Abstract
Oral squamous cell carcinoma (OSCC) is common in many Asian countries. The immunopathogenesis of OSCC is unclear. The authors analyzed the lymphocyte subtypes and surface activation markers in healthy Taiwanese people ( n = 130) and patients with OSCC ( n = 97)/oral leukoplakia (OL, n = 28) using flow cytometry. Univariate analysis found an elevation in the percentage of CD56+ NK cells, CD4+/CD69+ T cells, CD19+/CD69+ B cells and CD56+/CD69+ NK cells in OSCC patients relative to healthy people. The CD19+ and CD19+/CD25+ lymphocyte subtypes decreased in OSCC patients. CD56+ NK cells increased in OL patients. CD56+/CD69+ NK cells were elevated in recurrent and advanced OSCC. Multivariate analysis revealed an increase in CD56+ NK and CD19+/CD69+ cells in OL patients relative to controls. CD19+ B cells declined during progression from OL to OSCC. Betel quid chewing, alcohol, smoking, tumour location and staging showed little effect on lymphocyte subtypes. These results suggest that alterations and activation of NK cells, T and B cells are important and associated with disease status in oral carcinogenesis.
Oral cancer is the fourth highest cause of death for men in Taiwan . More than 90% of oral cancer patients chew betel quid (BQ) and some also smoke cigarettes and consume alcohol concomitantly. Individuals who have BQ chewing, smoking and drinking habits have a higher risk of oral leukoplakia (OL) and oral submucous fibrosis (OSF) . This is because chemical carcinogens in the BQ, tobacco and alcohol may attack the protein, lipid and DNA of mucosal cells, leading to gene mutation, chromosomal aberrations and clinical oral cancer . Most damaged DNA can be repaired and the transformed cells can be destroyed by the immune defense system, but some transformed cells may overcome the immune surveillance resulting in cancer development and progression .
Alterations of the cellular and humoral immune responses in affected tissues were observed in patients with cancers of the breast, lung, liver and oral cavity . Lymphocytes isolated from patients with potentially malignant oral disorders (PMOD) including OSF, OL and oral lichen planus (OLP) have higher division rates than those from healthy people . An evident increase in the cytogenetic damage of lymphocytes isolated from patients with oral cancer is reported. Patients with oral cancer have higher leukocyte and lymphocyte counts, B lymphocyte number and circulating immune complexes than healthy people . A reduction in total leukocyte and lymphocyte counts is seen in PMOD and oral cancer . The above results suggest the presence of marked alterations in the immune status of patients with PMOD and cancer. Little is known about the factors that modulate the immunological changes in different stages of oral carcinogenesis.
The aim of this cross-sectional study was to explore the distribution of different subtypes of peripheral blood lymphocyte (PBL) including CD8+ cytotoxic/suppressor T cells, CD4+ helper T cells, CD19+ B cells and CD56+ natural killer (NK) cells and their activation surface markers CD25 and CD69 in healthy adult Taiwanese as assessed by flow cytometry. The detailed biologic significance of these lymphocytic phenotypes is described in Table 1 . The authors analyzed whether alterations in these lymphocyte subtypes were obvious in peripheral blood mononuclear cells (PBMC) isolated from patients with oral squamous cell carcinoma (OSCC) and OL. They clarified the relationship between lymphocyte phenotypes and clinical variables in OSCC patients, to determine the influence of BQ chewing. Based on alterations in the distribution of lymphocyte phenotype in patients with OL and cancer, the authors established two models that can be used for clinical prediction of the nature and stages of oral carcinogenesis.
Official name a | Phenotype recognized | Biologic significance of this lymphocytic phenotype |
---|---|---|
CD3 | Surface marker of T cell lineage | CD3 is an antigen and cluster of differentiation protein, which is a part of the T cell receptor (TCR) complex on mature T lymphocytes to generate activation signals. |
CD4 | Surface marker of helper/inducer T cells | CD4+ T cells may regulate B lymphocyte activity and promote the bactericidal activity of macrophages. They may also activate and stimulate the proliferation of cytotoxic T cells. |
CD8 | Surface marker of cytotoxic/suppressor T cells | CD8 is a transmembrane glycoprotein that can bind to a major histocompatibility complex (MHC) molecule and serve as a co-receptor for the T cell receptor (TCR) on human cytotoxic/ suppressor T cell. CD8+ T cells may destroy virally infected cells, keratinocytes and tumour cells and may influence specific patterns of immune and inflammatory responses. |
CD19 | Surface marker of B cells | CD19 is present on the earliest recognizable B-lineage cells during development to mature B cells, which can serve as a co-receptor for response to antigens. But the surface expression of CD19 is lost when mature to plasma cells. |
CD25 | Surface of late activated T, B and NK cells | CD25 is the alpha chain of the IL-2 receptor present on the surface of activated T, B and NK cells. They are expressed in activated T cells, B cells and NK cells. They can bind and respond to IL-2 to induce signals to stimulate T cell proliferation. |
CD56 | Surface marker of natural killer (NK) cells | NK cells express CD56 and are important in mediating innate immune response by secretion of perforin and granzyme to kill tumour cells or virus infected cells. |
CD69 | Surface marker of early activated T, B and NK cell | CD69 is a cell surface glycoprotein and receptor involved in lymphocyte proliferation and signalling functions in lymphocytes, natural killer (NK) cells, and platelets. |
a Purchased from Coulter, Immunotech and Boehringer Ingelheim.
Materials and methods
From 2002 to 2004, 130 healthy volunteers (healthy control group), 27 patients with OL (disease control group) and 97 OSCC patients (experimental group) were included in this study after obtaining appropriate informed consent. The criteria for the healthy control group were: age 20–80 years; no history of oral cancer or PMOD; no obvious autoimmune disease; not taking any immunoactive medications at the time of enrolment; no drug abuse; no BQ chewing, smoking and drinking habits; no evident viral or bacterial infection at least 1 month prior to the study; and within normal limit for complete blood count and leukocyte classification. The OL cases included only those who had different dysplastic changes with or without OSF following histologic study. For cancer cases, only those with pathological diagnosis of squamous cell carcinoma (SCC) were chosen. Of the 130 healthy blood donors (aged 23–70 years), 80 were male (mean age 45.11 ± 13.15 years) and 50 female (mean age 44.78 ± 10.81 years). Of the 97 OSCC patients, 87 were male (mean age 54.18 ± 10.83 years), 10 were female (mean age 58.6 ± 14.51 years). All 27 patients with OL were male, with a mean age of 35.1 ± 10.83 years.
Questionnaire design
All cases of OSCC and OL were confirmed by pathology and the patients were interviewed by well-trained senior residents before enrolling in the study. For OSCC patients, a standardized, structured questionnaire was used and the clinical conditions before and after surgery (chest X ray, head and neck MRI, whole body Tc99m scintigraphy, abdominal sonography) were evaluated to obtain information on age, sex, clinical history, tumour site and TNM staging . Information on oral habits was collected, such as whether the interviewee was a habitual alcohol consumer (none, occasional or social, one or more drinks per day for at least one year), a BQ chewer (none, occasional or social, one or more quids per day for at least one year) or a smoker (none, occasional or social, one or more cigarettes per day for at least one year). For BQ chewers, the duration of habit and the average daily consumption were recorded. The duration of BQ chewing was categorized as less than 10, 10–20, 20–30 and more than 30 years. The daily consumption of BQ was categorized as less than 15, 15–30 and more than 30 quids/day. Patients who were newly diagnosed with OSCC and received no prior treatment were categorized as fresh cases. Recurrent cases included those with a history of OSCC, developing a new lesion in the adjacent mucosa at least 6 months after treatment. Terminal stage oral cancer patients, who had inoperable recurrent tumour, multiple distant metastases, cachexia and died within 1 month after enrolling in the study, were regrouped into advanced cases. The pathological findings were also recorded, including cervical lymph node (LN) metastasis, tumour margin and differentiation status of SCC of the biopsy specimens.
Isolation of PBMC
Briefly, 2 ml of peripheral blood was collected in the morning before surgery. The blood samples from patients with OL were obtained before biopsy. Healthy individuals were selected and their blood samples were taken for comparison.
Flow cytometric analysis
Identification of lymphocyte phenotypes and their activation markers was performed directly on blood samples anticoagulated K3 EDTA tubes using specific fluorescein-isothiocyoanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies against humans: CD3-FITC/CD4-PE; CD3-FITC/CD8-PE; CD4-PE/CD69-FITC; CD8-PE/CD69-FITC; CD19-PE/CD69-FITC; CD56-PE/CD69 -FITC; CD4-PE/CD25-FITC; CD8-PE/CD25-FITC; CD19-PE/CD25-FITC; CD56- PE/CD69-FITC. Details of the panel of antibodies used for detection of specific phenotypes of lymphocytes and their biologic significance are given in Table 1 . Briefly, every tubes contained 10 μl of each antibody and 50 μl of whole blood obtained from each blood donor. These tubes were vortexed and incubated at room temperature for 30 min in the dark. Then 750 μl of 1× BD FACS™ lysing solution was added to each tube, vortexed gently and incubated in the dark for 30 min. After centrifugation, the cell pellets were resuspended in 1 ml phosphate-buffered saline (PBS) with 0.5% bovine serum albumin (BSA) and analyzed for immunofluorescence on two-color FACScan flow cytometry (Becton Dickinson, San Jose, CA, USA) equipped with an argon ion laser (488 nm). Lymphocytes were gated using forward and side scatter to exclude dead cells and debris. 10,000 PBMC were analyzed and the percentage of lymphocytes expressing various CD markers was counted for each sample.
Statistical analysis
The counts of PBL populations were expressed as mean (%) ± standard deviation (SD). Bonferroni ANOVA and simple logistic regression models were used to test difference across the groups. The prediction models with a set of independent covariables identified by a stepwise method were established using a multi-variable series of logistic regression analysis. Age was considered an important factor affecting human immune status, so all models included this covariable regardless of its statistical significance. Reported P values are for a 2-sided hypothesis. The statistically significant level was set at 5% throughout test procedure. Statistical Analysis System Version 6.12 (SAS Institute, Cary, NC, USA) computer packages were used for statistical analysis.
Results
Lymphocyte subsets in healthy adults
Reference values for lymphocyte subtype percentages by gender are outlined in Table 2 . Gender-related differences were apparent for some lymphocyte phenotypes. Females generally had a higher proportion of helper T (CD4+/CD3+) cells ( p = 0.0007) and lower in CD19+/CD69+ ( p = 0.0452) and CD4+/CD25+ ( p = 0.0209) lymphocytes ( Table 2 ). Age-related fluctuation was observed for males. Older healthy men (>60 years) had a marked increase in NK (CD56+) cells ( p = 0.001) and CD4/CD8 ratio ( p = 0.0003). Younger healthy men (<30 years) had higher percentages of cytotoxic T (CD8+/CD3+) cells ( p < 0.0001) and activated CD8+ cells that express CD69 ( p = 0.0037) and CD25 ( p = 0.0258) ( Table 3 ). The only significant age-related difference for females was found in CD56+/CD69+ values ( p = 0.0331) ( Table 4 ).
Male ( n = 80) | Female ( n = 50) | p value * | |
---|---|---|---|
Age | 45.11 ± 13.15 | 44.78 ± 10.81 | 0.5461 |
CD4+/CD3+ | 37.03 ± 8.42% | 42.53 ± 7.91% | 0.0007 |
CD8+/CD3+ | 24.42 ± 6.88% | 25.45 ± 7.42% | 0.4184 |
CD19+ | 14.09 ± 5.17% | 12.87 ± 3.97% | 0.1579 |
CD56+ | 9.87 ± 4.74% | 8.58 ± 4.20% | 0.1206 |
CD4+/CD8+ ratio | 1.70 ± 0.86% | 1.85 ± 0.72% | 0.3111 |
CD4+/CD69+ | 0.38 ± 0.30% | 0.41 ± 0.27% | 0.6381 |
CD8+/CD69+ | 2.64 ± 2.48% | 2.69 ± 1.86% | 0.9121 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.08 ± 0.08% | 0.0452 |
CD56+/CD69+ | 0.57 ± 0.80% | 0.37 ± 0.45% | 0.1271 |
CD4+/CD25+ | 6.12 ± 2.03% | 5.25 ± 2.07% | 0.0209 |
CD8+/CD25+ | 1.99 ± 1.98% | 1.88 ± 1.83% | 0.7556 |
CD19+/CD25+ | 0.43 ± 0.33% | 0.34 ± 0.24% | 0.1213 |
CD56+/CD25+ | 0.24 ± 0.25% | 0.27 ± 0.29% | 0.6250 |
Years | p value * | |||||
---|---|---|---|---|---|---|
20–29 | 30–39 | 40–49 | 50–59 | ≥60 | ||
N | 13 | 12 | 23 | 18 | 14 | |
CD4+/CD3+ | 34.67 ± 5.48% | 35.99 ± 6.89% | 37.52 ± 8.42% | 37.47 ± 9.60% | 39.34 ± 11.30% | 0.2990 |
CD8+/CD3+ | 29.69 ± 4.79% | 27.32 ± 7.22% | 22.97 ± 5.56% | 23.58 ± 6.06% | 19.12 ± 7.28% | <0.0001 |
CD19+ | 14.51 ± 4.47% | 13.27 ± 4.72% | 16.52 ± 4.90% | 12.69 ± 6.08% | 11.71 ± 4.23% | 0.0383 |
CD56+ | 9.18 ± 3.81% | 11.34 ± 5.77% | 8.26 ± 3.74% | 8.75 ± 3.56% | 13.51 ± 5.59% | 0.0010 |
CD4+/CD8+ ratio | 1.21 ± 0.35% | 1.50 ± 0.88% | 1.71 ± 0.53% | 1.68 ± 0.63% | 2.45 ± 1.45% | 0.0003 |
CD4+/CD69+ | 0.60 ± 0.42% | 0.39 ± 0.28% | 0.31 ± 0.24% | 0.35 ± 0.26% | 0.33 ± 0.30% | 0.0768 |
CD8+/CD69+ | 5.01 ± 3.31% | 2.86 ± 2.35% | 1.81 ± 1.84% | 1.56 ± 0.82% | 2.67 ± 2.60% | 0.0037 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.12 ± 0.11% | 0.09 ± 0.07% | 0.15 ± 0.11% | 0.13 ± 0.13% | 0.3493 |
CD56+/CD69+ | 0.59 ± 0.49% | 0.44 ± 0.55% | 0.49 ± 0.44% | 0.37 ± 0.25% | 1.17 ± 1.72% | 0.1369 |
CD4+/CD25+ | 5.94 ± 1.71% | 5.61 ± 1.95% | 6.61 ± 2.54% | 5.68 ± 1.36% | 6.33 ± 1.94% | 0.0704 |
CD8+/CD25+ | 3.62 ± 2.67% | 2.08 ± 2.31% | 1.47 ± 1.32% | 1.60 ± 1.58% | 1.90 ± 1.69% | 0.0258 |
CD19+/CD25+ | 0.46 ± 0.35% | 0.39 ± 0.23% | 0.44 ± 0.28% | 0.53 ± 0.51% | 0.27 ± 0.16% | 0.1050 |
CD56+/CD25+ | 0.22 ± 0.11% | 0.20 ± 0.25% | 0.29 ± 0.33% | 0.29 ± 0.22% | 0.19 ± 0.24% | 0.9469 |
Years | p value * | |||||
---|---|---|---|---|---|---|
20–29 | 30–39 | 40–49 | 50–59 | ≥60 | ||
N | 7 | 9 | 12 | 18 | 4 | |
CD4+/CD3+ | 40.79 ± 9.26% | 42.46 ± 7.94% | 41.12 ± 7.89% | 44.47 ± 7.52% | 41.26 ± 9.69% | 0.7680 |
CD8+/CD3+ | 30.40 ± 6.88% | 27.75 ± 7.82% | 26.85 ± 6.03% | 22.52 ± 7.60% | 20.65 ± 4.17% | 0.0563 |
CD19+ | 11.07 ± 2.70% | 12.56 ± 5.99% | 12.12 ± 3.07% | 13.19 ± 3.42% | 17.58 ± 2.54% | 0.0997 |
CD56+ | 6.44 ± 2.36% | 11.69 ± 4.71% | 8.06 ± 4.28% | 8.32 ± 3.82% | 8.13 ± 5.13% | 0.1291 |
CD4+/CD8+ ratio | 1.46 ± 0.66% | 1.68 ± 0.65% | 1.62 ± 0.56% | 2.17 ± 0.77% | 2.12 ± 0.87% | 0.0951 |
CD4+/CD69+ | 0.23 ± 0.13% | 0.52 ± 0.40% | 0.40 ± 0.26% | 0.38 ± 0.21% | 0.63 ± 0.23% | 0.1195 |
CD8+/CD69+ | 3.04 ± 2.28% | 3.04 ± 2.62% | 2.78 ± 1.63% | 2.12 ± 1.34% | 3.52 ± 2.17% | 0.5681 |
CD19+/CD69+ | 0.07 ± 0.04% | 0.11 ± 0.11% | 0.06 ± 0.07% | 0.08 ± 0.07% | 0.13 ± 0.09% | 0.4739 |
CD56+/CD69+ | 0.30 ± 0.21% | 0.69 ± 0.81% | 0.26 ± 0.15% | 0.23 ± 0.16% | 0.77 ± 0.78% | 0.0331 |
CD4+/CD25+ | 4.02 ± 1.86% | 5.27 ± 1.63% | 4.84 ± 1.82% | 5.76 ± 2.30% | 6.29 ± 2.06% | 0.2769 |
CD8+/CD25+ | 2.28 ± 2.33% | 2.25 ± 2.23% | 1.27 ± 0.76% | 1.71 ± 1.92% | 2.98 ± 1.88% | 0.4686 |
CD19+/CD25+ | 0.42 ± 0.27% | 0.42 ± 0.38% | 0.26 ± 0.14% | 0.33 ± 0.23% | 0.33 ± 0.04% | 0.5869 |
CD56+/CD25+ | 0.25 ± 0.33% | 0.28 ± 0.21% | 0.18 ± 0.15% | 0.28 ± 0.22% | 0.48 ± 0.76% | 0.5106 |
Lymphocyte phenotypes in OL and OSCC
Univariate analysis revealed significant differences in the distributions of lymphocyte subsets across three different groups: male OL and OSCC patients and healthy men ( Table 5 ). The percentages of NK (CD56+) cells and activated helper T (CD4+), B (CD19+) and NK (CD56+) cells that also expressed CD69 were elevated in male patients with OSCC or OL compared with healthy men. Male OSCC patients had a higher percentage of CD56+/CD69+ NK cells than patients with OL.
Normal | OL | OSCC | p value * | |
---|---|---|---|---|
N † | 80 | 28 | 87 | |
CD4+/CD3+ | 37.03 ± 8.42% | 39.57 ± 7.51% | 37.90 ± 10.66% | 0.4630 |
CD8+/CD3+ | 24.42 ± 6.88% | 24.14 ± 6.61% | 25.52 ± 9.69% | 0.6093 |
CD19+ | 14.09 ± 5.17% | 14.87 ± 5.12% | 11.27 ± 5.42% | 0.0004 |
CD56+ | 9.87 ± 4.74% | 15.15 ± 11.17% | 19.74 ± 10.74% | <0.0001 |
CD4+/CD8+ ratio | 1.70 ± 0.86% | 1.79 ± 0.68% | 1.81 ± 1.13% | 0.7565 |
CD4+/CD69+ | 0.38 ± 0.30% | 0.47 ± 0.33% | 0.65 ± 0.58% | 0.0010 |
CD8+/CD69+ | 2.64 ± 2.48% | 2.06 ± 1.81% | 2.31 ± 1.91% | 0.4013 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.19 ± 0.23% | 0.30 ± 0.45% | 0.0016 |
CD56+/CD69+ | 0.57 ± 0.80% | 0.69 ± 0.69% | 1.20 ± 1.18% | 0.0002 |
N | 80 | 25 | 64 | |
CD4+/CD25+ | 6.12 ± 2.03% | 6.66 ± 2.71% | 7.65 ± 3.90% | 0.4405 |
CD8+/CD25+ | 1.99 ± 1.98% | 1.75 ± 2.04% | 2.11 ± 2.21% | 0.1071 |
CD19+/CD25+ | 0.43 ± 0.33% | 0.55 ± 0.74% | 0.42 ± 0.46% | 0.0003 |
CD56+/CD25+ | 0.24 ± 0.25% | 0.36 ± 0.38% | 0.82 ± 1.38% | 0.7705 |
* Statistical analysis by ANOVA.
† All the OL patients were male and there may be a possible influence of gender on immune status; only the males in the healthy control and OSCC groups were included for comparison.
Regarding the differences between the two study groups, the proportion of total (CD19+) and activated B (CD19+/CD25+) cells declined significantly in male OSCC patients compared with those of healthy men or patients with OL. The percentage of NK cells, activated helper T (CD4+/CD69+), cytotoxic T cells (CD8+/CD25+), B (CD19+/CD69+) and NK (CD56+/CD69+) cells in OSCC patients were higher than in healthy patients ( Table 6 ).
Normal | OSCC | p value * | |
---|---|---|---|
N † | 80 | 87 | |
CD4+/CD3+ | 37.03 ± 8.42% | 37.90 ± 10.66% | 0.5609 |
CD8+/CD3+ | 24.42 ± 6.88% | 25.52 ± 9.69% | 0.4034 |
CD19+ | 14.09 ± 5.17% | 11.27 ± 5.42% | 0.0013 |
CD56+ | 9.87 ± 4.74% | 19.74 ± 10.74% | <0.0001 |
CD4+/CD8+ ratio | 1.70 ± 0.86% | 1.81 ± 1.13% | 0.4885 |
CD4+/CD69+ | 0.38 ± 0.30% | 0.65 ± 0.58% | 0.0012 |
CD8+/CD69+ | 2.64 ± 2.48% | 2.31 ± 1.91% | 0.3373 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.30 ± 0.45% | 0.0030 |
CD56+/CD69+ | 0.57 ± 0.80% | 1.20 ± 1.18% | 0.0004 |
N | 80 | 64 | |
CD4+/CD25+ | 6.12 ± 2.03% | 7.65 ± 3.90% | 0.2106 |
CD8+/CD25+ | 1.99 ± 1.98% | 2.11 ± 2.21% | 0.0443 |
CD19+/CD25+ | 0.43 ± 0.33% | 0.42 ± 0.46% | 0.0002 |
CD56+/CD25+ | 0.24 ± 0.25% | 0.82 ± 1.38% | 0.5587 |
The proportions of CD19+ B cells and activated B cells (CD19+/CD25+) in male patients with OL were higher than in OSCC patients, but the proportion of activated NK cells (CD56+/CD69+) in OSCC patients was higher than in OL males ( Table 7 ).
OL | OSCC | p value * | |
---|---|---|---|
N | 28 | 87 | |
CD4+/CD3+ | 39.57 ± 7.51% | 37.90 ± 10.66% | 0.4388 |
CD8+/CD3+ | 24.14 ± 6.61% | 25.52 ± 9.69% | 0.4822 |
CD19+ | 14.87 ± 5.12% | 11.27 ± 5.42% | 0.0044 |
CD56+ | 15.15 ± 11.17% | 19.74 ± 10.74% | 0.0591 |
CD4+/CD8+ ratio | 1.79 ± 0.68% | 1.81 ± 1.13% | 0.9532 |
CD4+/CD69+ | 0.47 ± 0.33% | 0.65 ± 0.58% | 0.1472 |
CD8+/CD69+ | 2.06 ± 1.81% | 2.31 ± 1.91% | 0.5402 |
CD19+/CD69+ | 0.19 ± 0.23% | 0.30 ± 0.45% | 0.2458 |
CD56+/CD69+ | 0.69 ± 0.69% | 1.20 ± 1.18% | 0.0316 |
N | 25 | 64 | |
CD4+/CD25+ | 6.66 ± 2.71% | 7.65 ± 3.90% | 0.6400 |
CD8+/CD25+ | 1.75 ± 2.04% | 2.11 ± 2.21% | 0.7267 |
CD19+/CD25+ | 0.55 ± 0.74% | 0.42 ± 0.46% | 0.0498 |
CD56+/CD25+ | 0.36 ± 0.38% | 0.82 ± 1.38% | 0.6604 |
Multivariate analysis revealed that after adjustment for age, no marked difference between healthy males and OSCC patients was seen in the population of CD8+/CD25+ T cells. Differences in various T cell populations between healthy males and OSCC patients were evident ( Table 8 ). A marked elevation in CD56+ NK cells and CD19+CD69+ T cells was observed in OL patients relative to healthy controls ( Table 9 ). After controlling for age, the differences in CD19+, CD56+/CD69+ and CD19+/CD25+ between the OL and OSCC groups became less evident. Only CD19+ B cells showed a marked difference between these two groups ( Table 10 ). Based on the above analysis, the authors tried to develop a risk prediction model.
Normal | OSCC | p value * | |
---|---|---|---|
N | 80 | 87 | |
CD4+/CD3+ | 37.03 ± 8.42% | 37.90 ± 10.66% | 0.6225 |
CD8+/CD3+ | 24.42 ± 6.88% | 25.52 ± 9.69% | 0.0610 |
CD19+ | 14.09 ± 5.17% | 11.27 ± 5.42% | 0.0136 |
CD56+ | 9.87 ± 4.74% | 19.74 ± 10.74% | <0.001 |
CD4+/CD8+ ratio | 1.70 ± 0.86% | 1.81 ± 1.13% | 0.9782 |
CD4+/CD69+ | 0.38 ± 0.30% | 0.65 ± 0.58% | 0.0003 |
CD8+/CD69+ | 2.64 ± 2.48% | 2.31 ± 1.91% | 0.7222 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.30 ± 0.45% | 0.0028 |
CD56+/CD69+ | 0.57 ± 0.80% | 1.20 ± 1.18% | 0.0030 |
N | 80 | 64 | |
CD4+/CD25+ | 6.12 ± 2.03% | 7.65 ± 3.90% | 0.3208 |
CD8+/CD25+ | 1.99 ± 1.98% | 2.11 ± 2.21% | 0.1972 |
CD19+/CD25+ | 0.43 ± 0.33% | 0.42 ± 0.46% | 0.0014 |
CD56+/CD25+ | 0.24 ± 0.25% | 0.82 ± 1.38% | 0.4277 |
Normal | OL | p value * | |
---|---|---|---|
N | 80 | 28 | |
CD4+/CD3+ | 37.03 ± 8.42% | 39.57 ± 7.51% | 0.1187 |
CD8+/CD3+ | 24.42 ± 6.88% | 24.14 ± 6.61% | 0.6454 |
CD19+ | 14.09 ± 5.17% | 14.87 ± 5.12% | 0.5302 |
CD56+ | 9.87 ± 4.74% | 15.15 ± 11.17% | 0.0038 |
CD4+/CD8+ ratio | 1.70 ± 0.86% | 1.79 ± 0.68% | 0.4118 |
CD4+/CD69+ | 0.38 ± 0.30% | 0.47 ± 0.33% | 0.2166 |
CD8+/CD69+ | 2.64 ± 2.48% | 2.06 ± 1.81% | 0.1759 |
CD19+/CD69+ | 0.12 ± 0.10% | 0.19 ± 0.23% | 0.0266 |
CD56+/CD69+ | 0.57 ± 0.80% | 0.69 ± 0.69% | 0.4099 |
N | 80 | 25 | |
CD4+/CD25+ | 6.12 ± 2.03% | 6.66 ± 2.71% | 0.6228 |
CD8+/CD25+ | 1.99 ± 1.98% | 1.75 ± 2.04% | 0.1680 |
CD19+/CD25+ | 0.43 ± 0.33% | 0.55 ± 0.74% | 0.7381 |
CD56+/CD25+ | 0.24 ± 0.25% | 0.36 ± 0.38% | 0.7555 |
OL | OSCC | p value * | |
---|---|---|---|
N | 28 | 87 | |
CD4+/CD3+ | 39.57 ± 7.51% | 37.90 ± 10.66% | 0.6143 |
CD8+/CD3+ | 24.14 ± 6.61% | 25.52 ± 9.69% | 0.1968 |
CD19+ | 14.87 ± 5.12% | 11.27 ± 5.42% | 0.0198 |
CD56+ | 15.15 ± 11.17% | 19.74 ± 10.74% | 0.2345 |
CD4+/CD8+ ratio | 1.79 ± 0.68% | 1.81 ± 1.13% | 0.9106 |
CD4+/CD69+ | 0.47 ± 0.33% | 0.65 ± 0.58% | 0.0919 |
CD8+/CD69+ | 2.06 ± 1.81% | 2.31 ± 1.91% | 0.5197 |
CD19+/CD69+ | 0.19 ± 0.23% | 0.30 ± 0.45% | 0.3167 |
CD56+/CD69+ | 0.69 ± 0.69% | 1.20 ± 1.18% | 0.1679 |
N | 25 | 64 | |
CD4+/CD25+ | 6.66 ± 2.71% | 7.65 ± 3.90% | 0.8123 |
CD8+/CD25+ | 1.75 ± 2.04% | 2.11 ± 2.21% | 0.9670 |
CD19+/CD25+ | 0.55 ± 0.74% | 0.42 ± 0.46% | 0.0828 |
CD56+/CD25+ | 0.36 ± 0.38% | 0.82 ± 1.38% | 0.4734 |
Multivariate analysis using a logistic regression model further identified significant differences between healthy men and OSCC patients regarding the percentage of CD56+ ( p < 0.0001), CD4+/CD69+ ( p < 0.0001) and CD19+/CD25+ ( p < 0.0001) cells after appropriate model selection. The percentages of CD19+ ( p = 0.0628), CD4+/CD69+ ( p = 0.0423) and CD19+/CD25+ ( p = 0.0851) cells were found to be the most influential factors after multiple regression analysis. No marked difference in lymphocyte populations between healthy men and OL patients was noted. For men, a formula based on age and percentage of lymphocyte subsets was established to predict the diagnosis between OL and cancer lesions: