OSCC represents the most common malignant epithelial tumour of the oral cavity and contribute to approximately 90% of malignancies of the oral cavity. Although diagnosis and treatment have advanced, OSCC continues to cause substantial morbidity and mortality, partly because of aggressive local invasion, nodal spread and biological heterogeneity (Tan et al., 2023). Histopathological differentiation remains an important component of tumour assessment, with OSCC conventionally grouped as Grade I (well differentiated), Grade II (moderately differentiated) or Grade III (poorly differentiated) (Muller & Tilakaratne, 2022). Increasing loss of differentiation reflects progressively greater cytological and architectural atypia and may accompany more aggressive tumour behaviour. Systemic inflammatory responses are also increasingly recognised as clinically relevant features of OSCC biology and outcome assessment (Zubair et al., 2022). Inflammation is closely integrated with cancer biology and can influence tumour growth, angiogenesis, immune evasion and metastatic progression (Hanahan, 2022). This has stimulated interest in inexpensive peripheral-blood indices that reflect systemic inflammatory activity. NLR and PLR are established markers derived from routine blood counts, whereas SII integrates neutrophil, platelet and lymphocyte counts and SIRI incorporates monocyte, neutrophil and lymphocyte counts. These indices have been evaluated in OSCC cohorts as practical indicators of systemic inflammatory status (Diao et al., 2018; Hung et al., 2021; Zhuang et al., 2022). In OSCC, NLR and PLR have been investigated extensively, although reported associations with clinicopathological characteristics and outcomes have not been uniform. More recent studies have expanded assessment to SII and SIRI. Cho et al. (2022), evaluating NLR, PLR, SII and SIRI simultaneously in surgically treated OSCC, demonstrated differing prognostic associations among the indices. Diao et al. (2018) and Hung et al. (2021) further showed that SII could provide clinically relevant information in independent OSCC cohorts, while Song et al. (2022) reported prognostic value for SIRI in early-stage OSCC. Meta-analytic evidence has also linked elevated SII with adverse clinicopathological features, including poorer tumour differentiation (Zhang & Dai, 2024), although broader evidence for SII and SIRI remains heterogeneous (Yang & Fei, 2024). Thus, although systemic inflammatory biomarkers have demonstrated prognostic potential in OSCC, their relationship with the biological gradient of histopathological differentiation remains incompletely characterised. In particular, evidence directly comparing the four indices NLR, PLR, SII and SIRI across well-, moderately- and poorly differentiated OSCC is limited. Recent 2024–2026 evidence has reported grade-related associations for SII and/or SIRI, but findings remain inconsistent across cohorts and indices, and few studies have directly compared NLR, PLR, SII and SIRI across all three conventional histopathological grades (Kaihereman et al., 2024; Wang et al., 2026; Kumar & Raju, 2026). Establishing whether these routinely obtainable biomarkers correlate with increasing histopathological grade could clarify the relationship between systemic inflammation and tumour differentiation. The aim was to determine the relationship between NLR, PLR, SII and SIRI and histopathological grade in OSCC by comparing biomarker levels across the three grades (I, II and III) and assessing the direction and strength of their associations with increasing grade. Study Objectives 1. To determine NLR, PLR, SII and SIRI levels among patients with oral squamous cell carcinoma across histopathological Grades I, II and III. 2. To compare values of NLR, PLR, SII and SIRI among well-differentiated, moderately differentiated and poorly differentiated OSCC. 3. To assess how NLR, PLR, SII and SIRI correlate with increasing histopathological grade in oral squamous cell carcinoma.

Study design and setting A prospective analytical cross-sectional investigation examined the relationship between systemic inflammatory biomarkers and histopathological grade among patients with oral squamous cell carcinoma (OSCC) at Niazi Welfare Foundation Teaching Hospital, Sargodha, Pakistan, from 15 January 2025 to 14 January 2026. Participant-derived clinicopathological and peripheral blood count data collected at the pretreatment assessment were analysed. Study population, sample size and sampling The study population consisted of patients with histopathologically confirmed OSCC categorised as Grade I (well differentiated), Grade II (moderately differentiated) or Grade III (poorly differentiated). The analytical sample included 150 cases: 65 in Grade I, 60 in Grade II and 25 in Grade III. Eligible participants had a valid Grade I-III classification and recorded neutrophil, lymphocyte, platelet and monocyte values permitting calculation of all four inflammatory indices. Eligible patients were recruited using consecutive sampling during the recruitment period until the required sample was reached. Inclusion criteria were histopathologically confirmed OSCC with Grade I–III classification and available pretreatment neutrophil, lymphocyte, platelet and monocyte counts sufficient to calculate all four inflammatory indices. Cases without a valid Grade I–III classification or without any of the required blood-cell counts were excluded from the analytical cohort. Data collection, variables and measurements Recorded variables were participant identifier, age, sex, tobacco-exposure status, histopathological grade and absolute neutrophil, lymphocyte, platelet and monocyte counts. Histopathological grade was coded ordinally as 1 for Grade I, 2 for Grade II and 3 for Grade III, consistent with conventional differentiation-based OSCC grading terminology (Muller & Tilakaratne, 2022). The primary predictors were NLR, PLR, SII and SIRI. NLR was calculated as neutrophils/lymphocytes; PLR as platelets/lymphocytes; SII as platelets × neutrophils/lymphocytes; and SIRI as neutrophils × monocytes/lymphocytes, consistent with published OSCC inflammatory-index studies (Diao et al., 2018; Cho et al., 2022; Song et al., 2022). Blood-cell counts were expressed as ×10⁹/L. Age, sex and tobacco exposure were descriptive covariates. Histopathological grading followed conventional differentiation-based OSCC criteria, with well-differentiated tumours designated Grade I, moderately differentiated tumours Grade II and poorly differentiated tumours Grade III; grading reflected the extent of squamous differentiation/keratinisation together with cytological and architectural atypia (Muller & Tilakaratne, 2022). Statistical analysis Analyses were performed in IBM SPSS Statistics version 29.0. Categorical data were described using counts and percentages, while age was summarised by the mean ± standard deviation (SD). NLR, PLR, SII and SIRI were summarised as median with interquartile range (IQR) and analysed non-parametrically. Biomarker distributions were described overall and by histopathological grade. Differences across Grades I-III were evaluated with the Kruskal-Wallis test. Pairwise Mann-Whitney U comparisons with Holm adjustment were calculated for all four biomarkers; pairwise comparisons following a non-significant omnibus test were treated as exploratory and did not alter the omnibus conclusion. Spearman rank correlation assessed the relationship between each biomarker and increasing histopathological grade. All testing was two-sided, and statistical significance was defined as p < 0.05. Primary histopathological-grade and inflammatory-biomarker data were complete for all 150 cases. Ethical considerations Ethical clearance was granted by the Institutional Ethics Committee of Niazi Welfare Foundation Teaching Hospital, Sargodha, Pakistan. The study followed the Declaration of Helsinki, with written informed consent obtained from all participants and confidentiality maintained throughout.

One hundred and fifty OSCC cases entered the analysis. Histopathological grade, sex and all four inflammatory-biomarker variables were complete across all 150 cases. Grade I (well differentiated) comprised 65 cases (43.3%), Moderately differentiated Grade II comprised 60 (40.0%) and poorly differentiated Grade III comprised 25 (16.7%). Age was recorded for all 150 cases; the overall mean age was 57.0 ± 12.7 years, with grade-specific means of 53.2 ± 10.3 years in Grade I, 58.8 ± 14.5 years in Grade II and 62.7 ± 11.0 years in Grade III. Ninety-six cases (64.0%) were male and 54 (36.0%) were female. Tobacco exposure was recorded as present in 98 cases (65.3%) and absent in 52 (34.7%). The demographic and histopathological characteristics of the full 150-case cohort appear in Table 1. Table 1. Demographic profile of the cohort (N = 150), stratified by histopathological grade Characteristic Overall, N=150 Grade I (n=65) Grade II (n=60) Grade III (n=25) Age (years), mean ± SD 57.0 ± 12.7 53.2 ± 10.3 58.8 ± 14.5 62.7 ± 11.0 Male, n (%) 96 (64.0) 41 (63.1) 37 (61.7) 18 (72.0) Female, n (%) 54 (36.0) 24 (36.9) 23 (38.3) 7 (28.0) Tobacco exposure: Yes, n (%) 98 (65.3) 45 (69.2) 38 (63.3) 15 (60.0) Tobacco exposure: No, n (%) 52 (34.7) 20 (30.8) 22 (36.7) 10 (40.0) Data are n (%) unless otherwise specified. Age means are based on all 150 recorded values (Grade I: n = 65, Grade II: n = 60, Grade III: n = 25). Percentages for tobacco exposure use the full group denominator. Biomarker levels across OSCC histopathological grades The distributions of NLR, PLR, SII and SIRI according to histopathological grade are presented in Table 2. The overall median NLR was 2.15 (IQR 1.60-2.85), with medians of 2.08 (1.56-2.71), 2.16 (1.58-3.07) and 2.34 (1.90-2.88) for Grades I, II and III, respectively. The overall median PLR was 137.7 (107.7-182.4); corresponding grade-specific medians were 135.2 (97.5-175.9), 136.1 (109.3-172.6) and 164.1 (127.2-195.6). Median SII was 516.5 (388.7-729.5) overall and 474.8 (361.8-698.6), 496.5 (383.6-732.5) and 669.2 (538.1-891.9) across Grades I-III. Median SIRI was 1.16 (0.87-1.70) overall, with grade-specific medians of 1.10 (0.79-1.32), 1.17 (0.86-1.72) and 1.59 (1.20-2.12), respectively. Table 2. Inflammatory biomarker levels according to histopathological grade Biomarker Overall, n=150 Grade I (n=65) Grade II (n=60) Grade III (n=25) NLR, median [IQR] 2.15 (1.60-2.85) 2.08 (1.56-2.71) 2.16 (1.58-3.07) 2.34 (1.90-2.88) PLR, median (IQR) 137.7 (107.7-182.4) 135.2 (97.5-175.9) 136.1 (109.3-172.6) 164.1 (127.2-195.6) SII, median (IQR) 516.5 (388.7-729.5) 474.8 (361.8-698.6) 496.5 (383.6-732.5) 669.2 (538.1-891.9) SIRI, median (IQR) 1.16 (0.87-1.70) 1.10 (0.79-1.32) 1.17 (0.86-1.72) 1.59 (1.20-2.12) NLR, PLR, SII, SIRI and IQR are defined in the main text. Comparison of inflammatory biomarkers across Grades I, II and III Kruskal-Wallis tests compared the four biomarker distributions across Grades I-III (Table 3). NLR did not differ significantly across grades (H = 3.460, p = 0.177). Exploratory Holm-adjusted pairwise comparisons were also non-significant for Grades I and II (p = 0.483), Grades I and III (p = 0.219) and Grades II and III (p = 0.483). PLR likewise showed no significant overall difference (H = 3.246, p = 0.197) and exploratory pairwise comparisons were non-significant for Grades I and II (p = 0.757), Grades I and III (p = 0.256) and Grades II and III (p = 0.256). In contrast, SII differed significantly across grades (H = 9.528, p = 0.009). Holm-adjusted comparisons showed no significant difference between Grades I and II (p = 0.451), while Grades I and III (p = 0.008) and Grades II and III (p = 0.025) were significant. SIRI also differed significantly across grades (H = 11.738, p = 0.003); Grades I and II did not differ significantly (p = 0.186), whereas Grades I and III (p = 0.002) and Grades II and III (p = 0.045) were significant. Table 3. Comparison of inflammatory biomarkers across histopathological grades Biomarker Kruskal-Wallis H Overall p-value Pairwise comparison Holm-adjusted p NLR 3.460 0.177 I vs II 0.483 I vs III 0.219 II vs III 0.483 PLR 3.246 0.197 I vs II 0.757 I vs III 0.256 II vs III 0.256 SII 9.528 0.009 I vs II 0.451 I vs III 0.008 II vs III 0.025 SIRI 11.738 0.003 I vs II 0.186 I vs III 0.002 II vs III 0.045 Pairwise Mann-Whitney U comparisons with Holm adjustment are shown for all biomarkers. For NLR and PLR, pairwise comparisons are exploratory because the corresponding omnibus Kruskal-Wallis tests were not statistically significant. A two-sided p value below 0.05 denoted statistical significance. Association of inflammatory biomarkers with increasing histopathological grade Spearman rank correlation assessed the association between each inflammatory biomarker and increasing histopathological grade, coded ordinally as 1 for Grade I, 2 for Grade II and 3 for Grade III (Table 4). NLR showed a positive but non-significant association with grade (ρ = 0.146, p = 0.074) and PLR also showed a positive association that was not statistically significant (ρ = 0.119, p = 0.147). SII demonstrated a positive association with increasing grade that reached statistical significance (ρ = 0.215, p = 0.008). SIRI likewise showed a positive association that was statistically significant (ρ = 0.260, p = 0.001). Thus, statistically significant grade-related associations were observed for SII and SIRI in both the three-group comparisons and the ordinal association analysis, whereas NLR and PLR did not meet the prespecified two-sided significance threshold of p < 0.05. Table 4. Spearman rank associations of inflammatory biomarkers with histopathological grade Biomarker Spearman rho p value NLR 0.146 0.074 PLR 0.119 0.147 SII 0.215 0.008 SIRI 0.260 0.001 Ordinal coding assigned 1 to Grade I, 2 to Grade II and 3 to Grade III. Statistical significance used a two-sided threshold of p < 0.05

The present study examined associations linking histopathological grade and four peripheral blood inflammatory indices: NLR, PLR, SII and SIRI in participants with OSCC. Across Grades I-III, all four indices increased numerically, but only SII and SIRI showed significant between-grade differences and positive correlations with increasing grade. NLR and PLR showed weaker, non-significant associations. The pairwise results further indicated that the significant SII and SIRI differences were driven primarily by Grade III versus Grades I and II, rather than by separation between Grades I and II. The SII finding is strongly concordant with published OSCC evidence. Median SII increased from 474.8 in Grade I to 669.2 in Grade III, showing significant between-grade differences (p = 0.009), alongside a positive association with grade (ρ = 0.215, p = 0.008). Zhang and Dai (2024), in their meta-analysis of 11 studies comprising 3,464 patients, reported that higher SII was linked to poor tumour differentiation (odds ratio (OR) = 1.74, 95% confidence interval (CI) 1.25-2.43), advanced T stage and advanced tumour-node-metastasis (TNM) stage. The present pairwise pattern, in which Grade III differed from Grades I and II while Grades I and II did not differ significantly, is consistent with that published poor-differentiation signal. Wang et al. (2026) also reported an association of SII with tumour differentiation among 201 patients with OSCC (p = 0.020). SIRI showed the largest rank correlation with histopathological grade among the four indices (ρ = 0.260, p = 0.001), increasing from a median of 1.10 in Grade I to 1.59 in Grade III. This is consistent with Wang et al. (2026), who found SIRI associated with tumour differentiation (p = 0.004) and with Kaihereman et al. (2024), who reported significant differentiation-group differences in preoperative SIRI. However, Kumar and Raju (2026), in a smaller cross-sectional study of 50 patients, found a significant grade association for SII but not SIRI. The numerically larger rho for SIRI than SII in this analysis should therefore be interpreted as hypothesis-generating only; the two correlation coefficients were not formally compared and current evidence does not establish SIRI as superior to SII for grading OSCC. NLR and PLR did not differ significantly across Grades I-III and their correlations with increasing grade were not statistically significant. The NLR result is compatible with Wang et al. (2026), in which tumour differentiation was associated with SII and SIRI rather than NLR or PLR, but differs from Kaihereman et al. (2024), who reported significant NLR differences among differentiation groups. This inconsistency suggests that grade-specific NLR associations may vary with population, case mix, sampling and analytical approach. PLR appears more consistently weak for histopathological differentiation in recent grade-specific studies, although both NLR and PLR remain relevant to other OSCC outcomes. Cho et al. (2022), Ruiz-Ranz et al. (2022), Zubair et al. (2022) and Zhuang et al. (2022) reported prognostic associations for systemic inflammatory markers, emphasising that prognostic value does not necessarily imply a monotonic relationship with histological grade. Taken together, the results from this participant cohort support a clearer grade-related signal for the composite SII and SIRI indices than for NLR and PLR. This pattern is biologically plausible because SII and SIRI integrate three circulating cell populations rather than two, but the present analyses establish association only. The modest correlation coefficients (SII ρ = 0.215; SIRI ρ = 0.260) indicate that grade is associated with only part of the variation in these biomarkers. Accordingly, neither marker should be considered a substitute for histopathological assessment and the apparent relative strength of SIRI over SII should not be interpreted as a proven superiority finding. Limitations This single-centre cross-sectional study included fewer Grade III participants than Grade I or II participants, which may reduce precision. Mean age increased across the three grade groups (53.2, 58.8 and 62.7 years), creating potential confounding because the analyses were unadjusted. Although the SII meta-analysis by Zhang and Dai (2024) found no statistically significant pooled relationship between SII and age, residual confounding cannot be excluded. Potential influences from tumour site, stage, infection, inflammatory comorbidity, medication use and blood-sampling conditions were not modelled. The cross-sectional framework cannot establish temporal or causal relationships.

In this participant cohort, SII and SIRI showed significant positive associations with poorer histopathological differentiation, whereas NLR and PLR were not significantly grade-associated. The strongest between-grade separation occurred for poorly differentiated Grade III tumours. These findings are consistent with aspects of recent OSCC literature but require validation in independent clinical cohorts before diagnostic, prognostic or treatment-related application can be considered. Future Implications and Recommendations Further research should validate these findings in adequately powered, preferably multicentre OSCC cohorts with balanced representation of histopathological grades. Future protocols should prespecify blood-sampling timing, exclude or adjust for competing inflammatory conditions, standardise histopathological grading and use multivariable ordinal or other appropriate regression models to determine whether SII and SIRI provide information independently of age, tobacco exposure, tumour site, stage and other established clinicopathological factors. Direct statistical comparison of the predictive performance of SII and SIRI would clarify whether either composite index offers incremental value

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