Assessment of Programmed Cell Death Ligand -1 (PD-L1) Expression in Oral Potentially Malignant Lesions and Surgical Margins of Oral Squamous Cell Carcinoma and its Association with 3-year Survival

Assessment of Programmed Cell Death Ligand -1 (PD-L1) Expression in Oral Potentially Malignant Lesions and Surgical Margins of Oral Squamous Cell Carcinoma and its Association with 3-year Survival | 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 Study protocol Assessment of Programmed Cell Death Ligand -1 (PD-L1) Expression in Oral Potentially Malignant Lesions and Surgical Margins of Oral Squamous Cell Carcinoma and its Association with 3-year Survival Archana Sonone, Alka Hande, Swati Patil, Aayushi Pakhale, Preethi Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6045096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Oral squamous cell carcinoma (OSCC) is the most prevalent type of epithelial cancer affecting the oral cavity. In India, it ranks as the third most common cancer, following breast and cervical cancer. Significant research is being conducted to explore various treatment approaches for OSCC, with immunotherapy emerging as an advanced option. Under normal circumstances, the immune system protects the body from infections and tumors. Key players in this process are cell surface proteins called "programmed cell death 1 (PD-1)" and "programmed cell death ligand 1 (PD-L1)," which are crucial for immune checkpoint regulation. Higher expression of PD-L1 is associated with worse prognosis and shows an inverse relationship with CD8 + T cell infiltration in tumors. This study aims to examine the differential expression of PD-L1 in oral potentially malignant disorders (OPMD) and surgical margins of OSCC patients, while also exploring its link to clinicopathological features and its influence on patient survival. By doing so, the research will provide insights into the progression of the disease from premalignant stages to OSCC recurrence. Material and methods It is a observation retrospective study that will include 20 cases of OPMD and 20 tumor-free surgical margins from OSCC patients The immunoexpression of PD-L1 will be assessed by CPS score under Leica DMLB2 (Leica microscope). Discussion The assessment of PD-L1 immunoexpression form OPMD patients to surgical margins of OSCC patients, it gives whole disease spectrum and helpful to identify the individuals having a higher risk of malignant potential Oral potentially premalignant disorder ( OPMD) Oral squamous cell carcinoma (OSCC) Combined-positive score programmed cell death – ligand 1 Background “Oral squamous cell carcinoma (OSCC)” is the most common malignancy affecting the oral and maxillofacial regions. Globally, head and neck cancers rank as the eighth most prevalent type of cancer [1], with an estimated 880,000 new OSCC cases diagnosed annually and over 440,000 deaths reported each year [2]. Most OSCC cases develop from pre-existing precancerous lesions or conditions categorized as oral potentially malignant disorders (OPMDs). Approximately 2.5% of the general population is believed to have such precancerous conditions in the oral cavity. Common examples of OPMDs include oral “leukoplakia”, oral submucous fibrosis (OSMF), and oral lichen planus ( OLP), each exhibiting varying rates of malignant transformation [3]. Despite advances in treatment, the mortality rate has not significantly improved over the years, The overall 5 year survival rate for OSCC remains around fifty percentage. The Early detection of OSCC, particularly in well-differentiated tumors that have not metastasized, generally leads to a more favorable prognosis. However, most OSCC cases are diagnosed at advanced stages. Factors which are responsible for prognosis of OSCC are the patient's age , co-morbidities . Even with negative surgical margins, local recurrence is common, possibly due to the presence of minimal residual cancer (MRC) cells that evade detection by standard histological methods [4]. Immunohistochemical (IHC) analysis of tumor free surgical margins may provide a more precise method for detecting minimal residual cancer (MRC), as genetic changes often occur before visible phenotypic alterations in the epithelium[5]. For an IHC marker to be effective, it must accurately identify tumor cells and distinguish them from normal tissue [6]. Current research focuses on identifying reliable IHC markers that can improve the clinical management of OSCC patients and provide important prognostic insights. Routine IHC analysis of negative margins could be a more effective approach than conventional histology in identifying patients at higher risk of local recurrence, potentially enhancing survival rates for OSCC patients. Of the numerous IHC markers studied, “PD-L1 has emerged” as one of the most promising. It is utilized as a diagnostic biomarker for immunotherapy in various types of cancers. Under physiological conditions, the immune system defends the body against various infections and cancers, playing a key role in eliminating diseased or harmful cells. Immunological checkpoints, which serve as inhibitory regulators of the immune system of our body. They are very essential for maintaining self-tolerance. These checkpoints help control the duration, intensity, and extent of immune responses, preventing “autoimmunity” and reducing “collateral tissue damage”. One of the immunoinhibitory check is PD-L1 [7]. Programmed death-ligand 1 (PD-L1), also called as CD274 or B7-H1. It is a 290-amino acid protein in the B7 family associated with type I transmembrane receptors. It is formed by a cytoplasmic domain, a transmembrane domain, and two extracellular domains same as IgV and IgC. There are various cell types, including T cells, B cells antigen-presenting cells (APCs), monocytes, and epithelial cells which shows the expression of PD-L1. Its expression is upregulated in response to proinflammatory cytokines, which leads to increase PD-1 levels. The interaction between “PD-L1 and PD-1” triggers downstream signaling in T cells, suppressing their proliferation, cytotoxic function and cytokine secretion[8]. Despite growing interest in PD-L1 and PD-1 signaling, there remains limited comprehensive research on their clinical relevance and prognostic significance in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma (OSCC) [9]. The precise role of this pathway in the malignant transformation of PD-L1 is still under investigation. In this era where the incidence of OPMD and OSCC is increasing, it is necessary to identify specific biomarkers for both OPMD and OSCC that would help in clinical decision-making, early diagnosis, treatment strategies and prognosis prediction in OPMD and OSCC [3]. Therefore, the study will investigate the differential expression of PD-L1 in OPMD and in surgical margins of OSCC patients along with its association with clinicopathological features and its impact on patient survival ,thus this study will provide the disease spectrum from premalignancy to recurrence of OSCC. Research Study Design Type or Nature of study- Retrospective, Observational study Aim: To assess the utility of PD-L1 expression in OPMD as a screening marker and in surgical margins of OSCC as a prognostic marker. OBJECTIVE(S) To evaluate the CPS score of PD-L1 immunoexpression in OPMD cases To evaluate the CPS Score of PD-L1immunoexpression in tumor free surgical margins of OSCC cases . To compare the CPS Score of PD-L1 immunoexpression in OPMD and surgical margins of OSCC. To correlate the CPS Score of PD-L1 immunoexpression surgical margins of OSCC with three-year-survival. Materials and methods The study will be conducted in the “Department of Oral and Pathology and Microbiology at Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research (Deemed to be University), located in Sawangi (M), Wardha, Maharashtra”, India. Archived records of surgically treated OSCC cases and histologically diagnosed cases of OPMD from 2018 to 2020 will be retrieved from the departmental archives for analysis. Sample size and sampling: It will be divided into three groups Group A : Histologically diagnosed OPMD cases. (20 samples) Group B : Histologically tumor free surgical margins of OSCC cases. (20 samples) Group C - Normal oral mucosa (20 samples) Inclusion Criteria - Patients with primary disease as OPMD and OSCC, tumor sites specific to the oral cavity, and patients with histologically tumor-free surgical margins Exclusion criteria - , Recurrent tumors, Patients who were lost at follow-up, Variants of squamous cell carcinoma, and patients with histologically positive surgical margins Analysis plan- Tumor free surgical margins of OSCC cases and histopathologically diagnosed cases of OPMD will be retrieved from the departmental archives. The samples will be retrieved from the archives from a period of 2018–2020. The current survival status of these patients at the start of study will be noted. A further follow up of 3 years will be carried out for each patient Immunohistochemistry The assessment of PD-L1 antigen will be performed by IHC . Universal Immuno-enzyme Polymer method” will be used for this assessment. After the deparaffinization with xylene, tissue sections will be immersed into grades of alcohol for rehydration. Microwave heat antigen retrieval technique will be used to retrieve the antigen. In this technique sections will be heated for 10 minutes in 0.01M sodium citrate buffer ( PH 6.0). Then its get cooled at room temperature for about 20 minutes. Repeatedly this procedure will be carried out until the enough antigen retrieved. After this sections will be washed with the help of phosphate - buffer saline ( PBS) and treated with 3% hydrogen peroxide in methnol for 30 minutes to block endogenous peroxidase activity. For nonspecific binding of antigen, the section will be rinsed with PBS for three times. Then primary antibody PD-L1 ill be applied to tissue section and incubated for 90 minutes for room temperature in humidifying chamber. Again section will be washed for three for 10 minutes with PBS and polymer anti-mouse antibody conjugated with horseradish peroxidase (HRP) for 30 minutes. The antigen – antibody reaction will be observed using 3,3'-diaminobenzidine (DAB) chromogen in a buffer solution. Mayer's haematoxylin counterstaining will be used. PD-L1 immunoexpression will be assessed by two independent observer in a blinded manner. PD-L1 expression will be assessed using the Combined Positive Score (CPS) under light microscopy. Two pathologists will conduct the evaluation blinded, without access to the clinical details. The CPS is calculated by dividing the number of PD-L1-immunoexpression positive cells, including cells of epithelium, macrophages, and lymphocytes, by the total number of viable neoplastic cells, and then multiplying the result by 100. Sample size: - Minimum sample size required Sample Size Calculation Sample size Formula Using Mean difference \(\:n1=n2=2\frac{({Z}_{\alpha\:}+{Z}_{\beta\:}{)}^{2}{\sigma\:}^{2}}{(\delta\:{)}^{2}}\) \(\:{Z}_{\alpha\:}==TypeIerrorat\:1\%\:\:\:\:at\:both\:sides\:two\:tailed\:\) \(\:{Z}_{\beta\:}=99\:\%=Powerat\:99\%\) Primary Variable Difference in (PD-L1) expression For α = 0.01, Z(1-α/2) = 2.576, For β = 0.01, Z(1-β) = 2.326 Mean in group 1 (µ₁): 4.59 Standard deviation in group 1 (σ₁): 1.965 Mean in group 2 (µ₂): 2.03 Standard deviation in group 2 (σ₂): 1.204 Ratio (Group 2 / Group 1): 1 Pooled std dev = \(\:\sigma\:\) =1.5845 Sample size N= \(\:n1=n2=2\frac{(1.96\:+0.84\:{)}^{2}({1.5845)}^{2}}{(0.21{)}^{2}}\approx\:\:19.34\:per\:group\) Round up to the nearest whole number for each group: Minimum sample size per group = 20 Total sample size = 40 Conclusion The minimum of 20 sample are required in each group to analysed the statistically significant difference between the two means with an alpha level of 0.01 and a beta level of 0.01. The total minimum sample size required is 40 participants Discussion Oral squamous cell carcinoma (OSCC) is a multifactorial disease that progresses through multiple stages. Most of the times OSCC preceded by oral potentially malignant disorders (OPMDs) such as leucoplakia OSMF, Oral Lichen planus etc. There are the malignant cytological and architectural changes in the tissue even in the absence of visible tissue changes. Advances in molecular oncology have highlighted the significant role of immune checkpoint inhibitors in cancer pathophysiology. An important immunosuppressive mechanism is the interaction between the ligand, PD-L1, on cancer cells and the PD-1 receptor on T cells. Increased PD-L1 expression has been associated with disease progression. There are numerous studies carried out on resected specimens of OSCC and OPMD. Kanan Dave et al. examined the immunoexpression of “PD-L1” in oral dysplasia, which progresses to OSCC. They studied 49 cases by immunohistochemistry. It showed increase in “PD-L1” expression in progressing than in non-progressing epithelial dysplasia. So they concluded that the immunomodulation through “PD-L1” pathways happens earlier to the malignant transformation [ 10 ]. The study carried out by Pachpande et al. on OPMD and OSCC cases, where they analysed PD-L1 expression. They observed elevated PD-L1 in OSCC than OPMD. PD-L1 is associated with a higher likelihood of malignant transformation, So suggested that evaluating PD-L1 levels in OPMD patients able to identify the individuals at risk for malignant progression [ 3 ]. The assessing PD-L1 expression in OPMD and the surgical margins of OSCC may serve as a screening and prognostic marker. This study aims to analyze the disease spectrum by examining PD-L1 expression from premalignant stages to malignancy. Conclusion The PD-L1 is an important biomarker for immunotherapy in various cancers including the OSCC, which is measured by the CPS score. The assessment of PD-L1 expression in OPMD cases may be used as a screening marker for the presence or absence of malignant transformation risk, as an increase in PD-L1 expression signifies an increased risk of malignant change. The assessment of PD-L1 in surgical margins of OSCC may predict the molecular invasion. The calculation of the CPS score of PD-L1 in surgical margins of OSCC may predict prognostic indicators. The assessment of PD-L1 expression in OPMD and in the surgical margin of OSCC cases may be used as a screening marker and prognostic marker. It provides the whole disease spectrum from premalignancy to malignancy. Abbreviations OSCC Oral Squamous Cell Carcinoma OPMD Oral Premalignant Disorders PD L1-programmed cell death ligand 1 CPS Combined Positive Score Declarations Ethics approval and consent to participate Ethical reference number: DMIHER(DU)/IEC/2025/42. Every participant in the study has given their informed consent. Consent for publication – Not Applicable Availability of data and materials Data will be taken from the archival of the department. Competing interests The authors declare that they have no competing interests. Funding Declaration - No Funding. Author Contribution Dr. Archana Sonone- Principal investigator of study, formulation or evolution of overarching research goals and aims, writing an original script Dr. Alka Hande-​Preparation, creation, and/or presentation of the published work, specifically visualization/data presentation. editing of manuscript Dr Swati Patil-Data analysis Dr. Aayushi Pakhale-Review of the manuscript Dr. Preethi Sharma-Review of the manuscript References Ghapanchi J, Ranjbar Z, Mokhtari MJ, Koohpeima F, Derakhshan M, Khademi B, et al. The LncRNA H19 rs217727 Polymorphism Is Associated with Oral Squamous Cell Carcinoma Susceptibility in Iranian Population. Biomed Res Int. 2020;2020:e1634252. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. Pachpande PS, Mandale MS, Bhavthankar JD, Humbe JG, Zanwar P. Assessment of programmed cell death ligand- 1 (PD-L1) expression in oral potentially malignant disorders and oral squamous cell carcinoma - An immunohistochemical study. IP Arch Cytol Histopathol Res. 2023;8(3):180–8. Braakhuis BJ, Bloemena E, Leemans CR, Brakenhoff RH. Molecular analysis of surgical margins in head and neck cancer: more than a marginal issue. Oral Oncol. 2010;46:485–91. Ranka R, Chaudhary M, Sharma P. Role of Immunohistochemical Markers in Surgical Margins of Patients with Head and Neck Carcinoma-A Systematic Review. Int J Cur Res Rev. 2020;12:9–17. Roncella S, Ferro P, Bacigalupo B, Tognoni A, Falco E, Gianquinto D, et al. Human mammaglobin mRNA is a reliable molecular marker for detecting occult breast cancer cells in peripheral blood. J Exp Clin Cancer Res. 2005;24:265–71. Mildner F, Sopper S, Amann A, Pircher A, Pall G, Köck S, Naismith E, Wolf D, Gamerith G. Systematic review: soluble immunological biomarkers in advanced non-small-cell lung cancer (NSCLC). Crit Rev Oncol Hematol. 2020;153:102948. https://doi.org/10.1016/j.critrevonc.2020.102948 . Epub 2020 May 5. PMID: 32645684. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12(4):252–64. https://doi.org/10.1038/nrc3239 . PMID: 22437870; PMCID: PMC4856023. He X, Xu C. Immune checkpoint signaling and cancer immunotherapy. Cell Res. 2020;30(8):660–9. https://doi.org/10.1038/s41422-020-0343-4 . Epub 2020 May 28. PMID: 32467592; PMCID: PMC7395714. Dave K, Ali A, Magalhaes M. Increased expression of PD-1 and PD-L1 in oral lesions progressing to oral squamous cell carcinoma: a pilot study. Sci Rep. 2020;10(1):9705. 10.1038/s41598-020-66257-6 . PMID: 32546692; PMCID: PMC7297711. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-6045096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":421754405,"identity":"143f8946-da10-4cda-ab10-9fb42d43bafa","order_by":0,"name":"Archana Sonone","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYHACNiS2gY0cP4hOKCBaS0GasWQDSIsB0Vo+HErccABsHW71/LMPP3vwc4edPb9087EHHwwOJG4+vzrxwwMDBnl+sQNYtUicSzM37D2TnDhzzrF0wxkGd4y33Xi7WQLoMMOZsxOwW3OGh02Ct405weBGjpk0j8Ez2W03zm4AaUkwuI1dizxQi+Tftnp7sJY/BocZN884u/kHPi0GQC3SvG2HGTeAtDAYHFbcwN+7Da8thmfYzKRl244nzpyRlibZY5BmLHGDd5tFgoEETr/InWF+Jvm2rdqeXyL5mMSPP8Co7D+7+eaPCht5fmkc3scEEmCVEsQqBwH+A6SoHgWjYBSMghEAAPJcYAADNKEkAAAAAElFTkSuQmCC","orcid":"","institution":"Sharad Pawar Dental College \u0026 Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe)","correspondingAuthor":true,"prefix":"","firstName":"Archana","middleName":"","lastName":"Sonone","suffix":""},{"id":421754406,"identity":"9c012186-5110-42c0-80b4-7024df372509","order_by":1,"name":"Alka Hande","email":"","orcid":"","institution":"Sharad Pawar Dental College \u0026 Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe)","correspondingAuthor":false,"prefix":"","firstName":"Alka","middleName":"","lastName":"Hande","suffix":""},{"id":421754407,"identity":"f8688ad2-9648-4bf2-b352-a95da4c89ca3","order_by":2,"name":"Swati Patil","email":"","orcid":"","institution":"Sharad Pawar Dental College \u0026 Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe)","correspondingAuthor":false,"prefix":"","firstName":"Swati","middleName":"","lastName":"Patil","suffix":""},{"id":421754408,"identity":"56eb9eec-9bbb-4366-b79e-ac5b58e29199","order_by":3,"name":"Aayushi Pakhale","email":"","orcid":"","institution":"Sharad Pawar Dental College \u0026 Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe)","correspondingAuthor":false,"prefix":"","firstName":"Aayushi","middleName":"","lastName":"Pakhale","suffix":""},{"id":421754409,"identity":"fe3c1b53-5f84-433d-9591-6cccea2ebb8e","order_by":4,"name":"Preethi Sharma","email":"","orcid":"","institution":"Sharad Pawar Dental College \u0026 Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe)","correspondingAuthor":false,"prefix":"","firstName":"Preethi","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-02-17 06:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6045096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6045096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79674877,"identity":"6af0d62f-89f1-4231-b594-daf6f47b2c75","added_by":"auto","created_at":"2025-04-01 11:55:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":524708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6045096/v1/31590753-b13c-4f4a-a1c7-71a02c072159.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAssessment of Programmed Cell Death Ligand -1 (PD-L1) Expression in Oral Potentially Malignant Lesions and Surgical Margins of Oral Squamous Cell Carcinoma and its Association with 3-year Survival\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e\u0026ldquo;Oral squamous cell carcinoma (OSCC)\u0026rdquo; is the most common malignancy affecting the oral and maxillofacial regions. Globally, head and neck cancers rank as the eighth most prevalent type of cancer [1], with an estimated 880,000 new OSCC cases diagnosed annually and over 440,000 deaths reported each year [2]. Most OSCC cases develop from pre-existing precancerous lesions or conditions categorized as oral potentially malignant disorders (OPMDs). Approximately 2.5% of the general population is believed to have such precancerous conditions in the oral cavity. Common examples of OPMDs include oral \u0026ldquo;leukoplakia\u0026rdquo;, oral submucous fibrosis (OSMF), and oral lichen planus ( OLP), each exhibiting varying rates of malignant transformation [3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite advances in treatment, the mortality rate has not significantly improved over the years, The overall 5 year survival rate for OSCC remains around fifty percentage. The Early detection of OSCC, particularly in well-differentiated tumors that have not metastasized, generally leads to a more favorable prognosis. However, most OSCC cases are diagnosed at advanced stages. Factors which are responsible for \u0026nbsp;prognosis of OSCC are \u0026nbsp;the patient\u0026apos;s age , co-morbidities . Even with negative surgical margins, local recurrence is common, possibly due to the presence of minimal residual cancer (MRC) cells that evade detection by standard histological methods [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemical (IHC) analysis of tumor free \u0026nbsp;surgical margins may provide a more precise method for detecting minimal residual cancer (MRC), as genetic changes often occur before visible phenotypic alterations in the epithelium[5]. \u0026nbsp;For an IHC marker to be effective, it must accurately identify tumor cells and distinguish them from normal tissue [6]. Current research focuses on identifying reliable IHC markers that can improve the clinical management of OSCC patients and provide important prognostic insights. Routine IHC analysis of negative margins could be a more effective approach than conventional histology in identifying patients at higher risk of local recurrence, potentially enhancing survival rates for OSCC patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOf the numerous IHC markers studied, \u0026ldquo;PD-L1 has emerged\u0026rdquo; as one of the most promising. It is utilized as a diagnostic biomarker for immunotherapy in various types of cancers.\u003c/p\u003e\n\u003cp\u003eUnder physiological conditions, the immune system defends the body against various infections and cancers, playing a key role in eliminating diseased or harmful cells. Immunological checkpoints, which serve as inhibitory regulators of the immune system of our body. They are very essential for maintaining self-tolerance. These checkpoints help control the duration, intensity, and extent of immune responses, preventing \u0026ldquo;autoimmunity\u0026rdquo; and reducing \u0026ldquo;collateral tissue damage\u0026rdquo;. One of the immunoinhibitory check \u0026nbsp;is PD-L1 [7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProgrammed death-ligand 1 (PD-L1), also called as \u0026nbsp;CD274 or \u0026nbsp; B7-H1. It is \u0026nbsp; a 290-amino acid protein in the B7 family associated with \u0026nbsp;type I transmembrane receptors. It is formed by a cytoplasmic domain, \u0026nbsp;a transmembrane domain, \u0026nbsp;and two extracellular domains same as \u0026nbsp;IgV and IgC. There are various cell types, including T cells, B cells antigen-presenting cells (APCs), \u0026nbsp;monocytes, and epithelial cells which shows the expression of \u0026nbsp;PD-L1. Its expression is upregulated in response to proinflammatory cytokines, which leads to increase PD-1 levels. The interaction between \u0026ldquo;PD-L1 and PD-1\u0026rdquo; triggers downstream signaling in T cells, suppressing their proliferation, cytotoxic function and cytokine secretion[8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite growing interest in PD-L1 and PD-1 signaling, there remains limited comprehensive research on their clinical relevance and prognostic significance in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma (OSCC) [9]. \u0026nbsp;The precise role of this pathway in the malignant transformation of PD-L1 is still under investigation.\u003c/p\u003e\n\u003cp\u003eIn this era where the incidence of OPMD and OSCC is increasing, it is necessary to identify specific biomarkers for both OPMD and OSCC that would help in clinical decision-making, early diagnosis, treatment strategies and prognosis prediction in OPMD and OSCC [3]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, the study will investigate the differential expression of PD-L1 in OPMD and in surgical margins of OSCC patients \u0026nbsp;along with its association with clinicopathological features and its impact on patient survival ,thus \u0026nbsp;this study will provide the disease spectrum from premalignancy to recurrence of OSCC. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch \u0026nbsp;Study Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType or Nature of study- \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRetrospective, Observational study\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAim:\u0026nbsp;\u003c/strong\u003eTo assess the utility of PD-L1 expression in \u0026nbsp;OPMD as a screening marker \u0026nbsp; and in surgical margins of OSCC as a prognostic marker.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOBJECTIVE(S)\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eTo evaluate the CPS score of \u0026nbsp;PD-L1 immunoexpression in OPMD cases\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTo evaluate the CPS Score of PD-L1immunoexpression in tumor free surgical \u0026nbsp;margins of OSCC cases .\u003c/li\u003e\n \u003cli\u003eTo compare the CPS Score of \u0026nbsp;PD-L1 immunoexpression \u0026nbsp;in \u0026nbsp;OPMD and surgical margins of OSCC.\u003c/li\u003e\n \u003cli\u003eTo correlate the CPS Score of PD-L1 immunoexpression \u0026nbsp;surgical margins of OSCC with three-year-survival.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe study will be conducted in the \u0026ldquo;Department of Oral and Pathology and Microbiology at Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research (Deemed to be University), located in Sawangi (M), Wardha, Maharashtra\u0026rdquo;, India. Archived records of surgically treated OSCC cases and histologically diagnosed cases of OPMD from 2018 to 2020 will be retrieved from the departmental archives for analysis. Sample size and sampling: It will be divided into three groups\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGroup A\u003c/span\u003e: Histologically diagnosed OPMD cases. (20 samples)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGroup B\u003c/span\u003e: Histologically tumor free surgical margins of OSCC cases. (20 samples)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGroup C\u003c/span\u003e- Normal oral mucosa (20 samples)\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion Criteria\u003c/strong\u003e- Patients with primary disease as OPMD and OSCC, tumor sites specific to the oral cavity, and patients with histologically tumor-free surgical margins\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria -\u003c/strong\u003e, Recurrent tumors, Patients who were lost at follow-up, Variants of squamous cell carcinoma, and patients with histologically positive surgical margins\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis plan-\u003c/strong\u003e Tumor free surgical margins of OSCC cases and histopathologically diagnosed cases of OPMD will be retrieved from the departmental archives. The samples will be retrieved from the archives from a period of 2018\u0026ndash;2020. The current survival status of these patients at the start of study will be noted. A further follow up of 3 years will be carried out for each patient\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assessment of PD-L1 antigen will be performed by IHC \u003cstrong\u003e.\u003c/strong\u003e Universal Immuno-enzyme Polymer method\u0026rdquo; will be used for this assessment. After the deparaffinization with xylene, tissue sections will be immersed into grades of alcohol for rehydration. Microwave heat antigen retrieval technique will be used to retrieve the antigen. In this technique sections will be heated for 10 minutes in 0.01M sodium citrate buffer ( PH 6.0). Then its get cooled at room temperature for about 20 minutes. Repeatedly this procedure will be carried out until the enough antigen retrieved. After this sections will be washed with the help of phosphate - buffer saline ( PBS) and treated with 3% hydrogen peroxide in methnol for 30 minutes to block endogenous peroxidase activity. For nonspecific binding of antigen, the section will be rinsed with PBS for three times. Then primary antibody PD-L1 ill be applied to tissue section and incubated for 90 minutes for room temperature in humidifying chamber. Again section will be washed for three for 10 minutes with PBS and polymer anti-mouse antibody conjugated with horseradish peroxidase (HRP) for 30 minutes. The antigen \u0026ndash; antibody reaction will be observed using 3,3\u0026apos;-diaminobenzidine (DAB) chromogen in a buffer solution. Mayer\u0026apos;s haematoxylin counterstaining will be used. PD-L1 immunoexpression will be assessed by two independent observer in a blinded manner.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePD-L1 expression will be assessed using the Combined Positive Score (CPS) under light microscopy. Two pathologists will conduct the evaluation blinded, without access to the clinical details.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eThe CPS is calculated by dividing the number of PD-L1-immunoexpression positive cells, including cells of epithelium, macrophages, and lymphocytes, by the total number of viable neoplastic cells, and then multiplying the result by 100.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003ch3\u003e\u003cimg 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\"\u003e\u003c/h3\u003e\n\u003ch3\u003eSample size: - Minimum sample size required\u003c/h3\u003e\n\u003cp\u003eSample Size Calculation\u003c/p\u003e\n\u003cp\u003eSample size\u003c/p\u003e\n\u003cp\u003eFormula Using Mean difference\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\:n1=n2=2\\frac{({Z}_{\\alpha\\:}+{Z}_{\\beta\\:}{)}^{2}{\\sigma\\:}^{2}}{(\\delta\\:{)}^{2}}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\:{Z}_{\\alpha\\:}==TypeIerrorat\\:1\\%\\:\\:\\:\\:at\\:both\\:sides\\:two\\:tailed\\:\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\:{Z}_{\\beta\\:}=99\\:\\%=Powerat\\:99\\%\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003ePrimary Variable Difference in (PD-L1) expression\u003c/p\u003e\n\u003cp\u003eFor \u0026alpha;\u0026thinsp;=\u0026thinsp;0.01, Z(1-\u0026alpha;/2)\u0026thinsp;=\u0026thinsp;2.576, For \u0026beta;\u0026thinsp;=\u0026thinsp;0.01, Z(1-\u0026beta;)\u0026thinsp;=\u0026thinsp;2.326\u003c/p\u003e\n\u003cp\u003eMean in group 1 (\u0026micro;₁): 4.59\u003c/p\u003e\n\u003cp\u003eStandard deviation in group 1 (\u0026sigma;₁): 1.965\u003c/p\u003e\n\u003cp\u003eMean in group 2 (\u0026micro;₂): 2.03\u003c/p\u003e\n\u003cp\u003eStandard deviation in group 2 (\u0026sigma;₂): 1.204\u003c/p\u003e\n\u003cp\u003eRatio (Group 2 / Group 1): 1\u003c/p\u003e\n\u003cp\u003ePooled std dev = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sigma\\:\\)\u003c/span\u003e\u003c/span\u003e =1.5845\u003c/p\u003e\n\u003cp\u003eSample size N= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n1=n2=2\\frac{(1.96\\:+0.84\\:{)}^{2}({1.5845)}^{2}}{(0.21{)}^{2}}\\approx\\:\\:19.34\\:per\\:group\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eRound up to the nearest whole number for each group:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eMinimum sample size per group\u0026thinsp;=\u0026thinsp;20\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eTotal sample size\u0026thinsp;=\u0026thinsp;40\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003ch3\u003eConclusion\u003c/h3\u003e\n\u003cp\u003eThe minimum of 20 sample are required in each group to analysed the statistically significant difference between the two means with an alpha level of 0.01 and a beta level of 0.01. The total minimum sample size required is 40 participants\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOral squamous cell carcinoma (OSCC) is a multifactorial disease that progresses through multiple stages. Most of the times OSCC preceded by oral potentially malignant disorders (OPMDs) such as leucoplakia OSMF, Oral Lichen planus etc. There are the malignant cytological and architectural changes in the tissue even in the absence of visible tissue changes. Advances in molecular oncology have highlighted the significant role of immune checkpoint inhibitors in cancer pathophysiology. An important immunosuppressive mechanism is the interaction between the ligand, PD-L1, on cancer cells and the PD-1 receptor on T cells. Increased PD-L1 expression has been associated with disease progression. There are numerous studies carried out on resected specimens of OSCC and OPMD. Kanan Dave et al. examined the immunoexpression of \u0026ldquo;PD-L1\u0026rdquo; in oral dysplasia, which progresses to OSCC. They studied 49 cases by immunohistochemistry. It showed increase in \u0026ldquo;PD-L1\u0026rdquo; expression in progressing than in non-progressing epithelial dysplasia. So they concluded that the immunomodulation through \u0026ldquo;PD-L1\u0026rdquo; pathways happens earlier to the malignant transformation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The study carried out by Pachpande et al. on OPMD and OSCC cases, where they analysed PD-L1 expression. They observed elevated PD-L1 in OSCC than OPMD. PD-L1 is associated with a higher likelihood of malignant transformation, So suggested that evaluating PD-L1 levels in OPMD patients able to identify the individuals at risk for malignant progression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The assessing PD-L1 expression in OPMD and the surgical margins of OSCC may serve as a screening and prognostic marker. This study aims to analyze the disease spectrum by examining PD-L1 expression from premalignant stages to malignancy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe PD-L1 is an important biomarker for immunotherapy in various cancers including the OSCC, which is measured by the CPS score. The assessment of PD-L1 expression in OPMD cases may be used as a screening marker for the presence or absence of malignant transformation risk, as an increase in PD-L1 expression signifies an increased risk of malignant change. The assessment of PD-L1 in surgical margins of OSCC may predict the molecular invasion. The calculation of the CPS score of PD-L1 in surgical margins of OSCC may predict prognostic indicators. The assessment of PD-L1 expression in OPMD and in the surgical margin of OSCC cases may be used as a screening marker and prognostic marker. It provides the whole disease spectrum from premalignancy to malignancy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOSCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOral Squamous Cell Carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPMD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOral Premalignant Disorders\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eL1-programmed cell death ligand 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCombined Positive Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical reference number: DMIHER(DU)/IEC/2025/42.\u003c/p\u003e\n\u003cp\u003eEvery participant in the study has given their informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026ndash;\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be taken from the archival of the department.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration -\u003c/strong\u003eNo Funding.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDr. Archana Sonone- Principal investigator of study, formulation or evolution of overarching research goals and aims, writing an original script Dr. Alka Hande-​Preparation, creation, and/or presentation of the published work, specifically visualization/data presentation. editing of manuscript Dr Swati Patil-Data analysis Dr. Aayushi Pakhale-Review of the manuscript Dr. Preethi Sharma-Review of the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGhapanchi J, Ranjbar Z, Mokhtari MJ, Koohpeima F, Derakhshan M, Khademi B, et al. 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PMID: 32546692; PMCID: PMC7297711.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oral potentially premalignant disorder ( OPMD), Oral squamous cell carcinoma (OSCC), Combined-positive score, programmed cell death – ligand 1","lastPublishedDoi":"10.21203/rs.3.rs-6045096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6045096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOral squamous cell carcinoma (OSCC) is the most prevalent type of epithelial cancer affecting the oral cavity. In India, it ranks as the third most common cancer, following breast and cervical cancer. Significant research is being conducted to explore various treatment approaches for OSCC, with immunotherapy emerging as an advanced option. Under normal circumstances, the immune system protects the body from infections and tumors. Key players in this process are cell surface proteins called \"programmed cell death 1 (PD-1)\" and \"programmed cell death ligand 1 (PD-L1),\" which are crucial for immune checkpoint regulation. Higher expression of PD-L1 is associated with worse prognosis and shows an inverse relationship with CD8\u0026thinsp;+\u0026thinsp;T cell infiltration in tumors. This study aims to examine the differential expression of PD-L1 in oral potentially malignant disorders (OPMD) and surgical margins of OSCC patients, while also exploring its link to clinicopathological features and its influence on patient survival. By doing so, the research will provide insights into the progression of the disease from premalignant stages to OSCC recurrence.\u003c/p\u003e\u003ch2\u003eMaterial and methods\u003c/h2\u003e \u003cp\u003eIt is a observation retrospective study that will include 20 cases of OPMD and 20 tumor-free surgical margins from OSCC patients The immunoexpression of PD-L1 will be assessed by CPS score under Leica DMLB2 (Leica microscope).\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eThe assessment of PD-L1 immunoexpression form OPMD patients to surgical margins of OSCC patients, it gives whole disease spectrum and helpful to identify the individuals having a higher risk of malignant potential\u003c/p\u003e","manuscriptTitle":"Assessment of Programmed Cell Death Ligand -1 (PD-L1) Expression in Oral Potentially Malignant Lesions and Surgical Margins of Oral Squamous Cell Carcinoma and its Association with 3-year Survival","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 10:51:09","doi":"10.21203/rs.3.rs-6045096/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"298b2a79-247b-4f9d-80b6-539dd96e168e","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-01T11:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 10:51:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6045096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6045096","identity":"rs-6045096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}