell carcinoma: Insights regarding specimens, heterogeneity and therapy. Pathol Res Pract, 2021. 226 : p. 153605. Greeshma, L.R., et al., Correlation of PD-1 and PD-L1 expression in oral leukoplakia and oral squamous cell carcinoma: an immunohistochemical study. Sci Rep, 2023. 13 (1): p. 21698. Zhao, S., et al., Gastric cancer immune microenvironment score predicts neoadjuvant chemotherapy efficacy and prognosis. J Pathol Clin Res, 2024. 10 (3): p. e12378. Li, W., et al., PD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT. BMC Cancer, 2024. 24 (1): p. 633. Evrard, D., et al., PD-L1 expression in the microenvironment and the response to checkpoint inhibitors in head and neck squamous cell carcinoma. Oncoimmunology, 2020. 9 (1): p. 1844403. Peña-Cardelles, J.F., et al., Prognosis Value of Immunoregulatory Molecules in Oral Cancer Microenvironment: An Immunohistochemical Study. Biomedicines, 2022. 10 (3). Kulangara, K., et al., Clinical Utility of the Combined Positive Score for Programmed Death Ligand-1 Expression and the Approval of Pembrolizumab for Treatment of Gastric Cancer. Arch Pathol Lab Med, 2019. 143 (3): p. 330-337. Scheerens, H., et al., Current Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch. Clin Transl Sci, 2017. 10 (2): p. 84-92. Saeed, S., et al., Comparison of PD-L1 Expression in Oral Squamous Cell Carcinoma and Premalignant Lesions of Oral Cavity. Asian Pac J Cancer Prev, 2022. 23 (12): p. 4039-4045. Nokovitch, L., et al., Oral Cavity Squamous Cell Carcinoma Risk Factors: State of the Art. J Clin Med, 2023. 12 (9). Hashmi, A.A., et al., Risk Factors of Oral Squamous Cell Carcinoma with Special Emphasis on Areca Nut Usage and Its Association with Clinicopathological Parameters and Recurrence. Int J Surg Oncol, 2024. 2024 : p. 9725822. Chang, W.C., et al., A histopathological evaluation and potential prognostic implications of oral squamous cell carcinoma with adverse features. Oral Oncol, 2019. 95 : p. 65-73. Cho, Y.A., et al., Relationship between the expressions of PD-L1 and tumor-infiltrating lymphocytes in oral squamous cell carcinoma. Oral Oncol, 2011. 47 (12): p. 1148-53. Watanabe, Y., et al., Tumor-infiltrating lymphocytes, particularly the balance between CD8(+) T cells and CCR4(+) regulatory T cells, affect the survival of patients with oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2010. 109 (5): p. 744-52. Cui, Y.X. and X.S. Su, Clinicopathological Features of Programmed Cell Death-ligand 1 Expression in Patients with Oral Squamous Cell Carcinoma. Open Med (Wars), 2020. 15 : p. 292-301. Al-Azzawi, H.M.A., et al., PD-L1/PD-1 Expression in the Treatment of Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders: An Overview of Reviews. J Pers Med, 2025. 15 (4). Pollock, M., et al., Preferred Reporting Items for Overviews of Reviews (PRIOR): a protocol for development of a reporting guideline for overviews of reviews of healthcare interventions. Syst Rev, 2019. 8 (1): p. 335. Shea, B.J., et al., AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. Bmj, 2017. 358 : p. j4008. Warnakulasuriya, S., et al., Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer. Oral Dis, 2021. 27 (8): p. 1862-1880. Caponio, V.C.A., et al., The Immune Cells in the Development of Oral Squamous Cell Carcinoma. Cancers (Basel), 2023. 15 (15). Venkatesiah, S.S., et al., Immunology of Oral Squamous Cell Carcinoma-A Comprehensive Insight with Recent Concepts. Life (Basel), 2022. 12 (11). Li, C., X. Dong, and B. Li, Tumor microenvironment in oral squamous cell carcinoma. Front Immunol, 2024. 15 : p. 1485174. Wu, T., et al., PD-L1-Mediated Immunosuppression in Oral Squamous Cell Carcinoma: Relationship With Macrophage Infiltration and Epithelial to Mesenchymal Transition Markers. Front Immunol, 2021. 12 : p. 693881. Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2026 Read the published version in BMC Oral Health → Version 1 posted Editorial decision: Revision requested 12 Nov, 2025 Reviews received at journal 09 Nov, 2025 Reviews received at journal 01 Nov, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers invited by journal 16 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Submission checks completed at journal 10 Oct, 2025 First submitted to journal 03 Oct, 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. 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-7777087","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":535121115,"identity":"9866124b-c1f4-4e6c-80ec-9ecb5391f590","order_by":0,"name":"Hui-Zhu Yang","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hui-Zhu","middleName":"","lastName":"Yang","suffix":""},{"id":535121116,"identity":"1e863753-79a4-45ec-8ffb-3e0d71f5630f","order_by":1,"name":"Ling-Yu Kung","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ling-Yu","middleName":"","lastName":"Kung","suffix":""},{"id":535121117,"identity":"02188704-4e7d-4032-9f76-8f2d82190243","order_by":2,"name":"Yu-Chun Lin","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chun","middleName":"","lastName":"Lin","suffix":""},{"id":535121118,"identity":"2d982692-a89c-462e-97a1-fa291a4eb989","order_by":3,"name":"Cheng-Yu Yang","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Yu","middleName":"","lastName":"Yang","suffix":""},{"id":535121119,"identity":"684386f0-bfad-4504-af96-a285ea1aa6b4","order_by":4,"name":"Gu-Jiun Lin","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Gu-Jiun","middleName":"","lastName":"Lin","suffix":""},{"id":535121120,"identity":"ed382151-834a-41d4-902e-4c010ec6f49f","order_by":5,"name":"Yuan-Wu Chen","email":"","orcid":"","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Wu","middleName":"","lastName":"Chen","suffix":""},{"id":535121121,"identity":"6ddee147-5425-43ad-9c8b-db8e2fb3a30a","order_by":6,"name":"Wei-Chin Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDCCA0CcAGYxNzB8AFJs7IS1MDYkMBgAWYwNjDNAWpiJ0cIA1cLMA7aNgA6+473HHzz480feXLqx8bHNr23yfMwMjB8+5uDWInnmXGJDYpuB4c45B5uNc/tuG7YxMzBLztyGW4vBjRzDhsQGA8YNNxLbpHN7bjMCtbAx8+LTcv+NYUPCHwN7oJb235Y9t+0Ja7nBA9TCZpAIsoWZ4cftRIJaJM/kGM5IbDNOBvlFsrfhdnIbM2MzXr/wHT9j8PHHHznb7dLNBz/8+HPbdn47kPERjxaECyWABGMbiAmKKGIAWAvDH+IUj4JRMApGwcgCAHThWVYo8WUXAAAAAElFTkSuQmCC","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Wei-Chin","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2025-10-04 03:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7777087/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7777087/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-026-07845-2","type":"published","date":"2026-02-09T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94781413,"identity":"8519231e-451b-47c1-bb42-8a268fdebd6d","added_by":"auto","created_at":"2025-10-30 15:46:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1320542,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptforBMCversion1.1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/231a54abf273ac866dc7fbf5.docx"},{"id":94781410,"identity":"34c7b021-3f53-44e2-9a54-1ae93c81076f","added_by":"auto","created_at":"2025-10-30 15:46:31","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9386,"visible":true,"origin":"","legend":"","description":"","filename":"230448a1875542e5916c409507951e14.json","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/6d223a29fa270e136987722b.json"},{"id":94825130,"identity":"7feb005e-2cd0-4e02-9b53-47a7a9b8ecbc","added_by":"auto","created_at":"2025-10-31 06:49:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":528173,"visible":true,"origin":"","legend":"\u003cp\u003ePD-L1 negative expression.\u003c/p\u003e\n\u003cp\u003e(A\u0026amp;B) The HE stains in OSCC tissues. Magnification: ×10 (left), ×20 (right).\u003c/p\u003e\n\u003cp\u003e(C\u0026amp;D) The immunohistochemistry of PD-L1 expression stained with 22c3 in OSCC tissues. TPS:\u0026lt;1% and CPS:\u0026lt;1. Magnification: ×10 (left), ×20 (right).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/7353c8f0e8672f2ceb0fd737.png"},{"id":94781412,"identity":"92c54da4-ef17-4082-b3d0-1d6eb8c4d0d4","added_by":"auto","created_at":"2025-10-30 15:46:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":533708,"visible":true,"origin":"","legend":"\u003cp\u003ePD-L1 overexpression.\u003c/p\u003e\n\u003cp\u003e(E\u0026amp;F) The HE stains in OSCC tissues. Magnification: ×10 (left), ×20 (right).\u003c/p\u003e\n\u003cp\u003e(G\u0026amp;H) The immunohistochemistry of PD-L1 expression stained with 22c3 in OSCC tissues. TPS:60% and CPS:65. Magnification: ×10 (left), ×20 (right).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/aa75b60a770837901a70c9e0.png"},{"id":102785539,"identity":"dcc6775e-d3b8-482f-90aa-f12082e8410b","added_by":"auto","created_at":"2026-02-16 16:07:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1458193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/a23b6668-a58a-466c-b4ba-0139170dc83b.pdf"},{"id":94781409,"identity":"8f06d75b-8a76-4add-b6d1-59936b54b1f4","added_by":"auto","created_at":"2025-10-30 15:46:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34998,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7777087/v1/c20834872ef05bec76a73dc1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prognostic Significance of Programmed Death-Ligand 1 Expression in Betel Nut Chewing Patients with Oral Squamous Cell Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOral squamous cell carcinoma (OSCC), one of the most prevalent and aggressive malignancies in the head and neck region, results in high morbidity and mortality worldwide [1, 2]. Despite advancements in surgical techniques and adjuvant therapies, the prognosis of OSCC remains poor with a 5-year survival rate of approximately 60%, particularly in patients with advanced-stage disease or those with other pathologically adverse factors [3-5]. Thus, identifying reliable prognostic factors for risk stratification and informing therapeutic decision-making is necessary.\u003c/p\u003e\n\u003cp\u003eProgrammed death-ligand 1 (PD-L1), also known as CD274 or B7-H1, is a ligand of programmed death-protein 1 (PD-1) [6]. It is a transmembrane protein commonly expressed on the surface of tumor epithelial and immune cells [6, 7] and suppresses T-cell-mediated immune responses by binding to PD-1, allowing tumor cells to evade immune surveillance [3]. Elevated PD-L1 expression is associated with poor outcomes in several malignancies, including non-small cell lung cancer, advanced-stage melanoma, and gastrointestinal tumors [3, 8, 9]. However, its prognostic value in OSCC remains an area of active investigation, with several studies yielding conflicting results. Evaluation of PD-L1 expression may provide insights into the potential utility of immune checkpoint inhibitors (ICIs) as adjuvant or neoadjuvant strategies in surgically managed OSCC.\u003c/p\u003e\n\u003cp\u003eSeveral scoring systems have been developed for quantifying PD-L1 expression in tumor specimens, including Tumor Proportion Score (TPS), Combined Positive Score (CPS), and Tumor Cell scoring [6, 10, 11]. TPS measures the percentage of PD-L1–positive tumor cells relative to all viable tumor cells, whereas CPS indicates the sum of PD-L1–positive tumor cells and immune cells (e.g., lymphocytes and macrophages). Conversely, the Tumor Cell score specifically evaluates PD-L1 expression\u0026nbsp;in the tumor cells. These scoring systems are critical for predicting responses to ICIs and guiding cancer immunotherapy strategies\u0026nbsp;[6, 12]. The choice of scoring method is often influenced by the monoclonal antibody clone used for staining, because different clones (such as 22C3 or 28-8) may result in distinct staining intensities and cellular localization patterns\u0026nbsp;[6, 13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study aimed to elucidate the prognostic significance of PD-L1 expression in patients who underwent surgery for OSCC. By synthesizing\u0026nbsp;the current\u0026nbsp;evidence, we sought to clarify its role in predicting oncological outcomes and explore its implications for personalized treatment strategies.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cp\u003e\u003cem\u003eSelection Criteria\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective observational study included 118 patients with OSCC diagnosed and managed at the Tri-Service General Hospital between January 2017 and December 2023 and followed up until death or December 2024. Patients with advanced-stage disease in whom surgery was infeasible and those with poor physical conditions, distant metastases, or incomplete data were excluded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample Preparation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed, paraffin-embedded oral cancer tissue specimens were sectioned at 4 µm thickness using a microtome and mounted on positively charged glass slides to enhance tissue adhesion. The slides were then baked at 60°C for at least 1 h to ensure proper fixation before deparaffinization and rehydration using xylene and graded ethanol, respectively. Antigen retrieval was performed using a heat-induced method in citrate or ethylenediaminetetraacetic acid buffer to unmask PD-L1 epitopes. Immunohistochemical (IHC) staining was performed using a validated PD-L1 antibody (22c3) and an automated or manual staining procedure. The slides were counterstained with hematoxylin for tissue contrast. After dehydration and application of a cover slip, the stained slides were examined by two board-certified pathologists under a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIHC Evaluation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor CPS calculations, the two pathologists independently evaluated PD-L1 expression at 200× magnification. PD-L1–positive tumor cells, lymphocytes, and macrophages were counted across multiple high-power fields to estimate their proportions relative to the total number of viable tumor cells. Necrotic regions and artifacts were excluded, and disagreements between the two pathologists were resolved through consensus. CPS\u0026lt;1, ≥1, and ≥20 were defined as negative PD-L1 expression (Figure 1), positive PD-L1 expression, and PD-L1 overexpression, respectively (Figure 2). An additional score was obtained by subtracting CPS from TPS to determine further\u0026nbsp;whether PD-L1\u0026nbsp;positivity was highly concentrated in tumor cells and\u0026nbsp;whether immune cells affect\u0026nbsp;the prognosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026lt;Figure 1 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u0026lt;Figure 2 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurvival\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePatient survival data were obtained from the clinical records. The follow-up period was from the date of diagnosis to death or until December 2024. Overall survival (OS) was defined as the time from the date of diagnosis to death due to any cause. Disease-free survival was defined as the time from the date of curative treatment to the first instance of disease recurrence (local, regional, or distant metastasis) or death from any cause.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using the SPSS (version 20.0; IBM Corp., Armonk, NY, USA). The chi-square test was used to compare categorical variables, including age, sex, site, adverse factors for OSCC, alcohol consumption, cigarette smoking, and PD-L1 marker expression. Univariate Kaplan–Meier and multivariate Cox regression analyses were performed to determine the correlation of PD-L1 overexpression with OS, and disease-free survival after treatment. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eClinicopathological characteristics of the patients\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study included 118 patients, and the detailed clinicopathological characteristics of the study cohort are presented in Table 1. The cohort consisted predominantly of males (105 [89%]) and 13 females (11%). The median age at diagnosis was 57.9 years (range, 34–94 years), and the mean follow-up duration was 22.54 months (range, 0–76 months). According to the American Joint Committee on Cancer, 8th edition, most patients had stage IV disease (62/118, 52.5%) at diagnosis, followed by stages I (20/118, 16.9%) and III (20/118, 16.9%). The most common primary tumor subsites were the tongue (40/118, 33.9%) and buccal mucosa (37/118, 31.4%).\u003c/p\u003e\n\u003cp\u003e\u0026lt;Table 1 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePD-L1 expression in OSCC was stained using 22c3\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIHC staining for PD-L1 was observed in the membranes, tumor cells, lymphocytes, and macrophages. Representative images of IHC staining for PD-L1 are shown in Figures 1 and 2. PD-L1 positivity and overexpression were observed in 56.8% (67/118) and 31.4% (37/118) of tumors, respectively. PD-L1 overexpression was observed in 55.2% (37/67) of all cases with PD-L1 positivity.\u003c/p\u003e\n\u003cp\u003e\u0026lt;Figure 1 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u0026lt;Figure 2 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparative analysis of PD-L1 expression and association with clinicopathological characteristics in OSCC.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe clinicopathological characteristics of patients with OSCC, grouped according to PD-L1 expression level, are shown in Table 2. PD-L1 overexpression was not significantly associated with age, sex, primary site, adverse pathological factors, disease stage, cigarette smoking, or alcohol consumption.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026lt;Table 2 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurvival analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eKaplan–Meier curves revealed that OS was significantly better in patients with PD-L1-positive tumors than in those with PD-L1-negative tumors (\u003cem\u003ep=0.022\u003c/em\u003e). Univariate analysis showed that disease stage and PD-L1 overexpression were significantly correlated with OS (\u003cem\u003ep\u003c/em\u003e=0.001 and \u003cem\u003ep\u003c/em\u003e=0.022, respectively). Multivariate analysis (Cox regression model) of these factors showed that disease stage and PD-L1 overexpression were significantly correlated with OS (\u003cem\u003ep\u003c/em\u003e=0.001 and \u003cem\u003ep\u003c/em\u003e=0.010, respectively; Table 3). Furthermore, TPS-CPS≥10 was significantly associated with improved OS (\u003cem\u003ep\u003c/em\u003e=0.035).\u003c/p\u003e\n\u003cp\u003e\u0026lt;Table 3 near here\u0026gt;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study focused on a clinically and culturally distinct patient population. All participants were Taiwanese individuals with a history of betel nut chewing, which is a well-established risk factor for OSCC in Southeast Asia [4]. The inclusion of this relatively homogeneous cohort provides a unique opportunity to evaluate the prognostic significance of PD-L1 overexpression in high-risk groups with common environmental and behavioral exposures [14]. Considering the strong etiological association between betel nut use and oral carcinogenesis, our findings may reflect a tumor immune microenvironment specific to this subgroup, offering perspectives that differ from those of other studies based on populations without this exposure. Apart from betel nut chewing, cigarette smoking and alcohol consumption are common risk factors for OSCC [15, 16]. Some studies have reported lower PD-L1 expression in smokers due to reduced interferon-γ signaling, which is a potent PD-L1 inducer [2]. However, our results showed no significant association between cigarette smoking and OS.\u003c/p\u003e\n\u003cp\u003eGenerally, the prognosis of oral cancer is evaluated based on the presence of adverse pathological factors including extranodal extension, perineural invasion, lymphovascular invasion, depth of invasion, positive or close surgical margins, and metastatic lymph nodes [5, 17]. These histopathological features are associated with a higher risk of recurrence and poorer OS and are critical in guiding postoperative treatment decisions and prognosis assessment [3, 5, 17]. Additionally, advanced disease stage represents a worse prognosis, and the multivariate analysis in our study showed that staging was significantly correlated with OS.\u003c/p\u003e\n\u003cp\u003eIn this study, we evaluated PD-L1 expression using IHC in patients with locally advanced OSCC who underwent surgical resection. Notably, patients with PD-L1 overexpression had significantly better survival outcomes than those without. This observation contrasts with previous reports suggesting that PD-L1 overexpression correlates with poor prognosis in various malignancies, such as renal cell carcinoma, colorectal cancer, and lung cancer [14]. However, studies on patients with metastatic melanoma, non-small cell lung cancer, Merkel cell carcinoma, and laryngeal cancer have shown that PD-L1 overexpression, particularly when accompanied by a high density of tumor-infiltrating lymphocytes, is associated with prolonged survival [18, 19]. To emphasize the influence of tumor-infiltrating lymphocytes, we analyzed the differences between TPS and CPS.\u003c/p\u003e\n\u003cp\u003eIn this study, the survival rate was better in patients with PD-L1 overexpression than in those without. Conversely, most previous studies have reported an association between high PD-L1 expression and poor prognosis. Therefore, we examined the contribution of immune-cell staining to this outcome because the difference between TPS and CPS might reflect the influence of PD-L1 expression on immune cells more clearly [20]. This approach could help illustrate whether PD-L1 positivity within the tumor microenvironment, independent of tumor-cell staining, correlates with improved clinical outcomes. Previous studies have suggested that PD-L1 expression in tumor-infiltrating immune cells may reflect a preexisting antitumor immune response, which could partly explain the observed survival benefit. These findings highlight the importance of considering tumor-intrinsic and immune-related PD-L1 expression when evaluating its prognostic relevance in OSCC [11]. In this study, TPS-CPS≥10 was significantly correlated with OS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA key distinguishing feature of this study was the more stringent definition of PD-L1 overexpression adopted for patient stratification. Although most previous studies have used relatively low thresholds, commonly CPS\u0026gt;1 or 5, to define PD-L1 positivity, our analysis applied a higher cut-off value of CPS≥20 [2, 3, 6]. The higher threshold was selected to identify cases with biologically and clinically meaningful PD-L1 expression, thereby potentially improving the specificity of its prognostic value. By setting stricter criteria, our study aimed to better delineate the subset of patients who may truly benefit from ICIs and identify a distinct correlation between PD-L1 overexpression and clinical outcomes [21-23]. This methodological distinction strengthens the validity of our findings and contributes to ongoing discussions regarding the optimal CPS cutoff in head and neck cancer immuno-oncology research [23, 24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmune oncology, an evolving field that uses the body’s own immune system to fight cancer, has gained significant attention as a novel treatment approach, particularly for patients with recurrent or metastatic head and neck squamous cell carcinoma [25, 26]. ICIs targeting the PD-1/PD-L1 axis have emerged as important second-line therapies, especially for patients who do not respond to standard concurrent chemoradiotherapy [27]. Nonetheless, despite its growing clinical application, the prognostic value of PD-L1 expression in advanced head and neck squamous cell carcinoma remains controversial [28].\u003c/p\u003e\n\u003cp\u003eThis study had some limitations. First, the relatively small sample size of 118 patients may have limited the statistical power and generalizability of the findings. Therefore, the association between PD-L1 overexpression and survival outcomes should be interpreted with caution. Second, the study population consisted exclusively of Taiwanese individuals with a habit of chewing betel nuts. Although this homogeneity reduced confounding factors and allowed for focused analysis, it introduced the risk of selection bias that may limit the applicability of the results to broader and more diverse populations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings suggest that PD-L1 overexpression, defined as CPS ≥20, may serve as a favorable prognostic marker in patients with OSCC with a habit of chewing betel nuts. This finding highlights the relevance of immune checkpoint markers for risk stratification and treatment planning. Based on the unique exposure to betel nut chewing, our results offer population-specific insights. Further prospective studies are required to validate the prognostic value of PD-L1 and explore its potential as a therapeutic guide.\u003c/p\u003e"},{"header":"Abbreviation","content":"\u003cp\u003ePD-L1, programmed death-ligand 1; OSCC, oral squamous cell carcinoma; CPS, Combined Positive Score; TPS, Tumor Proportion Score; OS, overall survival; ICI, immune checkpoint inhibitor; IHC, Immunohistochemical.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical consulting company Estat provided consultation and editing services for this research. The authors also appreciated the team of Cancer Registry Group and Medical Records Room of Tri-Service General Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are not publicly available due to institutional and ethical restrictions. Still, they are available from the corresponding author on reasonable request and with approval from the Tri-Service General Hospital Institutional Review Board.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Institutional Review Board of Tri-Service General Hospital (IRB no: C202405205), and informed consent was obtained from all participants. The study was retrospective; clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that no generative AI tools were used for data collection, analysis, or figure creation in this study. The research was conducted independently by the authors, and all interpretations and conclusions are solely those of the authors and do not necessarily reflect the views of the affiliated institutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Tri-Service General Hospital, Republic of China [Grant Nos. TSGH-D-114061].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui-Zhu Yang: Writing – Original draft preparation. Ling-Yu Kung, Yu-Chun Lin: Writing – Figure and figure legends. Cheng-Yu Yang: Data curation. Gu-Jiun Lin, Yuan-Wu Chen: Validation. Wei-Chin Chang: Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiang, S., et al., \u003cem\u003ePrognostic value of PD-1, PD-L1 and PD-L2 deserves attention in head and neck cancer.\u003c/em\u003e Front Immunol, 2022. \u003cstrong\u003e13\u003c/strong\u003e: p. 988416.\u003c/li\u003e\n\u003cli\u003eLeporace-Jim\u0026eacute;nez, F., et al., \u003cem\u003eRevisiting the Role of PD-L1 Overexpression in Prognosis and Clinicopathological Features in Patients with Oral Squamous Cell Carcinoma.\u003c/em\u003e Onco, 2024. \u003cstrong\u003e4\u003c/strong\u003e(3): p. 131-142.\u003c/li\u003e\n\u003cli\u003eLenouvel, D., et al., \u003cem\u003ePrognostic and clinicopathological significance of PD-L1 overexpression in oral squamous cell carcinoma: A systematic review and comprehensive meta-analysis.\u003c/em\u003e Oral Oncol, 2020. \u003cstrong\u003e106\u003c/strong\u003e: p. 104722.\u003c/li\u003e\n\u003cli\u003eKilaru, S., et al., \u003cem\u003ePD-L1 expression in head and neck squamous cell carcinoma and its clinical significance: A prospective observational study from a tertiary care centre.\u003c/em\u003e J Cancer Res Ther, 2024. \u003cstrong\u003e20\u003c/strong\u003e(1): p. 46-51.\u003c/li\u003e\n\u003cli\u003eCaudell, J.J., et al., \u003cem\u003eNCCN Guidelines\u0026reg; Insights: Head and Neck Cancers, Version 1.2022.\u003c/em\u003e J Natl Compr Canc Netw, 2022. \u003cstrong\u003e20\u003c/strong\u003e(3): p. 224-234.\u003c/li\u003e\n\u003cli\u003ePaolino, G., et al., \u003cem\u003ePD-L1 evaluation in head and neck squamous cell carcinoma: Insights regarding specimens, heterogeneity and therapy.\u003c/em\u003e Pathol Res Pract, 2021. \u003cstrong\u003e226\u003c/strong\u003e: p. 153605.\u003c/li\u003e\n\u003cli\u003eGreeshma, L.R., et al., \u003cem\u003eCorrelation of PD-1 and PD-L1 expression in oral leukoplakia and oral squamous cell carcinoma: an immunohistochemical study.\u003c/em\u003e Sci Rep, 2023. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 21698.\u003c/li\u003e\n\u003cli\u003eZhao, S., et al., \u003cem\u003eGastric cancer immune microenvironment score predicts neoadjuvant chemotherapy efficacy and prognosis.\u003c/em\u003e J Pathol Clin Res, 2024. \u003cstrong\u003e10\u003c/strong\u003e(3): p. e12378.\u003c/li\u003e\n\u003cli\u003eLi, W., et al., \u003cem\u003ePD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT.\u003c/em\u003e BMC Cancer, 2024. \u003cstrong\u003e24\u003c/strong\u003e(1): p. 633.\u003c/li\u003e\n\u003cli\u003eEvrard, D., et al., \u003cem\u003ePD-L1 expression in the microenvironment and the response to checkpoint inhibitors in head and neck squamous cell carcinoma.\u003c/em\u003e Oncoimmunology, 2020. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 1844403.\u003c/li\u003e\n\u003cli\u003ePe\u0026ntilde;a-Cardelles, J.F., et al., \u003cem\u003ePrognosis Value of Immunoregulatory Molecules in Oral Cancer Microenvironment: An Immunohistochemical Study.\u003c/em\u003e Biomedicines, 2022. \u003cstrong\u003e10\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eKulangara, K., et al., \u003cem\u003eClinical Utility of the Combined Positive Score for Programmed Death Ligand-1 Expression and the Approval of Pembrolizumab for Treatment of Gastric Cancer.\u003c/em\u003e Arch Pathol Lab Med, 2019. \u003cstrong\u003e143\u003c/strong\u003e(3): p. 330-337.\u003c/li\u003e\n\u003cli\u003eScheerens, H., et al., \u003cem\u003eCurrent Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch.\u003c/em\u003e Clin Transl Sci, 2017. \u003cstrong\u003e10\u003c/strong\u003e(2): p. 84-92.\u003c/li\u003e\n\u003cli\u003eSaeed, S., et al., \u003cem\u003eComparison of PD-L1 Expression in Oral Squamous Cell Carcinoma and Premalignant Lesions of Oral Cavity.\u003c/em\u003e Asian Pac J Cancer Prev, 2022. \u003cstrong\u003e23\u003c/strong\u003e(12): p. 4039-4045.\u003c/li\u003e\n\u003cli\u003eNokovitch, L., et al., \u003cem\u003eOral Cavity Squamous Cell Carcinoma Risk Factors: State of the Art.\u003c/em\u003e J Clin Med, 2023. \u003cstrong\u003e12\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eHashmi, A.A., et al., \u003cem\u003eRisk Factors of Oral Squamous Cell Carcinoma with Special Emphasis on Areca Nut Usage and Its Association with Clinicopathological Parameters and Recurrence.\u003c/em\u003e Int J Surg Oncol, 2024. \u003cstrong\u003e2024\u003c/strong\u003e: p. 9725822.\u003c/li\u003e\n\u003cli\u003eChang, W.C., et al., \u003cem\u003eA histopathological evaluation and potential prognostic implications of oral squamous cell carcinoma with adverse features.\u003c/em\u003e Oral Oncol, 2019. \u003cstrong\u003e95\u003c/strong\u003e: p. 65-73.\u003c/li\u003e\n\u003cli\u003eCho, Y.A., et al., \u003cem\u003eRelationship between the expressions of PD-L1 and tumor-infiltrating lymphocytes in oral squamous cell carcinoma.\u003c/em\u003e Oral Oncol, 2011. \u003cstrong\u003e47\u003c/strong\u003e(12): p. 1148-53.\u003c/li\u003e\n\u003cli\u003eWatanabe, Y., et al., \u003cem\u003eTumor-infiltrating lymphocytes, particularly the balance between CD8(+) T cells and CCR4(+) regulatory T cells, affect the survival of patients with oral squamous cell carcinoma.\u003c/em\u003e Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2010. \u003cstrong\u003e109\u003c/strong\u003e(5): p. 744-52.\u003c/li\u003e\n\u003cli\u003eCui, Y.X. and X.S. Su, \u003cem\u003eClinicopathological Features of Programmed Cell Death-ligand 1 Expression in Patients with Oral Squamous Cell Carcinoma.\u003c/em\u003e Open Med (Wars), 2020. \u003cstrong\u003e15\u003c/strong\u003e: p. 292-301.\u003c/li\u003e\n\u003cli\u003eAl-Azzawi, H.M.A., et al., \u003cem\u003ePD-L1/PD-1 Expression in the Treatment of Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders: An Overview of Reviews.\u003c/em\u003e J Pers Med, 2025. \u003cstrong\u003e15\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003ePollock, M., et al., \u003cem\u003ePreferred Reporting Items for Overviews of Reviews (PRIOR): a protocol for development of a reporting guideline for overviews of reviews of healthcare interventions.\u003c/em\u003e Syst Rev, 2019. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 335.\u003c/li\u003e\n\u003cli\u003eShea, B.J., et al., \u003cem\u003eAMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both.\u003c/em\u003e Bmj, 2017. \u003cstrong\u003e358\u003c/strong\u003e: p. j4008.\u003c/li\u003e\n\u003cli\u003eWarnakulasuriya, S., et al., \u003cem\u003eOral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer.\u003c/em\u003e Oral Dis, 2021. \u003cstrong\u003e27\u003c/strong\u003e(8): p. 1862-1880.\u003c/li\u003e\n\u003cli\u003eCaponio, V.C.A., et al., \u003cem\u003eThe Immune Cells in the Development of Oral Squamous Cell Carcinoma.\u003c/em\u003e Cancers (Basel), 2023. \u003cstrong\u003e15\u003c/strong\u003e(15).\u003c/li\u003e\n\u003cli\u003eVenkatesiah, S.S., et al., \u003cem\u003eImmunology of Oral Squamous Cell Carcinoma-A Comprehensive Insight with Recent Concepts.\u003c/em\u003e Life (Basel), 2022. \u003cstrong\u003e12\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eLi, C., X. Dong, and B. Li, \u003cem\u003eTumor microenvironment in oral squamous cell carcinoma.\u003c/em\u003e Front Immunol, 2024. \u003cstrong\u003e15\u003c/strong\u003e: p. 1485174.\u003c/li\u003e\n\u003cli\u003eWu, T., et al., \u003cem\u003ePD-L1-Mediated Immunosuppression in Oral Squamous Cell Carcinoma: Relationship With Macrophage Infiltration and Epithelial to Mesenchymal Transition Markers.\u003c/em\u003e Front Immunol, 2021. \u003cstrong\u003e12\u003c/strong\u003e: p. 693881.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are 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":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oral squamous cell carcinoma, programmed death-ligand 1, immunohistochemistry, prognosis, betel nut, immune checkpoint, Combined Positive Score","lastPublishedDoi":"10.21203/rs.3.rs-7777087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7777087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eObjective:\u003c/em\u003e This study aimed to investigate the prognostic significance of programmed death-ligand 1 (PD-L1) expression in Taiwanese patients with oral squamous cell carcinoma (OSCC) who underwent surgical treatment, particularly in those with a history of betel-nut chewing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMaterials and Methods:\u003c/em\u003e We retrospectively analyzed the medical records of patients with OSCC diagnosed and treated at a single tertiary hospital between 2017 and 2023. Immunohistochemical evaluation of formalin-fixed, paraffin-embedded tumor specimens was performed using the validated anti–PD-L1 antibody clone 22C3. PD-L1 expression was evaluated using the Combined Positive Score (CPS), and CPS≥1 and ≥20 were defined as PD-L1 positivity and overexpression, respectively. An additional score was obtained by subtracting the CPS from the Tumor Proportion Score (TPS) to clarify the effect of the immune cells. The associations between PD-L1 expression and overall survival (OS), and disease-free survival were assessed using Kaplan–Meier and Cox regression analyses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults:\u003c/em\u003e Of the 118 OSCCs, 56.8% and 31.4% exhibited PD-L1 positivity and overexpression, respectively. PD-L1 overexpression and TPS-CPS≥10 were significantly associated with improved OS (\u003cem\u003ep\u003c/em\u003e=0.022 and \u003cem\u003ep\u003c/em\u003e=0.035, respectively). Multivariate analysis confirmed that PD-L1 overexpression and cancer stage were independent prognostic factors for OS (\u003cem\u003ep\u003c/em\u003e=0.010 and \u003cem\u003ep\u003c/em\u003e=0.001, respectively). However, PD-L1 overexpression was not significantly correlated with tumor stage, primary site, or adverse pathological features.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusion:\u003c/em\u003e High PD-L1 expression is a favorable prognostic biomarker in patients with surgically treated OSCC, particularly in populations with prevalent betel nut use. These findings suggest that PD-L1 expression can guide immunotherapy decisions and risk stratification in OSCC management.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClinical relevance\u003c/em\u003e: Patients with OSCC exhibiting high PD-L1 expression have a better survival rate after surgery. PD-L1 testing may help in risk stratification and guiding immunotherapy decisions.\u003c/p\u003e","manuscriptTitle":"Prognostic Significance of Programmed Death-Ligand 1 Expression in Betel Nut Chewing Patients with Oral Squamous Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 15:46:26","doi":"10.21203/rs.3.rs-7777087/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T09:54:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T10:06:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-01T21:30:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T01:58:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141628565679898289574514096698951306811","date":"2025-10-19T08:33:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45429111389494076716048997622643053595","date":"2025-10-18T14:46:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66869320963655380894292080916613041372","date":"2025-10-18T12:35:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-16T14:31:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T06:03:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T06:00:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-10-04T03:24:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b40e161-c65f-4fc7-aa0d-e88fd82fd4e6","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:04:57+00:00","versionOfRecord":{"articleIdentity":"rs-7777087","link":"https://doi.org/10.1186/s12903-026-07845-2","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2026-02-09 15:57:57","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-10-30 15:46:26","video":"","vorDoi":"10.1186/s12903-026-07845-2","vorDoiUrl":"https://doi.org/10.1186/s12903-026-07845-2","workflowStages":[]},"version":"v1","identity":"rs-7777087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7777087","identity":"rs-7777087","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}