TO DETRIMINE THE IMMUNE CHECK POINT PROTEIN EXPRESSION CTLA-4 AND VISTA WITH CLINICAL AND PATHOLOGICAL STAGING OF POTENTIAL ORAL MALIGNANT LESIONS BY IMMUNO HISTO CHEMISTRY ANALYSIS

TO DETRIMINE THE IMMUNE CHECK POINT PROTEIN EXPRESSION CTLA-4 AND VISTA WITH CLINICAL AND PATHOLOGICAL STAGING OF POTENTIAL ORAL MALIGNANT LESIONS BY IMMUNO HISTO CHEMISTRY ANALYSIS | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 March 2025 V1 Latest version Share on TO DETRIMINE THE IMMUNE CHECK POINT PROTEIN EXPRESSION CTLA-4 AND VISTA WITH CLINICAL AND PATHOLOGICAL STAGING OF POTENTIAL ORAL MALIGNANT LESIONS BY IMMUNO HISTO CHEMISTRY ANALYSIS Authors : Kalaiselvi Santhosh [email protected] , Karthik Sigamani , Thalaimalai Saravanan , K.R.Shakila Ramalingam , Santhoshkumar Elango , and Aravindh Swamy Authors Info & Affiliations https://doi.org/10.22541/au.174131507.79218599/v1 237 views 79 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Abstract Introduction : The immune microenvironment is essential in the development of leukoplakia. Which is a potentially precancerous lesion with increased chances of evolving into oral squamous cell carcinoma (OSCC). The immune check point proteins V-domain Ig Suppressor of T-cell Activation (VISTA) and cytotoxic T lymphocyte antigen-4(CTLA-4) are the most important co stimulatory molecule the playa a key role in negative regulation of T cell during process of carcinogenesis. The VISTA and CTLA-4 serves as a emerging new biomarker in early detection and to determine malignant potential of leucoplakia Material and method: Tissue samples from patients with suspected oral malignant lesions were analyzed using immunohistochemistry (IHC). The expression levels of CTLA-4 and VISTA were evaluated using standardized scoring systems. Clinical finding and histopathological grading was established . The correlation between protein expression patterns and clinicopathological parameters was statistically analyzed. Result : VISTA expression varied among the different dysplasia grade with a non-linear relationship observed between VISTA expression and the severity of dysplasia.A distinct progressive rise in CTLA 4 positivity that paralleled the severity of dysplasia, indicating that CTLA 4 expression could serve as a valuable biomarker for evaluating the risk of malignant transformation in oral potentially malignant disorders Keywords: CTLA-4, VISTA, leukoplakia, immune checkpoint proteins, immunohistochemistry, inflammatory check points Introduction: According to the ICMR Globocan 2012 data , oral cancer is the most common cancer among men than women. Around 1 in 60 men and 1 in 141 women will acquire oral cavity and oropharyngeal cancer in their lifetimes. It is estimated that there are around 77,000 new cases of oral cancer each year in the country, Unfortunately, oral cancer is often diagnosed late in India, resulting in poorer outcomes and a lower survival rate 1 . The Immune checkpoints binds with the partner proteins bind and send an “off” signal to the T cells.Thereby preventing the immune system from destroying the cancer cells.Check point protein have shown clinical efficacy in several solid malignancies including head and neck squamous cell carcinoma 1 .OSCC is frequently preceded by the emergence of precancer lesions that have the potential to progress to invasive carcinoma. Theoretically, identifying and eliminating cancer precursors would eradicate the majority of human cancers 2 . Caner is a two-step process that starts with a potentially malignant precursor lesion and progresses to cancer.Among the precancerous conditions, oral leukoplakia is recognized as the most common potentially malignant oral disorders (PMODs). Notably, oral leukoplakia is distinguished as the most prevalent PMOD, exhibiting a considerable likelihood of progression to oral squamous cell carcinoma (OSCC) 3 . While current clinical practices predominantly depend on histological evaluations and assessments of dysplasia severity, recent studies indicate the promise of alternative prognostic indicators. DNA ploidy analysis has been suggested as a potential surrogate marker for forecasting the behavior of PMODs. Nevertheless, the expression of immune checkpoint proteins in OSCC remains largely unexamined, especially within our demographic. Few studies have thoroughly explored these molecular mechanisms and their possible implications for diagnosis, prognosis, and recurrence. Our present study aims is to evaluate the expression patterns of immune checkpoint proteins CTLA-4 and VISTA in oral potentially malignant lesions and Correlate immune checkpoint protein expression with clinical and pathological staging thereby exploring their potential as diagnostic and prognostic markers in OSCC. There by giving us a insights into the immunological mechanisms underlying oral cancer development and contribute to precise early detection and management strategies. Material and methods For this retrospective study, paraffin-embedded tissue samples were collected from Department of Oral Pathology, Karpaga Vinayaga Institute of Dental Sciences, Tamil Nadu. Samples between 2022 and 2024 were examined. A total of 60 speciment were evaluated by oral pathologist and Group 1 included tissue samples from normal healthy patients. Group 2 included oral leukoplakia with mild moderate and sever dysplasia s accordance with the World Health Organization (WHO) classification of tumors of the Head and Neck 2017 4 . The samples were further analysed according to demogrophic,clinical and histopathological data Qualitative Immunohistochemistry was performed in 4-μm tissue microarray sections of formalin fixed, charged slides . These cases were stained with monoclonal antibody against CTLA AND VISTA by BioGenex, USA (Ready-To-Use, New & Improved Super Sensitive™ Polymer-HRP Detection Kit, HRP/DAB). For Immunohistochemical staining of cases, first deparaffinization of formalin fixed paraffin embedded tissue was carried out. Antigen retrieval was done by inserting in citrate buffer and then heating in microwave oven at 95-100C for 20 minutes. Slides were allowed to cool at room temperature for 15-20 minutes. Slides were rinsed with distilled water and phosphate buffer saline (PBS). Peroxidase blocking solution was added to the sections of the slides incubated for 10 minutes at room temperature. Rinsed in PBS for 6 minutes. Primary antibody CTLA AND VISTA was applied to sections on the slides and incubated for 60 minutes (1 hour) in humidified chamber at room temperature. After 1 hour, the slides were washed again. Biotinylated secondary antibody was applied to the sections and incubated for 30 minutes at room temperature. Rinsed in PBS for 6 minutes. Chromogen/substrate was applied, and sections were incubated in peroxidase substrate solution to reveal color of the antibody. Color was allowed to develop for less than 5 minutes, and slides were washed again. Counterstaining was performed by immersing slides in hematoxylin for 1-2 minutes. Slides were again rinsed in running tap water for about 15 minutes. Tissue slides were dehydrated through four changes of alcohol (95%, 95% 100% and 100%) 5 minutes each. Slides were cleared in three changes of xylene and cover slip was placed using mounting solution and were stored at room temperature. PD-L1 immunoreactivity scoring was defined according to cell intensity for all OSCC and OPMD cases. The H-score is calculated by evaluating both the intensity of staining and the percentage of positive cells in immunohistochemistry (IHC) samples. the formula, H-score = 3 × the percentage of strong staining + 2 × the percentage of moderate staining + the percentage of weak staining.The following criteria was used to observe the intensity of CTLA AND VISTA stain. 0=Negative 1=Weak staining 2=Moderate staining 3=Strong staining 4,5 . RESULTS All statistical analyses were performed using IBM SPSS Statistics v26 (IBM, Armonk, NY, USA), including standard frequency, descriptive assessments as well as inferential statistics was done and Significance was determined with the Chi-square test . Demographic and clinical data of the study are shown in Table1. Histopathological grading and expression of CTLA -4 and VISTA shown in table 2 &3. Our demographic analysis revealed that the majority of subjects were in the 40-50-year age group (45%), followed by the 50-60-year age group (33.33%), which is consistent with the typical age distribution of oral potentially malignant disorders. The equal gender distribution in our study (50% male, 50% female) differs from some previous reports that suggest a male predominance in oral leukoplakia, possibly reflecting changing patterns of risk factor exposure. The anatomical site distribution showed a clear predilection for the buccal mucosa (30%), which correlates with common sites of tobacco placement in users of smokeless tobacco. This is further supported by our risk factor analysis, which identified tobacco chewing (30%), quid use (28.33%), and alcohol consumption (31.67%) as the most prevalent risk factors among the study participants. Pattern of expression of CTLA-4 in histopathological leukoplakia The primary outcome of our research highlights a string correlation between CTLA 4 expression and the extent of dysplasia in oral leukoplakia. A distinct progressive rise in CTLA 4 positivity that paralleled the severity of dysplasia, with 0% expression observed in mild dysplasia, 40% in moderate dysplasia, and 80% in severe dysplasia. This relationship was statistically significant (p=0.001273), indicating that CTLA 4 expression could serve as a valuable biomarker for evaluating the risk of malignant transformation in oral potentially malignant disorders. CTLA 4 negative expression in healthy control tissues further reinforces its potential as a specific marker for dysplastic alterations. These findings are consistent with emerging research in other cancers, where CTLA 4 has been associated with mechanisms of immune evasion, enabling neoplastic cells to evade immune detection. Pattern of expression of VISTA in histopathological leukoplakia The result analysis of 30 oral leukoplakia samples, there was an equal distribution of 10 cases across each degree of dysplasia. Although VISTA expression varied among the different dysplasia grades—recording 0% in mild cases, 20% in moderate cases, and 40% in severe cases—this correlation did not achieve statistical significance (p=0.00127726). The non-linear relationship observed between VISTA expression and the severity of dysplasia. Demographic and Risk Factor Considerations The epidemiological age distribution patterns of oral potentially malignant disorders (OPMDs) with predominance in the 40-50 year (45%) and 50-60 year (33.33%) age groups, this finding is consistent with Warnakulasuriya (2018), who reported peak incidence of leukoplakia in the fifth and sixth decades of life 5 . The equal gender distribution observed (50% male, 50% female) is a significant change from the earlier reported male predominance by Petti 6 . In particular, this could represent an increase in risk and health hazards, particularly tobacco smoking among women, as noted by Sridharan 7 . The anatomical predilection for buccal mucosa strongly correlates with 30% of habitual placement of smokeless tobacco products. This site specific association of tobacco consumption and lesion site was established by Hashibe et al 8 . Further analyzing the prevalence of chewing tobacco (30%), quid (28.33%), and alcohol (31.67%), corroborates the finding of Mello et al (2018) which attributed the increased rates of chewing, dribling and smoking to OPMDs 9 . CTLA-4 Expression pattern and Dysplasia Progression Gradual increase of CTLA-4 expression corresponding with (p = 0.001273) the severity of dysplasia (0% in mild, 40% in moderate, and 80% in severe) with p shows that dysplastic lesions are malignant. This pattern leads to the suggestion that CTLA-4 might serve as a biomarker in the risk of malignant transformation of OPMDs. The same correlations has been done in the CTLA-4 over expression in younger cases of esophageal dysplasia by Zhang et al. (2016) 10 . The negative result of CTLA-4 expression in control tissues strengthens its specificity for marker of dysplastic transformation. This is consistent with the results on in vitro investigations done by Stasikowska-Kanicka et al. (2018) who showed that ordinary oral epithelium doesn’t express CTLA-4, while oral squamous cell carcinoma manifests considerable expression of this molecule 11 . Increased CTLA-4 expression in severe forms of dysplasia has strong immunologic consequences that suggest tumor associated immune suppression. Teng et al. (2015) noted that upregulation of immune checkpoint molecules CTLA-4 creates an immune suppressive microenvironment and enhances tumor progression 12 . This fact adds to the concept that CTLA-4 is not a simple biomarker, but might contribute to carcinogenesis by suppressing anti-tumor immunity. The significant association between CTLA-4 expression and dysplasia severity has potential clinical applications. Wu et al. (2019) demonstrated that immune checkpoint expression profiles could stratify risk of malignant transformation in oral leukoplakia 13 . Our findings support the potential utility of CTLA-4 as a prognostic biomarker for identifying high-risk lesions requiring more aggressive management. VISTA Expression Patterns and Dysplasia Progression The lack of statistical significance (p=0.00127726) suggests that correlation between VISTA expression and the severity of dysplasia is a phenomenon with much intricacy. Clare et al reported similar findings that showed variable VISTA expression across stages of colorectal neoplasia 14 . This variation might capture the different potential roles of VISTA in immunological regulation. As explained by ElTanbouly and colleagues (2020), VISTA can function in both co-inhibition and co-stimulation depending on surrounding microenvironment 15 . The non-significant associations with grade of dysplasia may be capturing the variations of VISTA expression that are influenced by other factors beyond mere histopathological progression Furthermore, the differential expression patterns between CTLA-4 and VISTA highlight the complexity of immune checkpoint regulation in OPMDs. Keenan et al. (2019) emphasized the importance of comprehensive profiling of multiple immune checkpoints to fully understand the immunological landscape of premalignant lesions 16 .These insights may inform potential immunotherapeutic approaches. The established efficacy of anti-CTLA-4 therapies in various malignancies, as reviewed by Callahan et al 17 ., raises the possibility of exploring immune checkpoint inhibition as a preventive strategy in high-risk OPMDs showing elevated CTLA-4 expression. Future research should explore the functional consequences of CTLA-4 upregulation in oral dysplasia, potentially through in vitro models as employed by Li et al 18 . The PD-L1 positivity of subepithelial TAFs ( p < 0.001) increased with increasing grades of oral leukoplakia. Pearson’s correlation indicated a high positive correlation between the PD-L1 labelling index of epithelial tumour cells and the PD-1 labelling index of tumour infiltrating lymphocytes ( p value: 0.005) in OSCC 18 Integration of multiple immune checkpoint markers, including PD-1/PD-L1 alongside CTLA-4 and VISTA, could provide a more comprehensive immunological profile, as suggested by Feng et al and Kujan etal 19. . Conclusion Our research finding contribute to understand the immune evasion mechanisms in oral carcinogenesis and suggest that immunohistochemical evaluation of CTLA-4 expression could potentially enhance the histopathological assessment of oral leukoplakia, helping to identify high-risk lesions that may benefit from more aggressive management or closer surveillance. Further larger-scale studies and follow up is necessary to definitively correlate CTLA-4 expression with malignant transformation rates. their clinical applications. Table 1:Demographic and clinical date 30-40 5 8.33 40-50 27 45.00 50-60 20 33.33 60-70 8 13.33 GENDER MALE 30 50.00 FEMALE 30 50.00 DURATION LESS THSN 6months 21 35.00 LESS THAN1 YEAR 13 21.67 LESS THAN 3 YEAR 9 15.00 MORE THAN 5 YEAR 17 28.33 TOBACCO SMOKING 0.00 CIGRATTE 9 15.00 BIDI 6 10.00 SMOKELESS CHEWING 18 30.00 QUIDING 17 28.33 ALCOHOL 19 31.67 SITE BUCCAL MUCOSA 18 30.00 FLOOR OF THE MOUTH 1 1.67 GINGIVA 1 1.67 HARD PALATE 2 3.33 LABIAL MUCOSA 4 6.67 TONGUE 3 5.00 RETROMOLAR AREA 1 1.67 SIZE LESS THE 2 CM 12 20.00 GREATER THAN 2 CM 18 30.00 SURFACE HOMOGENEOUS 20 33.33 NON HOMOGENEOUS 10 16.67 SYMPTOMS BURNING SENSATION 8 13.33 NO SYMPTOMS 12 20.20 TABLE 2: Pattern of expression of CTLA- 4 in oral leukoplakia Mild CTLA- 4 positive 0 CTLA- 4 negative 10(100%) Moderate CTLA- 4 positive 4(20%) CTLA- 4 negative 6(80%) Severe CTLA- 4 positive 8(20%) CTLA- 4 negative 2(60%) Chi-square value: 13.33 and P-value: 0.0012726 (statistically zignificant) Table 3: Pattern of expression of VISTA in oral leukoplakia Mild VISTA positive 0 VISTA negative 10(100%) Moderate VISTA positive 1(10%) VISTA negative 9(90%) Severe CTLA positive 4(40%) CTLA negative 8(80%) Chi-square value: 5.00 and P-value: 0.082085 (not statistically significant) Reference 1. 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Authors Affiliations Kalaiselvi Santhosh [email protected] Karpaga Vinayaga Institute of Dental Sciences View all articles by this author Karthik Sigamani Karpaga Vinayaga Institute of Medical Science and Research Center View all articles by this author Thalaimalai Saravanan Karpaga Vinayaga Institute of Dental Sciences View all articles by this author K.R.Shakila Ramalingam Karpaga Vinayaga Institute of Dental Sciences View all articles by this author Santhoshkumar Elango Karpaga Vinayaga Institute of Medical Science and Research Center View all articles by this author Aravindh Swamy Karpaga Vinayaga Institute of Dental Sciences View all articles by this author Metrics & Citations Metrics Article Usage 237 views 79 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Kalaiselvi Santhosh, Karthik Sigamani, Thalaimalai Saravanan, et al. TO DETRIMINE THE IMMUNE CHECK POINT PROTEIN EXPRESSION CTLA-4 AND VISTA WITH CLINICAL AND PATHOLOGICAL STAGING OF POTENTIAL ORAL MALIGNANT LESIONS BY IMMUNO HISTO CHEMISTRY ANALYSIS. Authorea . 07 March 2025. DOI: https://doi.org/10.22541/au.174131507.79218599/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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