PD-L1 Expression in Undifferentiated Nasopharyngeal Carcinoma Is Associated with EBV-LMP1 and Tumor Immune Infiltration : Data from a Tunisian Cohort

PD-L1 Expression in Undifferentiated Nasopharyngeal Carcinoma Is Associated with EBV-LMP1 and Tumor Immune Infiltration : Data from a Tunisian Cohort | 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 PD-L1 Expression in Undifferentiated Nasopharyngeal Carcinoma Is Associated with EBV-LMP1 and Tumor Immune Infiltration : Data from a Tunisian Cohort Amani Sammoud, Nehla Mokni Baizig, Chaima Brirmi, Salma Kamoun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8584562/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 Programmed death-ligand 1 (PD-L1) is a key immune checkpoint involved in tumor immune evasion and a therapeutic target in immuno-oncology. Undifferentiated nasopharyngeal carcinoma (UCNT) is strongly linked to Epstein–Barr virus (EBV) infection, but data on PD-L1 expression and its relationship with tumor immunity and viral markers in North African populations are limited. Objective To assess PD-L1 expression in UCNT and its associations with clinicopathological features, tumor-infiltrating immune cells, and the viral protein LMP1. Methods We retrospectively analyzed 75 UCNT patients diagnosed at the Salah Azaïez Cancer Institute, Tunis. PD-L1 expression was evaluated by immunohistochemistry using the Tumor Proportion Score (TPS) and Combined Positive Score (CPS). CD8⁺ cytotoxic T cells, FOXP3⁺ regulatory T cells, CD163⁺ macrophages, and LMP1 expression were also assessed. Spearman’s correlation and χ² tests were used, with p < 0.05 considered significant. Results PD-L1 was positive in 70.7% of cases (TPS) and 73.3% (CPS), with strong correlation between the two scores (rho = 0.956 ; p < 0.001). PD-L1 expression was not associated with clinicopathological parameters but showed significant positive correlations with LMP1 (TPS : rho = 0.341; p = 0.003; CPS: rho = 0.345; p = 0.002) and with intratumoral CD8⁺ T cells (rho = 0.371; p = 0.002) and FOXP3⁺ regulatory T cells (rho = 0.253; p = 0.047). No correlation was observed with CD163⁺ macrophages. Conclusion PD-L1 is frequently expressed in UCNT and closely associated with EBV-LMP1 and tumor-infiltrating immune cells. These findings highlight PD-L1 as a potential biomarker and provide insight into the immune-viral interactions shaping the tumor microenvironment in this population. Nasopharyngeal carcinoma PD-L1 EBV LMP1 Tumor-infiltrating immune cells Figures Figure 1 Figure 2 Introduction Nasopharyngeal carcinoma (NPC) is a rare malignant epithelial tumor worldwide, but it exhibits a highly heterogeneous geographic distribution. Its incidence is particularly high in Southeast Asia and North Africa, where the predominant histological form is undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) [ 1 ]. This form is characterized by strong lymphocytic infiltration and a close association with latent Epstein–Barr virus (EBV) infection, making NPC a tumor with a significant immunobiological dimension. Due to the deep location of the nasopharynx and the atypical nature of early symptoms, the diagnosis of NPC is often delayed. Consequently, more than 70% of patients are diagnosed at a locally advanced stage, and approximately 6 to 15% already present with metastases at the time of diagnosis [ 2 – 4 ]. Despite advances brought by conformal radiotherapy and chemoradiotherapy, which constitute the standard treatment for locally advanced forms, the prognosis remains limited. About 20% of patients do not achieve a durable therapeutic response, due to interindividual variations in immune function and treatment tolerance [ 5 ]. Tumor recurrence occurs in 5 to 15% of patients, while distant metastases develop in 15 to 30% of cases [ 4 ]. This unfavorable course highlights the central role of immune escape mechanisms in tumor progression and justifies the growing interest in immunotherapy. Among the well-characterized immune escape mechanisms, the PD-1/PD-L1 axis has recently emerged as a central immunoregulatory pathway, whose functional implication makes it a major area of interest for the development of innovative therapeutic strategies. The expression of PD-L1 by tumor cells and infiltrating immune populations inhibits the cytotoxic activity of T lymphocytes, thereby contributing to the establishment of an immunosuppressive tumor microenvironment [ 6 , 7 ]. Immune checkpoint inhibitors targeting this pathway have demonstrated notable clinical efficacy in recurrent or metastatic disease, reinforcing the therapeutic relevance of this approach [ 8 , 5 ]. However, treatment responses remain heterogeneous, underscoring the need for reliable predictive biomarkers. This variability reflects the influence of multiple biological factors, including the structural organization of the tumor microenvironment and the presence of chronic EBV infection. EBV plays a central role in immune modulation, particularly through the viral oncoprotein LMP1. LMP1 activates oncogenic and inflammatory signaling pathways capable of upregulating PD-L1 expression, promoting the recruitment of regulatory T cells and M2-polarized macrophages, and ultimately reinforcing local immunosuppression [ 9 , 10 ]. These observations highlight the presence of a complex immunological network that facilitates tumor immune escape and contributes to therapeutic resistance. To date, most available data regarding PD-L1 expression and its interaction with the tumor microenvironment originate from Asian cohorts, while North African data remain limited despite the high incidence reported in this region. A more comprehensive characterization of the immune landscape of NPC in these populations is therefore essential to improve patient stratification and to optimize immunotherapy-based therapeutic strategies. In this context, the present study evaluates PD-L1 expression in a Tunisian cohort of UCNT and investigates its correlations with clinicopathological features, major tumor-infiltrating immune cell populations (CD8⁺ T cells, FOXP3⁺ regulatory T cells, and M2 macrophages), and EBV-LMP1 expression, aiming to better characterize the immunological determinants of NPC in our regional context. Materials and Methods This retrospective observational cohort study was conducted at the Salah Azaiz Institute in Tunis. A total of 75 patients with histologically confirmed undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) were included between 2017 and 2024, identified through the institution’s database. All tumor specimens were independently reviewed by an experienced pathologist to confirm the diagnosis. Demographic and clinical data were extracted from medical records, and clinical staging was assigned according to the eighth edition of the American Joint Committee on Cancer (AJCC) TNM classification. Inclusion Criteria : Patients were included if they had a nasopharyngeal tumor of the undifferentiated non-keratinizing type (UCNT), a histologically confirmed diagnosis, a biopsy performed prior to the initiation of treatment, and available tumor samples for analysis. Exclusion Criteria : Patients with incomplete or unavailable medical records in the Institute’s archives, as well as damaged or poorly preserved paraffin blocks and specimens of insufficient size for reliable immunohistochemical analysis, were excluded from the study. Immunohistochemical analysis : Immunohistochemical (IHC) analysis was performed on 75 formalin-fixed, paraffin-embedded (FFPE) tumor samples to assess the expression of PD-L1, LMP1, FoxP3, CD163, and CD8 using the Novolink™ Polymer Detection System (Leica Biosystems, USA). Paraffin-embedded tissues were sectioned at 4 µm, deparaffinized in toluene, and rehydrated through decreasing ethanol concentrations (100%, 95%, and 80%; 5 min each). Antigen retrieval was performed in Tris-EDTA buffer (pH 6 for CD163, PD-L1, and FoxP3 ; pH 9 for CD8 and LMP1) followed by heating at 98°C for 30 min in a water bath. After rinsing in phosphate-buffered saline (PBS), slides were incubated with primary antibodies under the following conditions : CD163 and CD8 for 30 min at room temperature, and FoxP3, PD-L1, and LMP1 for 60 min at room temperature. Following Tris-buffered saline (TBS) washes, slides were sequentially incubated with Post Primary Block and NovoLink Polymer for 30 min each. Antigen–antibody complexes were visualized with 3,3′-diaminobenzidine (DAB) for 10 min, counterstained with hematoxylin for 5 min, dehydrated through graded ethanol concentrations (80%, 95%, and 100%; 5 min each), cleared in toluene, and mounted with Eukitt. Evaluation of immunostaining : Slide evaluation and analysis were performed independently by two pathologists. Each section was first examined at low magnification (×10) to assess the overall tissue architecture, followed by high magnification (×400) for counting immunolabeled cells. In case of different scoring results by two pathologists, a consensus on final scoring was reached after joint review. Slides showing tissue detachment or significant background staining were excluded from the analysis. Cell counting was performed in the five fields with the highest density of positive cells, and the labeling rate was calculated as the mean percentage of positive cells relative to the total number of cells observed. PD-L1 evaluation : PD-L1 expression was assessed using two scores: the Tumor Proportion Score (TPS) and the Combined Positive Score (CPS). The TPS corresponds to the percentage of viable tumor cells showing partial or complete membranous staining relative to the total number of viable tumor cells in the sample. It is scored on a scale from 0% to 100% and is considered positive if detected in at least 1% of viable tumor cells, with low expression defined as a TPS of 1–49% and high expression as a TPS ≥ 50%. The CPS was calculated by dividing the number of PD-L1–positive cells (the sum of tumor cells, lymphocytes, and macrophages) by the total number of viable tumor cells on the slide and then multiplying the result by 100. It was considered negative if less than 1% and positive if ≥ 1%, with low expression for a CPS of 1–19% and high expression for a CPS ≥ 20%. Immun cell evaluation : The levels of CD8, CD163, and FoxP3 expression were assessed and classified into two categories: low (< 50% of positive cells) and high (≥ 50% of positive cells). LMP1 evaluation : LMP1 expression was assessed and categorized as negative (0% positive cells) or positive (≥ 1% positive cells). Statistical analysis : SPSS version 26.0 software was used for statistical analysis. Associations between PD-L1 expression profiles (high vs. low) and clinicopathological parameters, as well as immune markers (CD163, CD8, FoxP3) and LMP1, were evaluated using Pearson’s chi-squared (χ²) test. Correlations between the expression levels of cellular markers (CD163, CD8, FoxP3, and PD-L1) were analyzed using Spearman’s correlation coefficient (ρ). A p-value of < 0.05 was considered statistically significant, with a 95% confidence interval. Results Clinicopathological description of the study population : A cohort of 75 patients diagnosed with undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) and treated at the Salah Azaiez Institute was analyzed. The study population consisted of 58 men and 17 women, with a mean age of 57 years (median = 54; SD = 11.46; 95% CI: 39–86), corresponding to a male-to-female ratio of 3.41. According to the TNM classification, 48% of patients presented with early T stage disease (T1–T2), while 52% had advanced T stages (T3–T4). Lymph node involvement was absent in 5.33% of cases (N0), moderate in 54.67% (N1–N2), and advanced in 40% (N3). Distant metastases were identified in 17.33% of patients. In terms of overall disease stage, early stages (I–II) accounted for 20% of the cohort, whereas advanced stages (III–IV) predominated with 80%. Among the 34 patients assessed three months after completion of treatment, therapeutic response was characterized by a complete response in 17 cases (50%) and a partial response in 17 cases (50%) (Table 1 ). Table 1 Clinical-demographic characteristics Variable n = 75 Age ≤ 54 > 54 38 (50.67%) 37 (49.33%) Gender Female Male 17 (22.67%) 58 (77.33%) T stage T1 T2 T3 T4 11 (14.67%) 25 (33.33%) 18 (24%) 21 (28%) N stage N0 N1 N2 N3 4 (5.33%) 22 (29.33%) 19 (25.34%) 30 (40%) M stage M0 M1 62 (82.67%) 13 (17.33%) TNM stage I II III IV 1 (1.33%) 14 (18.67%) 26 (34.67%) 34 (45.33%) Treatment response (n = 34) PR CR 17 (50%) 17 (50%) PR: partial response, CR: complete response Expression of PD-L1 protein in NPC patients : PD-L1 expression was analyzed in 75 nasopharyngeal carcinoma (NPC) specimens by immunohistochemistry. The protein was predominantly localized to the cell membrane (Fig. 1 ). Immunohistochemical analysis showed that, based on the TPS, PD-L1 expression was positive (> 1%) in 53 cases (70.7%) and negative in 22 cases (29.3%). According to the CPS, high expression (> 20) was observed in 45 cases (60%), low expression (1–19) in 10 cases (13.3%), and negative staining in 20 cases (26.7%) (Table 2 ). Table 2 PD-L1 Expression Profile According to CPS and TPS Scores PD-L1 Expression Profile (n = 75) Number Percentage PD-L1 TPS Assessment Negative 22 29.3% Positive 53 70,7% Positive (1–49%) 21 28% Positive (> 50%) 32 42.7% PD-L1 CPS Assessment Negative 20 26.7% Positive 55 73.3% Positive (1–19) 10 13.3% Positive (> 20) 45 60% PD-L1 : programmed cell death ligand-1 ; TPS : Tumor proportion score ; CPS : Combined positive score PD-L1 expression and the clinicopathological features of NPC patients : The association between PD-L1 expression and clinicopathological parameters was assessed to clarify the role of PD-L1 in NPC. PD-L1 expression was evaluated using TPS and CPS scores and categorized as negative (< 1%), positive with low expression (< 50% for TPS, < 20 for CPS), or high expression (≥ 50% for TPS, ≥ 20 for CPS), as shown in Table 3 . No significant associations were observed between PD-L1 expression and sex, age, tumor size (T), lymph node status (N), distant metastasis (M), overall TNM stage, or treatment response. Spearman’s correlation was performed between the positive TPS and CPS variables. There was a strong correlation demonstrated through the coefficient value of 0,941 (p < 0,001) (Fig. 2 ). PD-L1, programmed cell death ligand-1; TPS, tumor proportion score; CPS, combined positive score Table 3 Statistical association between PD-L1 expression and patient clinicopathological parameters Variable CPS Score PD-L1 Negative PD-L1 Positive P-value TPS Score PD-L1 Negative PD-L1 Positive P-value Low (1–49%) High (> 50%) Low (1–49%) High (> 50%) Gender Male Female 16 (88.89%) 2 (11.11%) 9 (75%) 3 (25%) 33 (73.33%) 12 (26.67%) 0.403 17 (77.27%) 5 (22.73%) 15 (71.43%) 6 (28.57%) 26 (81.25%) 6 (18.75%) 0.705 Age (years) ≤ 54 > 54 9 (50%) 9 (50%) 6 (50%) 6 (50%) 26 (57.78%) 19 (42.22%) 0.803 12 (54.55%) 10 (45.45%) 9 (42.86%) 12 (57.14%) 20 (62.5%) 12 (37.5%) 0.373 T Stage T1 + T2 T3 + T4 8 (44.44%) 10 (55.56%) 5 (41.67%) 7 (58.33%) 23 (51.11%) 22 (48.89%) 0.795 11 (50%) 11 (50%) 8 (38.1%) 13 (61.90%) 17 (53.13%) 15 (46.87%) 0.550 N Stage N0 N1 + N2 N3 0 (0%) 12 (66.67%) 6 (33.33%) 1 (8.33%) 6 (50%) 5 (41.67%) 3 (6.67%) 23 (51.11%) 19 (42.22%) 0.699 2 (9.09%) 13 (59.09%) 7 (31.82%) 1 (4.76%) 13 (61.90%) 7 (33.34%) 1 (3.13%) 15 (46.87%) 16 (50%) 0.570 M Stage M0 M1 15 (83.33%) 3 (16.67%) 10 (83.33%) 2 (16.67%) 37 (82.22%) 8 (17.78%) 0.992 19 (86.36%) 3 (13.64%) 19 (90.48%) 2 (9.52%) 24 (75%) 8 (25%) 0.299 TNM Stage I + II III + IV 5 (27.78%) 13 (72.22%) 3 (25%) 9 (75%) 7 (15.56%) 38 (84.44%) 0.491 7 (31.82%) 15 (68.18%) 4 (19.05%) 17 (80.95%) 4 (12.5%) 28 (87.5%) 0.217 Treatment response (n = 34) PR CR 1 (20%) 4 (80%) 4 (57.14%) 3 (42.86%) 11 (50%) 11 (50%) 0.4 1 (14.29%) 6 (85.71%) 6 (50%) 6 (50%) 9 (60%) 6 (40%) 0.131 PD-L1 : programmed cell death ligand-1 ; TPS : Tumor proportion score ; CPS : Combined positive score ; CR : complete response ; PR : partial response ; Pearson’s χ2 test, p < 0.05. Correlation between PD-L1 Expression and Immune Cell Infiltration : Spearman’s correlation analysis was performed to evaluate the association between PD-L1 expression (TPS and CPS) and the infiltration of regulatory T cells (FoxP3), cytotoxic T cells (CD8), and M2 macrophages (CD163), with p < 0.05 considered significant. PD-L1 expression was positively correlated with FoxP3⁺ T cell infiltration (CPS : r = 0.253, p = 0.047 ; TPS : r = 0.254, p = 0.046) and CD8⁺ T cell infiltration (CPS : r = 0.371, p = 0.002 ; TPS : r = 0.355, p = 0.003). No significant correlation was observed between PD-L1 expression and M2 macrophage (CD163) infiltration (CPS : r = 0.232, p = 0.066 ; TPS : r = 0.190, p = 0.132) (Table 4 ). Table 4 Statistical correlation between PD-L1 expression and tumor-infiltrating immune cells CD8 FoxP3 CD163 TPS 0.355 ** 0.254 * 0,190 p = 0.003 p = 0.046 p = 0.132 CPS 0.371 ** 0.254 * 0.232 p = 0.002 p = 0.046 p = 0.066 TPS : Tumor proportion score ; CPS : Combined positive score ; Spearman’s rho test *. Correlation is significant at the 0.05 level (two-tailed). **. Correlation is significant at the 0.01 level (two-tailed). Correlation between PD-L1 and LMP1 expression : Spearman’s correlation analysis revealed a significant positive association between PD-L1 and LMP1 expression. Specifically, PD-L1 expression was positively correlated with LMP1 levels according to both TPS (r = 0.341, p = 0.003) and CPS (r = 0.345, p = 0.002) scores, indicating that higher PD-L1 expression is associated with increased LMP1 expression in nasopharyngeal carcinoma specimens (Table 5 ). Table 5 Statistical correlation between PD-L1 and LMP1 LMP1 TPS 0.341** p = 0.003 CPS 0.345** p = 0.002 TPS : Tumor proportion score ; CPS : Combined positive score ; Spearman’s rho test **. Correlation is significant at the 0.01 level (two-tailed). Discussion The regulation of the antitumor immune response relies on a delicate balance between activating and inhibitory signals, among which the PD-1/PD-L1 pathway plays a central role in modulating immune tolerance [ 11 ]. In nasopharyngeal carcinoma (NPC), which is strongly associated with Epstein–Barr virus (EBV) infection, activation of this pathway has been shown to promote tumor immune evasion mechanisms [ 12 ]. Recent studies have demonstrated that the viral protein LMP1 and certain pro-inflammatory cytokines, notably interferon-γ, induce PD-L1 expression and contribute to the establishment of an immunosuppressive microenvironment [ 13 , 14 ]. This dysregulation has led to the development of immune checkpoint inhibitors, whose clinical efficacy has been confirmed in several recent trials [ 15 , 16 ]. However, the biological heterogeneity of tumors, intertumoral and interindividual variability, as well as ethnic and geographic differences, can influence PD-L1 expression profiles, thereby limiting its universal prognostic value. Few data are available regarding North African patients with NPC prior to any therapeutic intervention. In this context, our study sought to characterize PD-L1 expression and its clinical relevance in a cohort of primary NPC biopsies from Tunisia using immunohistochemistry. We first evaluated PD-L1 expression in both tumor and immune cells and assessed its associations with key clinicopathological features to gauge its potential prognostic value. To further elucidate local immune dynamics, we examined correlations between PD-L1 status and the composition of tumor-infiltrating immune cells. Additionally, by investigating the relationship between PD-L1 and the viral oncogene LMP1, we explored how latent EBV infection may influence immune checkpoint regulation within the tumor microenvironment. In our cohort, PD-L1 expression was observed in 70.7% of cases according to TPS (≥ 1% of tumor cells) and in 73.3% according to CPS (≥ 1), with 60% showing high expression (CPS > 20). These findings highlight the combined contribution of tumor and immune cells in PD-L1 regulation and underscore the central role of the tumor microenvironment. These results are consistent with several previous studies reporting comparable PD-L1 expression rates in NPC [ 17 – 19 ]. Nevertheless, lower frequencies have also been described in the literature, which may be attributed to differences in immunohistochemical methods, antibody clones, scoring systems, or histological and population-related variations [ 8 , 20 , 21 ]. In line with this observation, a strong correlation between TPS and CPS was observed. Notably, the higher the TPS value, the higher the CPS value. This finding suggests that PD-L1 expression by tumor cells largely reflects the overall PD-L1 status of the tumor, including immune cell expression. Importantly, the inclusion of immune cells in the CPS calculation did not substantially modify the expression pattern observed with TPS alone. This supports the notion that TPS may serve as a reliable and reproducible surrogate marker of immune checkpoint activation in NPC, particularly in routine diagnostic settings. Despite the high prevalence of PD-L1 expression, no significant association was observed between PD-L1 levels (assessed by either TPS or CPS) and clinicopathological parameters in our cohort. These results are in agreement with several published studies, including those by Dias et al. and Şahinli et al., which also failed to demonstrate correlations between PD-L1 expression and variables such as age, sex, or TNM stage. Collectively, these findings suggest that PD-L1 expression primarily reflects local adaptive immune responses rather than intrinsic tumor aggressiveness or disease extent [ 22 – 24 ]. However, some studies have reported contrasting results. Zhang et al. identified a significant association between PD-L1 expression and tumor size (p = 0.002) as well as lymph node involvement (p = 0.015) [ 18 ], while Zhao et al. and Xu et al. similarly reported association with tumor size in larger cohorts [ 16 , 25 ]. Such discrepancies may be explained by methodological heterogeneity, differences in patient ethnicity, Epstein–Barr virus–related factors, or variations in tumor biology across populations. These results confirm that, although PD-L1 plays a central role in modulating the immune response, it does not serve as an independent prognostic marker. Its assessment is therefore most valuable when integrated into a combined approach that considers immune infiltrates (CD8⁺, FOXP3⁺, CD163⁺) and the expression of viral markers such as LMP1 [ 15 , 16 ]. In this context, our analysis revealed a high infiltration of FOXP3⁺ regulatory T cells in 62.9% of cases, supporting the presence of a strongly immunosuppressive tumor microenvironment in nasopharyngeal carcinoma. These findings are consistent with those reported by Zhou et al. and Feng et al. [ 25 , 26 ]. Importantly, we observed a significant positive intratumoral correlation between FOXP3⁺ cell density and PD-L1 expression, assessed by both TPS and CPS. This association supports the concept of PD-L1⁺/FOXP3⁺ co-accumulation, a well-described mechanism contributing to immune escape [ 27 ]. Mechanistically, regulatory T cells are known to promote and sustain PD-L1 expression through immunosuppressive cytokines such as IL-10 and TGF-β, thereby inhibiting effector T cell function and maintaining active immune tolerance. Similar patterns have been reported across multiple epithelial malignancies, where FOXP3⁺/PD-L1 co-expression is associated with an immunosuppressive microenvironment and poorer clinical outcomes [ 19 , 28 , 29 ]. Analysis of the cytotoxic compartment further highlighted the adaptive nature of immune regulation in NPC. CD8⁺ T cell infiltration was high in 27.3% of cases and low in 72.7%, indicating a quantitatively limited but functionally active cytotoxic response. A significant positive correlation between CD8⁺ T cell density and PD-L1 expression was observed, in line with the findings of Chan et al. (p < 0.05) [ 19 ]. This association is consistent with an adaptive immune resistance mechanism, whereby IFN-γ released by activated CD8⁺ T cells induces PD-L1 expression on tumor and immune cells. Engagement of the PD-1/PD-L1 axis subsequently dampens CD8⁺ cytotoxic activity, enabling tumor immune evasion. Consequently, high CD8⁺ infiltration may paradoxically coexist with elevated PD-L1 expression, reflecting a dynamic equilibrium between immune activation and immune suppression. In contrast, Yuan et al. reported an inverse association between CD8⁺ infiltration and PD-L1 expression in hypoxic tumor regions, suggesting that exosomal PD-L1 may contribute to CD8⁺ T cell exhaustion. In our cohort, the positive CD8⁺/PD-L1 correlation likely reflects an inflamed yet restrained immune microenvironment, which may be indicative of potential responsiveness to PD-1/PD-L1–targeted immunotherapy [ 14 ]. Regarding the myeloid compartment, CD163⁺ macrophage infiltration was relatively low, suggesting a moderate degree of M2 polarization in our series. No significant correlation was observed between CD163⁺ macrophage density and PD-L1 expression. These findings contrast with those reported by Deng et al., who demonstrated a strong positive association between CD163⁺ macrophages and PD-L1 expression in NPC (p < 0.001), as well as with observations by Guo et al. in other epithelial carcinomas implicating IL-10/TGF-β–PI3K/AKT signaling pathways in this interaction [141]. These discrepancies may reflect the functional heterogeneity of M2 macrophages, as conventional markers such as CD163 are insufficient to distinguish specific subpopulations, particularly C1q⁺ macrophages, which have been described as highly immunosuppressive and associated with increased PD-L1 expression [142]. Such discrepancies may be explained by the marked functional heterogeneity of M2 macrophages. Indeed, conventional markers such as CD163 do not adequately discriminate between distinct macrophage subpopulations, particularly C1q⁺ macrophages, which have been identified as highly immunosuppressive and strongly associated with PD-L1 expression [142]. Therefore, the absence of a significant CD163/PD-L1 correlation in EBV⁺ nasopharyngeal carcinoma does not exclude functional crosstalk. Rather, it likely reflects the plasticity of tumor-associated macrophages, whose PD-L1 expression is dynamically regulated by cytokine cues and microenvironmental conditions. This complexity underscores the need for more refined phenotypic and functional characterization of myeloid cells to better elucidate their role in immune regulation and tumor immune escape in NPC. Epstein–Barr virus (EBV) infection represents the principal etiological factor in nasopharyngeal carcinoma (NPC), and latent membrane protein 1 (LMP1) constitutes the main EBV-encoded oncogenic protein expressed in lymphoepithelial tissues. LMP1 functions as a constitutively active receptor-like molecule that drives multiple signaling cascades, including NF-κB, JAK/STAT, and MAPK/ERK pathways, thereby regulating genes involved in cell survival, proliferation, and immune evasion. Among these downstream targets, PD-L1 has emerged as a key mediator of tumor immune escape [ 26 ]. In this context, we investigated the relationship between LMP1 expression and PD-L1 status in our biopsy series. LMP1 expression was detected in approximately half of the cases, and a statistically significant positive correlation between LMP1 and PD-L1 expression was identified. These findings are consistent with those reported by Kase et al. and Chen et al., who similarly demonstrated a positive association between LMP1 and PD-L1 expression (p = 0.008 and p = 0.04, respectively), mediated through NF-κB– and STAT3-dependent transcriptional activation [ 12 , 13 , 30 ]. This concordance supports the hypothesis that LMP1 actively contributes to PD-L1 upregulation through multiple mechanisms, including direct transcriptional control and the release of PD-L1–enriched exosomes. Nevertheless, PD-L1 expression remains dynamically modulated by additional microenvironmental factors, such as cytokine signaling and immune cell interactions, underscoring the multifactorial regulation of immune checkpoints in EBV-associated NPC. Taken together, these findings emphasize that PD-L1 expression in nasopharyngeal carcinoma does not reflect a single immunological parameter but rather arises from complex and dynamic interactions among tumor cells, cytotoxic CD8⁺ T lymphocytes, FOXP3⁺ regulatory T cells, tumor-associated macrophages, and EBV-driven oncogenic signaling. This complexity reinforces the concept that PD-L1 should not be interpreted as an isolated biomarker. Instead, its assessment should be integrated within a comprehensive, multiparametric immune framework that incorporates both lymphoid and myeloid infiltrates, as well as viral determinants such as EBV-related markers. Such an integrated approach may provide a more accurate basis for prognostic stratification and for identifying patients most likely to benefit from immune checkpoint blockade. Despite the strengths of this study, several limitations must be acknowledged. First, the relatively small sample size may have limited the statistical power of the analyses and reduced the generalizability of the results. Larger, multicenter studies are required to validate these findings and to better account for the heterogeneity of the immune microenvironment in NPC. Second, the retrospective design and limited follow-up duration preclude definitive conclusions regarding the prognostic and predictive significance of PD-L1 expression and immune infiltrates, particularly in relation to treatment response and long-term survival. Prospective studies with extended follow-up are therefore warranted. Additional technical limitations should also be considered. The use of formalin-fixed, paraffin-embedded biopsy specimens may have affected antigen preservation and immunostaining quality, potentially influencing immunohistochemical evaluation. Moreover, the reliance on small biopsy samples may not fully capture the spatial heterogeneity of the tumor immune microenvironment, a well-recognized feature of NPC. Although standardized scoring systems and expert pathological review were applied, the semi-quantitative nature of immunohistochemistry introduces an inherent degree of subjectivity, and inter-observer variability cannot be entirely excluded for markers such as PD-L1, FOXP3⁺, CD8⁺, and CD163⁺. Finally, the exclusive use of immunohistochemistry limits functional and molecular characterization of immune cell subpopulations. Advanced approaches, including multiplex immunofluorescence, spatial transcriptomics, and single-cell sequencing, could provide deeper insights into immune cell interactions, macrophage heterogeneity, and EBV-driven immunoregulatory mechanisms in nasopharyngeal carcinoma. Conclusion PD-L1 is frequently expressed in nasopharyngeal carcinoma and is closely associated with EBV-LMP1 and tumor-infiltrating immune cells. These findings highlight the role of EBV in driving PD-L1 expression, contributing to the establishment of an immunosuppressive tumor microenvironment and facilitating tumor immune escape. By demonstrating significant correlations with both cytotoxic CD8⁺ T cells and FOXP3⁺ regulatory T cells, our results provide insight into how viral oncoproteins modulate immune cell composition and function within the tumor. As the first study in a Tunisian cohort, this work offers novel insights into the regional immune landscape of UCNT and underscores the importance of considering population-specific biological characteristics when evaluating biomarkers. Collectively, these observations reinforce PD-L1 as a potential biomarker for patient stratification and a therapeutic target. Future studies integrating clinical outcomes and prognostic parameters will be critical to clarify the functional and clinical relevance of PD-L1 expression in nasopharyngeal carcinoma and to guide its potential application in immunologically informed treatment strategies. Declarations Conflicts of interest : The authors declare that they have no conflicts of interest related to this work. Funding statement : This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors Data availability statement : The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Ethics approval : This study was approved by the Ethics Committee of the Salah Azaiz Institute, Tunis (approval no. [ISA/2025/19] ). Consent to participate : Informed consent was obtained from all participants included in the study. 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Microorganisms 15 mars 9(3):603 Yuan X, Liu X, Jiang D, Zheng Z, Ma X, Wu S et al (2025) Exosomal PD-L1 derived from hypoxia nasopharyngeal carcinoma cell exacerbates CD8 + T cell suppression by promoting PD-L1 upregulation in macrophages. Cancer Immunol Immunother. 24 mai. ;74(7):220 Liu X, Shen H, Zhang L, Huang W, Zhang S, Zhang B (2024) Immunotherapy for recurrent or metastatic nasopharyngeal carcinoma. Npj Precis Oncol. 16 mai. ;8(1):101 Xu R, Wong CHL, Chan KSK, Chiang CL (2024) PD-L1 expression as a potential predictor of immune checkpoint inhibitor efficacy and survival in patients with recurrent or metastatic nasopharyngeal cancer: a systematic review and meta-analysis of prospective trials. Front Oncol. 3 juin. ;14:1386381 Kilaru S, Panda SS, Moharana L, Mohapatra D, Mohapatra SSG, Panda A et al (2024) PD-L1 expression in head and neck squamous cell carcinoma and its clinical significance: A prospective observational study from a tertiary care centre. 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Int J Mol Sci 3 23(19):11720 Sahinli H, Akyürek N, Yılmaz M, Kandemir O, Duran A, Kulaçoğlu S et al (2021) PD-L1 expression in immune cells is a favorable prognostic factor for nasopharyngeal carcinoma. Indian J Cancer 58(4):561 Liu X, Shan C, Song Y, Du J (oct 2019) Prognostic Value of Programmed Cell Death Ligand-1 Expression in Nasopharyngeal Carcinoma: A Meta-Analysis of 1,315 Patients. Front Oncol 25:9:1111 Zhao J, Yang H, Hu H, Liu C, Wei M, Zhao Y et al (2022) Prognostic value of PD-L1 and Siglec-15 expression in patients with nasopharyngeal carcinoma. Sci Rep 21 juin 12(1):10401 Feng E, Yang Y, Yang J, Hu R, Tian L, Yang X et al (2025) Tumor-infiltrating CD4 + CD25 + FOXP3 + Treg is associated with plasma EBV DNA and disease progression in nasopharyngeal carcinoma. Infect Agent Cancer. 9 mai. ;20(1):29 Gong L, Luo J, Zhang Y, Yang Y, Li S, Fang X et al (2023) Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction. Nat Commun 6 avr 14(1):1912 Wang W, Ding M, Wang Q, Song Y, Huo K, Chen X et al (2024) Advances in Foxp3 + regulatory T cells (Foxp3 + Treg) and key factors in digestive malignancies. Front Immunol 11 juin 15:1404974 Nagib R, Refat S, Eladl AE, Emarah Z, Elnaghi K (2019) Potential prognostic value of PD-L1 and FOXP3 as predictors of relapse in breast cancer. J Solid Tumors 29 août 9(2):38 Chan ATC, Lee VHF, Hong RL, Ahn MJ, Chong WQ, Kim SB et al (2023) Pembrolizumab monotherapy versus chemotherapy in platinum-pretreated, recurrent or metastatic nasopharyngeal cancer (KEYNOTE-122): an open-label, randomized, phase III trial. Ann Oncol mars 34(3):251–261 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-8584562","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576577527,"identity":"93242e12-9554-42b4-a331-57446369e4a1","order_by":0,"name":"Amani 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14:54:02","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111989,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8584562/v1/4207ff468199d5a8a1e7ea85.html"},{"id":100695269,"identity":"093fc8ab-bd20-4398-96e3-049de25e6be9","added_by":"auto","created_at":"2026-01-20 14:52:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1842682,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic image of a histological section from an undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) showing positive PD-L1 expression (Magnification : (a) ×400 ; (b) ×200)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8584562/v1/c256a8a034bd7c0ae85990aa.png"},{"id":100695219,"identity":"51ceae3a-dc3a-4744-a0a6-20cabc31f22a","added_by":"auto","created_at":"2026-01-20 14:52:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43675,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot showing the Spearman correlation between TPS and CPS for PD-L1 expression\u003c/p\u003e\n\u003cp\u003ePD-L1, programmed cell death ligand-1; TPS, tumor proportion score; CPS, combined positive score\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8584562/v1/a4741431518bf915437cb5df.png"},{"id":103529413,"identity":"0209c6f8-c45c-4532-8cc6-c962f6cdac1d","added_by":"auto","created_at":"2026-02-26 16:40:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3084176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8584562/v1/6d5a2258-81d0-4170-94d0-c96da912d3ff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PD-L1 Expression in Undifferentiated Nasopharyngeal Carcinoma Is Associated with EBV-LMP1 and Tumor Immune Infiltration : Data from a Tunisian Cohort","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNasopharyngeal carcinoma (NPC) is a rare malignant epithelial tumor worldwide, but it exhibits a highly heterogeneous geographic distribution. Its incidence is particularly high in Southeast Asia and North Africa, where the predominant histological form is undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This form is characterized by strong lymphocytic infiltration and a close association with latent Epstein\u0026ndash;Barr virus (EBV) infection, making NPC a tumor with a significant immunobiological dimension.\u003c/p\u003e \u003cp\u003eDue to the deep location of the nasopharynx and the atypical nature of early symptoms, the diagnosis of NPC is often delayed. Consequently, more than 70% of patients are diagnosed at a locally advanced stage, and approximately 6 to 15% already present with metastases at the time of diagnosis [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite advances brought by conformal radiotherapy and chemoradiotherapy, which constitute the standard treatment for locally advanced forms, the prognosis remains limited. About 20% of patients do not achieve a durable therapeutic response, due to interindividual variations in immune function and treatment tolerance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Tumor recurrence occurs in 5 to 15% of patients, while distant metastases develop in 15 to 30% of cases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This unfavorable course highlights the central role of immune escape mechanisms in tumor progression and justifies the growing interest in immunotherapy.\u003c/p\u003e \u003cp\u003eAmong the well-characterized immune escape mechanisms, the PD-1/PD-L1 axis has recently emerged as a central immunoregulatory pathway, whose functional implication makes it a major area of interest for the development of innovative therapeutic strategies. The expression of PD-L1 by tumor cells and infiltrating immune populations inhibits the cytotoxic activity of T lymphocytes, thereby contributing to the establishment of an immunosuppressive tumor microenvironment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Immune checkpoint inhibitors targeting this pathway have demonstrated notable clinical efficacy in recurrent or metastatic disease, reinforcing the therapeutic relevance of this approach [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, treatment responses remain heterogeneous, underscoring the need for reliable predictive biomarkers. This variability reflects the influence of multiple biological factors, including the structural organization of the tumor microenvironment and the presence of chronic EBV infection.\u003c/p\u003e \u003cp\u003eEBV plays a central role in immune modulation, particularly through the viral oncoprotein LMP1. LMP1 activates oncogenic and inflammatory signaling pathways capable of upregulating PD-L1 expression, promoting the recruitment of regulatory T cells and M2-polarized macrophages, and ultimately reinforcing local immunosuppression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These observations highlight the presence of a complex immunological network that facilitates tumor immune escape and contributes to therapeutic resistance.\u003c/p\u003e \u003cp\u003eTo date, most available data regarding PD-L1 expression and its interaction with the tumor microenvironment originate from Asian cohorts, while North African data remain limited despite the high incidence reported in this region. A more comprehensive characterization of the immune landscape of NPC in these populations is therefore essential to improve patient stratification and to optimize immunotherapy-based therapeutic strategies.\u003c/p\u003e \u003cp\u003e In this context, the present study evaluates PD-L1 expression in a Tunisian cohort of UCNT and investigates its correlations with clinicopathological features, major tumor-infiltrating immune cell populations (CD8⁺ T cells, FOXP3⁺ regulatory T cells, and M2 macrophages), and EBV-LMP1 expression, aiming to better characterize the immunological determinants of NPC in our regional context.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis retrospective observational cohort study was conducted at the Salah Azaiz Institute in Tunis. A total of 75 patients with histologically confirmed undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) were included between 2017 and 2024, identified through the institution\u0026rsquo;s database. All tumor specimens were independently reviewed by an experienced pathologist to confirm the diagnosis. Demographic and clinical data were extracted from medical records, and clinical staging was assigned according to the eighth edition of the American Joint Committee on Cancer (AJCC) TNM classification.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInclusion Criteria :\u003c/h2\u003e \u003cp\u003ePatients were included if they had a nasopharyngeal tumor of the undifferentiated non-keratinizing type (UCNT), a histologically confirmed diagnosis, a biopsy performed prior to the initiation of treatment, and available tumor samples for analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExclusion Criteria :\u003c/h3\u003e\n\u003cp\u003ePatients with incomplete or unavailable medical records in the Institute\u0026rsquo;s archives, as well as damaged or poorly preserved paraffin blocks and specimens of insufficient size for reliable immunohistochemical analysis, were excluded from the study.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical analysis :\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical (IHC) analysis was performed on 75 formalin-fixed, paraffin-embedded (FFPE) tumor samples to assess the expression of PD-L1, LMP1, FoxP3, CD163, and CD8 using the Novolink\u0026trade; Polymer Detection System (Leica Biosystems, USA). Paraffin-embedded tissues were sectioned at 4 \u0026micro;m, deparaffinized in toluene, and rehydrated through decreasing ethanol concentrations (100%, 95%, and 80%; 5 min each). Antigen retrieval was performed in Tris-EDTA buffer (pH 6 for CD163, PD-L1, and FoxP3 ; pH 9 for CD8 and LMP1) followed by heating at 98\u0026deg;C for 30 min in a water bath. After rinsing in phosphate-buffered saline (PBS), slides were incubated with primary antibodies under the following conditions : CD163 and CD8 for 30 min at room temperature, and FoxP3, PD-L1, and LMP1 for 60 min at room temperature. Following Tris-buffered saline (TBS) washes, slides were sequentially incubated with Post Primary Block and NovoLink Polymer for 30 min each. Antigen\u0026ndash;antibody complexes were visualized with 3,3\u0026prime;-diaminobenzidine (DAB) for 10 min, counterstained with hematoxylin for 5 min, dehydrated through graded ethanol concentrations (80%, 95%, and 100%; 5 min each), cleared in toluene, and mounted with Eukitt.\u003c/p\u003e\n\u003ch3\u003eEvaluation of immunostaining :\u003c/h3\u003e\n\u003cp\u003eSlide evaluation and analysis were performed independently by two pathologists. Each section was first examined at low magnification (\u0026times;10) to assess the overall tissue architecture, followed by high magnification (\u0026times;400) for counting immunolabeled cells. In case of different scoring results by two pathologists, a consensus on final scoring was reached after joint review.\u003c/p\u003e \u003cp\u003eSlides showing tissue detachment or significant background staining were excluded from the analysis. Cell counting was performed in the five fields with the highest density of positive cells, and the labeling rate was calculated as the mean percentage of positive cells relative to the total number of cells observed.\u003c/p\u003e\n\u003ch3\u003ePD-L1 evaluation :\u003c/h3\u003e\n\u003cp\u003ePD-L1 expression was assessed using two scores: the Tumor Proportion Score (TPS) and the Combined Positive Score (CPS). The TPS corresponds to the percentage of viable tumor cells showing partial or complete membranous staining relative to the total number of viable tumor cells in the sample. It is scored on a scale from 0% to 100% and is considered positive if detected in at least 1% of viable tumor cells, with low expression defined as a TPS of 1\u0026ndash;49% and high expression as a TPS\u0026thinsp;\u0026ge;\u0026thinsp;50%.\u003c/p\u003e \u003cp\u003eThe CPS was calculated by dividing the number of PD-L1\u0026ndash;positive cells (the sum of tumor cells, lymphocytes, and macrophages) by the total number of viable tumor cells on the slide and then multiplying the result by 100. It was considered negative if less than 1% and positive if\u0026thinsp;\u0026ge;\u0026thinsp;1%, with low expression for a CPS of 1\u0026ndash;19% and high expression for a CPS\u0026thinsp;\u0026ge;\u0026thinsp;20%.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmun cell evaluation :\u003c/h2\u003e \u003cp\u003eThe levels of CD8, CD163, and FoxP3 expression were assessed and classified into two categories: low (\u0026lt;\u0026thinsp;50% of positive cells) and high (\u0026ge;\u0026thinsp;50% of positive cells).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLMP1 evaluation :\u003c/h3\u003e\n\u003cp\u003eLMP1 expression was assessed and categorized as negative (0% positive cells) or positive (\u0026ge;\u0026thinsp;1% positive cells).\u003c/p\u003e\n\u003ch3\u003eStatistical analysis :\u003c/h3\u003e\n\u003cp\u003eSPSS version 26.0 software was used for statistical analysis. Associations between PD-L1 expression profiles (high vs. low) and clinicopathological parameters, as well as immune markers (CD163, CD8, FoxP3) and LMP1, were evaluated using Pearson\u0026rsquo;s chi-squared (χ\u0026sup2;) test. Correlations between the expression levels of cellular markers (CD163, CD8, FoxP3, and PD-L1) were analyzed using Spearman\u0026rsquo;s correlation coefficient (ρ). A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant, with a 95% confidence interval.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eClinicopathological description of the study population :\u003c/h2\u003e\n \u003cp\u003eA cohort of 75 patients diagnosed with undifferentiated non-keratinizing nasopharyngeal carcinoma (UCNT) and treated at the Salah Azaiez Institute was analyzed. The study population consisted of 58 men and 17 women, with a mean age of 57 years (median\u0026thinsp;=\u0026thinsp;54; SD\u0026thinsp;=\u0026thinsp;11.46; 95% CI: 39\u0026ndash;86), corresponding to a male-to-female ratio of 3.41. According to the TNM classification, 48% of patients presented with early T stage disease (T1\u0026ndash;T2), while 52% had advanced T stages (T3\u0026ndash;T4). Lymph node involvement was absent in 5.33% of cases (N0), moderate in 54.67% (N1\u0026ndash;N2), and advanced in 40% (N3). Distant metastases were identified in 17.33% of patients. In terms of overall disease stage, early stages (I\u0026ndash;II) accounted for 20% of the cohort, whereas advanced stages (III\u0026ndash;IV) predominated with 80%. Among the 34 patients assessed three months after completion of treatment, therapeutic response was characterized by a complete response in 17 cases (50%) and a partial response in 17 cases (50%) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" style=\"width: 39%; margin-right: calc(61%);\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical-demographic characteristics\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;75\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;54\u003c/p\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e38 (50.67%)\u003c/p\u003e\n \u003cp\u003e37 (49.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17 (22.67%)\u003c/p\u003e\n \u003cp\u003e58 (77.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eT stage\u003c/p\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 (14.67%)\u003c/p\u003e\n \u003cp\u003e25 (33.33%)\u003c/p\u003e\n \u003cp\u003e18 (24%)\u003c/p\u003e\n \u003cp\u003e21 (28%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eN stage\u003c/p\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003cp\u003eN1\u003c/p\u003e\n \u003cp\u003eN2\u003c/p\u003e\n \u003cp\u003eN3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (5.33%)\u003c/p\u003e\n \u003cp\u003e22 (29.33%)\u003c/p\u003e\n \u003cp\u003e19 (25.34%)\u003c/p\u003e\n \u003cp\u003e30 (40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eM stage\u003c/p\u003e\n \u003cp\u003eM0\u003c/p\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e62 (82.67%)\u003c/p\u003e\n \u003cp\u003e13 (17.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (1.33%)\u003c/p\u003e\n \u003cp\u003e14 (18.67%)\u003c/p\u003e\n \u003cp\u003e26 (34.67%)\u003c/p\u003e\n \u003cp\u003e34 (45.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52.6801%;\" align=\"left\"\u003e\n \u003cp\u003eTreatment response (n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003cp\u003eCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.0686%;\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17 (50%)\u003c/p\u003e\n \u003cp\u003e17 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 242px;\" colspan=\"2\"\u003ePR: partial response, CR: complete response\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eExpression of PD-L1 protein in NPC patients :\u003c/h2\u003e\n \u003cp\u003ePD-L1 expression was analyzed in 75 nasopharyngeal carcinoma (NPC) specimens by immunohistochemistry. The protein was predominantly localized to the cell membrane (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Immunohistochemical analysis showed that, based on the TPS, PD-L1 expression was positive (\u0026gt;\u0026thinsp;1%) in 53 cases (70.7%) and negative in 22 cases (29.3%). According to the CPS, high expression (\u0026gt;\u0026thinsp;20) was observed in 45 cases (60%), low expression (1\u0026ndash;19) in 10 cases (13.3%), and negative staining in 20 cases (26.7%) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePD-L1 Expression Profile According to CPS and TPS Scores\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePD-L1 Expression Profile (n\u0026thinsp;=\u0026thinsp;75)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePD-L1 TPS Assessment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70,7%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive (1\u0026ndash;49%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive (\u0026gt;\u0026thinsp;50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD-L1 CPS Assessment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive (1\u0026ndash;19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive (\u0026gt;\u0026thinsp;20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003ePD-L1 : programmed cell death ligand-1 ; TPS : Tumor proportion score ; CPS : Combined positive score\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003ePD-L1 expression and the clinicopathological features of NPC patients :\u003c/h2\u003e\n \u003cp\u003eThe association between PD-L1 expression and clinicopathological parameters was assessed to clarify the role of PD-L1 in NPC. PD-L1 expression was evaluated using TPS and CPS scores and categorized as negative (\u0026lt;\u0026thinsp;1%), positive with low expression (\u0026lt;\u0026thinsp;50% for TPS, \u0026lt;\u0026thinsp;20 for CPS), or high expression (\u0026ge;\u0026thinsp;50% for TPS, \u0026ge;\u0026thinsp;20 for CPS), as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. No significant associations were observed between PD-L1 expression and sex, age, tumor size (T), lymph node status (N), distant metastasis (M), overall TNM stage, or treatment response.\u003c/p\u003e\n \u003cp\u003eSpearman\u0026rsquo;s correlation was performed between the positive TPS and CPS variables. There was a strong correlation demonstrated through the coefficient value of 0,941 (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003ePD-L1, programmed cell death ligand-1; TPS, tumor proportion score; CPS, combined positive score\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistical association between PD-L1 expression and patient clinicopathological parameters\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"4\" align=\"left\"\u003e\n \u003cp\u003eCPS Score\u003c/p\u003e\n \u003cp\u003ePD-L1 Negative PD-L1 Positive P-value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"5\" align=\"left\"\u003e\n \u003cp\u003eTPS Score\u003c/p\u003e\n \u003cp\u003ePD-L1 Negative PD-L1 Positive P-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow (1\u0026ndash;49%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh (\u0026gt;\u0026thinsp;50%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow (1\u0026ndash;49%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh (\u0026gt;\u0026thinsp;50%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e16 (88.89%)\u003c/p\u003e\n \u003cp\u003e2 (11.11%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (75%)\u003c/p\u003e\n \u003cp\u003e3 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e33 (73.33%)\u003c/p\u003e\n \u003cp\u003e12 (26.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17 (77.27%)\u003c/p\u003e\n \u003cp\u003e5 (22.73%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15 (71.43%)\u003c/p\u003e\n \u003cp\u003e6 (28.57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26 (81.25%)\u003c/p\u003e\n \u003cp\u003e6 (18.75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.705\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;54\u003c/p\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (50%)\u003c/p\u003e\n \u003cp\u003e9 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26 (57.78%)\u003c/p\u003e\n \u003cp\u003e19 (42.22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.803\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12 (54.55%)\u003c/p\u003e\n \u003cp\u003e10 (45.45%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (42.86%)\u003c/p\u003e\n \u003cp\u003e12 (57.14%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20 (62.5%)\u003c/p\u003e\n \u003cp\u003e12 (37.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.373\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT Stage\u003c/p\u003e\n \u003cp\u003eT1\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e\n \u003cp\u003eT3\u0026thinsp;+\u0026thinsp;T4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8 (44.44%)\u003c/p\u003e\n \u003cp\u003e10 (55.56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 (41.67%)\u003c/p\u003e\n \u003cp\u003e7 (58.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23 (51.11%)\u003c/p\u003e\n \u003cp\u003e22 (48.89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 (50%)\u003c/p\u003e\n \u003cp\u003e11 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8 (38.1%)\u003c/p\u003e\n \u003cp\u003e13 (61.90%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17 (53.13%)\u003c/p\u003e\n \u003cp\u003e15 (46.87%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.550\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN Stage\u003c/p\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003cp\u003eN1\u0026thinsp;+\u0026thinsp;N2\u003c/p\u003e\n \u003cp\u003eN3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003cp\u003e12 (66.67%)\u003c/p\u003e\n \u003cp\u003e6 (33.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (8.33%)\u003c/p\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003cp\u003e5 (41.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (6.67%)\u003c/p\u003e\n \u003cp\u003e23 (51.11%)\u003c/p\u003e\n \u003cp\u003e19 (42.22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2 (9.09%)\u003c/p\u003e\n \u003cp\u003e13 (59.09%)\u003c/p\u003e\n \u003cp\u003e7 (31.82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (4.76%)\u003c/p\u003e\n \u003cp\u003e13 (61.90%)\u003c/p\u003e\n \u003cp\u003e7 (33.34%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (3.13%)\u003c/p\u003e\n \u003cp\u003e15 (46.87%)\u003c/p\u003e\n \u003cp\u003e16 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.570\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM Stage\u003c/p\u003e\n \u003cp\u003eM0\u003c/p\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15 (83.33%)\u003c/p\u003e\n \u003cp\u003e3 (16.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10 (83.33%)\u003c/p\u003e\n \u003cp\u003e2 (16.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e37 (82.22%)\u003c/p\u003e\n \u003cp\u003e8 (17.78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19 (86.36%)\u003c/p\u003e\n \u003cp\u003e3 (13.64%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19 (90.48%)\u003c/p\u003e\n \u003cp\u003e2 (9.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e24 (75%)\u003c/p\u003e\n \u003cp\u003e8 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.299\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNM Stage\u003c/p\u003e\n \u003cp\u003eI\u0026thinsp;+\u0026thinsp;II\u003c/p\u003e\n \u003cp\u003eIII\u0026thinsp;+\u0026thinsp;IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 (27.78%)\u003c/p\u003e\n \u003cp\u003e13 (72.22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (25%)\u003c/p\u003e\n \u003cp\u003e9 (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7 (15.56%)\u003c/p\u003e\n \u003cp\u003e38 (84.44%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7 (31.82%)\u003c/p\u003e\n \u003cp\u003e15 (68.18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (19.05%)\u003c/p\u003e\n \u003cp\u003e17 (80.95%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (12.5%)\u003c/p\u003e\n \u003cp\u003e28 (87.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.217\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment response (n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003cp\u003eCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (20%)\u003c/p\u003e\n \u003cp\u003e4 (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (57.14%)\u003c/p\u003e\n \u003cp\u003e3 (42.86%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 (50%)\u003c/p\u003e\n \u003cp\u003e11 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (14.29%)\u003c/p\u003e\n \u003cp\u003e6 (85.71%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003cp\u003e6 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9 (60%)\u003c/p\u003e\n \u003cp\u003e6 (40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003ePD-L1 : programmed cell death ligand-1 ; TPS : Tumor proportion score ; CPS : Combined positive score ; CR : complete response ; PR : partial response ; Pearson\u0026rsquo;s \u0026chi;2 test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCorrelation between PD-L1 Expression and Immune Cell Infiltration\u003c/strong\u003e :\u003c/p\u003e\n \u003cp\u003eSpearman\u0026rsquo;s correlation analysis was performed to evaluate the association between PD-L1 expression (TPS and CPS) and the infiltration of regulatory T cells (FoxP3), cytotoxic T cells (CD8), and M2 macrophages (CD163), with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant. PD-L1 expression was positively correlated with FoxP3⁺ T cell infiltration (CPS : r\u0026thinsp;=\u0026thinsp;0.253, p\u0026thinsp;=\u0026thinsp;0.047 ; TPS : r\u0026thinsp;=\u0026thinsp;0.254, p\u0026thinsp;=\u0026thinsp;0.046) and CD8⁺ T cell infiltration (CPS : r\u0026thinsp;=\u0026thinsp;0.371, p\u0026thinsp;=\u0026thinsp;0.002 ; TPS : r\u0026thinsp;=\u0026thinsp;0.355, p\u0026thinsp;=\u0026thinsp;0.003). No significant correlation was observed between PD-L1 expression and M2 macrophage (CD163) infiltration (CPS : r\u0026thinsp;=\u0026thinsp;0.232, p\u0026thinsp;=\u0026thinsp;0.066 ; TPS : r\u0026thinsp;=\u0026thinsp;0.190, p\u0026thinsp;=\u0026thinsp;0.132) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistical correlation between PD-L1 expression and tumor-infiltrating immune cells\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCD8\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFoxP3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCD163\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eTPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.355\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.254\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,190\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.371\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.254\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eTPS : Tumor proportion score ; CPS : Combined positive score ; Spearman\u0026rsquo;s rho test\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*. Correlation is significant at the 0.05 level (two-tailed).\u003c/p\u003e\n \u003cp\u003e**. Correlation is significant at the 0.01 level (two-tailed).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCorrelation between PD-L1 and LMP1 expression :\u003c/h2\u003e\n \u003cp\u003eSpearman\u0026rsquo;s correlation analysis revealed a significant positive association between PD-L1 and LMP1 expression. Specifically, PD-L1 expression was positively correlated with LMP1 levels according to both TPS (r\u0026thinsp;=\u0026thinsp;0.341, p\u0026thinsp;=\u0026thinsp;0.003) and CPS (r\u0026thinsp;=\u0026thinsp;0.345, p\u0026thinsp;=\u0026thinsp;0.002) scores, indicating that higher PD-L1 expression is associated with increased LMP1 expression in nasopharyngeal carcinoma specimens (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistical correlation between PD-L1 and LMP1\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLMP1\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.341**\u003c/p\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.345**\u003c/p\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTPS : Tumor proportion score ; CPS : Combined positive score ; Spearman\u0026rsquo;s rho test **. Correlation is significant at the 0.01 level (two-tailed).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe regulation of the antitumor immune response relies on a delicate balance between activating and inhibitory signals, among which the PD-1/PD-L1 pathway plays a central role in modulating immune tolerance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In nasopharyngeal carcinoma (NPC), which is strongly associated with Epstein\u0026ndash;Barr virus (EBV) infection, activation of this pathway has been shown to promote tumor immune evasion mechanisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies have demonstrated that the viral protein LMP1 and certain pro-inflammatory cytokines, notably interferon-γ, induce PD-L1 expression and contribute to the establishment of an immunosuppressive microenvironment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This dysregulation has led to the development of immune checkpoint inhibitors, whose clinical efficacy has been confirmed in several recent trials [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the biological heterogeneity of tumors, intertumoral and interindividual variability, as well as ethnic and geographic differences, can influence PD-L1 expression profiles, thereby limiting its universal prognostic value. Few data are available regarding North African patients with NPC prior to any therapeutic intervention.\u003c/p\u003e \u003cp\u003eIn this context, our study sought to characterize PD-L1 expression and its clinical relevance in a cohort of primary NPC biopsies from Tunisia using immunohistochemistry. We first evaluated PD-L1 expression in both tumor and immune cells and assessed its associations with key clinicopathological features to gauge its potential prognostic value. To further elucidate local immune dynamics, we examined correlations between PD-L1 status and the composition of tumor-infiltrating immune cells. Additionally, by investigating the relationship between PD-L1 and the viral oncogene LMP1, we explored how latent EBV infection may influence immune checkpoint regulation within the tumor microenvironment.\u003c/p\u003e \u003cp\u003eIn our cohort, PD-L1 expression was observed in 70.7% of cases according to TPS (\u0026ge;\u0026thinsp;1% of tumor cells) and in 73.3% according to CPS (\u0026ge;\u0026thinsp;1), with 60% showing high expression (CPS\u0026thinsp;\u0026gt;\u0026thinsp;20). These findings highlight the combined contribution of tumor and immune cells in PD-L1 regulation and underscore the central role of the tumor microenvironment. These results are consistent with several previous studies reporting comparable PD-L1 expression rates in NPC [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, lower frequencies have also been described in the literature, which may be attributed to differences in immunohistochemical methods, antibody clones, scoring systems, or histological and population-related variations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn line with this observation, a strong correlation between TPS and CPS was observed. Notably, the higher the TPS value, the higher the CPS value. This finding suggests that PD-L1 expression by tumor cells largely reflects the overall PD-L1 status of the tumor, including immune cell expression. Importantly, the inclusion of immune cells in the CPS calculation did not substantially modify the expression pattern observed with TPS alone. This supports the notion that TPS may serve as a reliable and reproducible surrogate marker of immune checkpoint activation in NPC, particularly in routine diagnostic settings.\u003c/p\u003e \u003cp\u003eDespite the high prevalence of PD-L1 expression, no significant association was observed between PD-L1 levels (assessed by either TPS or CPS) and clinicopathological parameters in our cohort. These results are in agreement with several published studies, including those by Dias et al. and Şahinli et al., which also failed to demonstrate correlations between PD-L1 expression and variables such as age, sex, or TNM stage. Collectively, these findings suggest that PD-L1 expression primarily reflects local adaptive immune responses rather than intrinsic tumor aggressiveness or disease extent [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, some studies have reported contrasting results. Zhang et al. identified a significant association between PD-L1 expression and tumor size (p\u0026thinsp;=\u0026thinsp;0.002) as well as lymph node involvement (p\u0026thinsp;=\u0026thinsp;0.015) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while Zhao et al. and Xu et al. similarly reported association with tumor size in larger cohorts [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Such discrepancies may be explained by methodological heterogeneity, differences in patient ethnicity, Epstein\u0026ndash;Barr virus\u0026ndash;related factors, or variations in tumor biology across populations.\u003c/p\u003e \u003cp\u003eThese results confirm that, although PD-L1 plays a central role in modulating the immune response, it does not serve as an independent prognostic marker. Its assessment is therefore most valuable when integrated into a combined approach that considers immune infiltrates (CD8⁺, FOXP3⁺, CD163⁺) and the expression of viral markers such as LMP1 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, our analysis revealed a high infiltration of FOXP3⁺ regulatory T cells in 62.9% of cases, supporting the presence of a strongly immunosuppressive tumor microenvironment in nasopharyngeal carcinoma. These findings are consistent with those reported by Zhou et al. and Feng et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Importantly, we observed a significant positive intratumoral correlation between FOXP3⁺ cell density and PD-L1 expression, assessed by both TPS and CPS. This association supports the concept of PD-L1⁺/FOXP3⁺ co-accumulation, a well-described mechanism contributing to immune escape [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Mechanistically, regulatory T cells are known to promote and sustain PD-L1 expression through immunosuppressive cytokines such as IL-10 and TGF-β, thereby inhibiting effector T cell function and maintaining active immune tolerance. Similar patterns have been reported across multiple epithelial malignancies, where FOXP3⁺/PD-L1 co-expression is associated with an immunosuppressive microenvironment and poorer clinical outcomes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of the cytotoxic compartment further highlighted the adaptive nature of immune regulation in NPC. CD8⁺ T cell infiltration was high in 27.3% of cases and low in 72.7%, indicating a quantitatively limited but functionally active cytotoxic response. A significant positive correlation between CD8⁺ T cell density and PD-L1 expression was observed, in line with the findings of Chan et al. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This association is consistent with an adaptive immune resistance mechanism, whereby IFN-γ released by activated CD8⁺ T cells induces PD-L1 expression on tumor and immune cells. Engagement of the PD-1/PD-L1 axis subsequently dampens CD8⁺ cytotoxic activity, enabling tumor immune evasion. Consequently, high CD8⁺ infiltration may paradoxically coexist with elevated PD-L1 expression, reflecting a dynamic equilibrium between immune activation and immune suppression. In contrast, Yuan et al. reported an inverse association between CD8⁺ infiltration and PD-L1 expression in hypoxic tumor regions, suggesting that exosomal PD-L1 may contribute to CD8⁺ T cell exhaustion. In our cohort, the positive CD8⁺/PD-L1 correlation likely reflects an inflamed yet restrained immune microenvironment, which may be indicative of potential responsiveness to PD-1/PD-L1\u0026ndash;targeted immunotherapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the myeloid compartment, CD163⁺ macrophage infiltration was relatively low, suggesting a moderate degree of M2 polarization in our series. No significant correlation was observed between CD163⁺ macrophage density and PD-L1 expression. These findings contrast with those reported by Deng et al., who demonstrated a strong positive association between CD163⁺ macrophages and PD-L1 expression in NPC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as well as with observations by Guo et al. in other epithelial carcinomas implicating IL-10/TGF-β\u0026ndash;PI3K/AKT signaling pathways in this interaction [141]. These discrepancies may reflect the functional heterogeneity of M2 macrophages, as conventional markers such as CD163 are insufficient to distinguish specific subpopulations, particularly C1q⁺ macrophages, which have been described as highly immunosuppressive and associated with increased PD-L1 expression [142]. Such discrepancies may be explained by the marked functional heterogeneity of M2 macrophages. Indeed, conventional markers such as CD163 do not adequately discriminate between distinct macrophage subpopulations, particularly C1q⁺ macrophages, which have been identified as highly immunosuppressive and strongly associated with PD-L1 expression [142]. Therefore, the absence of a significant CD163/PD-L1 correlation in EBV⁺ nasopharyngeal carcinoma does not exclude functional crosstalk. Rather, it likely reflects the plasticity of tumor-associated macrophages, whose PD-L1 expression is dynamically regulated by cytokine cues and microenvironmental conditions. This complexity underscores the need for more refined phenotypic and functional characterization of myeloid cells to better elucidate their role in immune regulation and tumor immune escape in NPC.\u003c/p\u003e \u003cp\u003eEpstein\u0026ndash;Barr virus (EBV) infection represents the principal etiological factor in nasopharyngeal carcinoma (NPC), and latent membrane protein 1 (LMP1) constitutes the main EBV-encoded oncogenic protein expressed in lymphoepithelial tissues. LMP1 functions as a constitutively active receptor-like molecule that drives multiple signaling cascades, including NF-κB, JAK/STAT, and MAPK/ERK pathways, thereby regulating genes involved in cell survival, proliferation, and immune evasion. Among these downstream targets, PD-L1 has emerged as a key mediator of tumor immune escape [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this context, we investigated the relationship between LMP1 expression and PD-L1 status in our biopsy series.\u003c/p\u003e \u003cp\u003eLMP1 expression was detected in approximately half of the cases, and a statistically significant positive correlation between LMP1 and PD-L1 expression was identified. These findings are consistent with those reported by Kase et al. and Chen et al., who similarly demonstrated a positive association between LMP1 and PD-L1 expression (p\u0026thinsp;=\u0026thinsp;0.008 and p\u0026thinsp;=\u0026thinsp;0.04, respectively), mediated through NF-κB\u0026ndash; and STAT3-dependent transcriptional activation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This concordance supports the hypothesis that LMP1 actively contributes to PD-L1 upregulation through multiple mechanisms, including direct transcriptional control and the release of PD-L1\u0026ndash;enriched exosomes. Nevertheless, PD-L1 expression remains dynamically modulated by additional microenvironmental factors, such as cytokine signaling and immune cell interactions, underscoring the multifactorial regulation of immune checkpoints in EBV-associated NPC.\u003c/p\u003e \u003cp\u003eTaken together, these findings emphasize that PD-L1 expression in nasopharyngeal carcinoma does not reflect a single immunological parameter but rather arises from complex and dynamic interactions among tumor cells, cytotoxic CD8⁺ T lymphocytes, FOXP3⁺ regulatory T cells, tumor-associated macrophages, and EBV-driven oncogenic signaling. This complexity reinforces the concept that PD-L1 should not be interpreted as an isolated biomarker. Instead, its assessment should be integrated within a comprehensive, multiparametric immune framework that incorporates both lymphoid and myeloid infiltrates, as well as viral determinants such as EBV-related markers. Such an integrated approach may provide a more accurate basis for prognostic stratification and for identifying patients most likely to benefit from immune checkpoint blockade.\u003c/p\u003e \u003cp\u003eDespite the strengths of this study, several limitations must be acknowledged. First, the relatively small sample size may have limited the statistical power of the analyses and reduced the generalizability of the results. Larger, multicenter studies are required to validate these findings and to better account for the heterogeneity of the immune microenvironment in NPC. Second, the retrospective design and limited follow-up duration preclude definitive conclusions regarding the prognostic and predictive significance of PD-L1 expression and immune infiltrates, particularly in relation to treatment response and long-term survival. Prospective studies with extended follow-up are therefore warranted.\u003c/p\u003e \u003cp\u003eAdditional technical limitations should also be considered. The use of formalin-fixed, paraffin-embedded biopsy specimens may have affected antigen preservation and immunostaining quality, potentially influencing immunohistochemical evaluation. Moreover, the reliance on small biopsy samples may not fully capture the spatial heterogeneity of the tumor immune microenvironment, a well-recognized feature of NPC. Although standardized scoring systems and expert pathological review were applied, the semi-quantitative nature of immunohistochemistry introduces an inherent degree of subjectivity, and inter-observer variability cannot be entirely excluded for markers such as PD-L1, FOXP3⁺, CD8⁺, and CD163⁺. Finally, the exclusive use of immunohistochemistry limits functional and molecular characterization of immune cell subpopulations. Advanced approaches, including multiplex immunofluorescence, spatial transcriptomics, and single-cell sequencing, could provide deeper insights into immune cell interactions, macrophage heterogeneity, and EBV-driven immunoregulatory mechanisms in nasopharyngeal carcinoma.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePD-L1 is frequently expressed in nasopharyngeal carcinoma and is closely associated with EBV-LMP1 and tumor-infiltrating immune cells. These findings highlight the role of EBV in driving PD-L1 expression, contributing to the establishment of an immunosuppressive tumor microenvironment and facilitating tumor immune escape. By demonstrating significant correlations with both cytotoxic CD8⁺ T cells and FOXP3⁺ regulatory T cells, our results provide insight into how viral oncoproteins modulate immune cell composition and function within the tumor. As the first study in a Tunisian cohort, this work offers novel insights into the regional immune landscape of UCNT and underscores the importance of considering population-specific biological characteristics when evaluating biomarkers. Collectively, these observations reinforce PD-L1 as a potential biomarker for patient stratification and a therapeutic target. Future studies integrating clinical outcomes and prognostic parameters will be critical to clarify the functional and clinical relevance of PD-L1 expression in nasopharyngeal carcinoma and to guide its potential application in immunologically informed treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have\u0026nbsp;\u003cstrong\u003eno conflicts of interest\u003c/strong\u003e related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement :\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement :\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval :\u0026nbsp;\u003c/strong\u003eThis study was approved by the Ethics Committee of the Salah Azaiz Institute, Tunis (approval no. \u003cstrong\u003e[ISA/2025/19]\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate :\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Ann Oncol mars 34(3):251\u0026ndash;261\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":"Nasopharyngeal carcinoma, PD-L1, EBV, LMP1, Tumor-infiltrating immune cells","lastPublishedDoi":"10.21203/rs.3.rs-8584562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8584562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eProgrammed death-ligand 1 (PD-L1) is a key immune checkpoint involved in tumor immune evasion and a therapeutic target in immuno-oncology. Undifferentiated nasopharyngeal carcinoma (UCNT) is strongly linked to Epstein\u0026ndash;Barr virus (EBV) infection, but data on PD-L1 expression and its relationship with tumor immunity and viral markers in North African populations are limited.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess PD-L1 expression in UCNT and its associations with clinicopathological features, tumor-infiltrating immune cells, and the viral protein LMP1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe retrospectively analyzed 75 UCNT patients diagnosed at the Salah Aza\u0026iuml;ez Cancer Institute, Tunis. PD-L1 expression was evaluated by immunohistochemistry using the Tumor Proportion Score (TPS) and Combined Positive Score (CPS). CD8⁺ cytotoxic T cells, FOXP3⁺ regulatory T cells, CD163⁺ macrophages, and LMP1 expression were also assessed. Spearman\u0026rsquo;s correlation and χ\u0026sup2; tests were used, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePD-L1 was positive in 70.7% of cases (TPS) and 73.3% (CPS), with strong correlation between the two scores (rho\u0026thinsp;=\u0026thinsp;0.956 ; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PD-L1 expression was not associated with clinicopathological parameters but showed significant positive correlations with LMP1 (TPS : rho\u0026thinsp;=\u0026thinsp;0.341; p\u0026thinsp;=\u0026thinsp;0.003; CPS: rho\u0026thinsp;=\u0026thinsp;0.345; p\u0026thinsp;=\u0026thinsp;0.002) and with intratumoral CD8⁺ T cells (rho\u0026thinsp;=\u0026thinsp;0.371; p\u0026thinsp;=\u0026thinsp;0.002) and FOXP3⁺ regulatory T cells (rho\u0026thinsp;=\u0026thinsp;0.253; p\u0026thinsp;=\u0026thinsp;0.047). No correlation was observed with CD163⁺ macrophages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePD-L1 is frequently expressed in UCNT and closely associated with EBV-LMP1 and tumor-infiltrating immune cells. These findings highlight PD-L1 as a potential biomarker and provide insight into the immune-viral interactions shaping the tumor microenvironment in this population.\u003c/p\u003e","manuscriptTitle":"PD-L1 Expression in Undifferentiated Nasopharyngeal Carcinoma Is Associated with EBV-LMP1 and Tumor Immune Infiltration : Data from a Tunisian Cohort","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 12:22:25","doi":"10.21203/rs.3.rs-8584562/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":"444736ef-322a-4167-8808-3f0e3e18a78a","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-26T16:40:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 12:22:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8584562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8584562","identity":"rs-8584562","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}