Evaluation of lymphocyte apoptosis in patients with oral cancer

Abstract Objectives To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). Methodology The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. Results The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. Conclusions The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.


Introduction
Oral cancer (OC) is a worldwide common type of malignancy. 1 This cancer originates from the stratified squamous epithelium of the oral cavity and is called oral squamous cell carcinoma (OSCC). 2 It is often diagnosed at advanced stages, entailing fatalities and low survival rates. 3 During metastasis (stage IV), OSCC patients experience weakness and fatigue, are susceptible to infection, and commonly present leukopenia. [4][5][6] Low lymphocyte counts (lymphopenia) may lead to a decrease in immune system's ability to inhibit cancer development, promoting tumor growth and worsening prognosis. 7 OC has an immune-escape mechanism. Malignant cells are associated with immune suppression, enabling cancer cells to evade the host's immune surveillance. 7,8 The impaired function of the immune system might be directly associated with head and neck squamous cell carcinomas (HNSCC) growth and metastasis. 8 It has also been reported that tumor cells can escape immune surveillance, inhibit immune function, 9 and induce immunogenic cell death and lymphocyte apoptosis, changing lymphocyte homeostasis. 7 Apoptosis is a form of programmed cell death that plays a critical role in normal development and homeostasis of adult tissues, including cell turnover, immune system development, embryonic development, and chemical-induced cell death. 10,11 Bcl-2-associated X (Bax), a pro-apoptotic protein, and B-cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, are interrelated members of the Bcl-2 family proteins, associated with mechanisms that regulate the permeabilization of the mitochondrial outer membrane, a critical step of apoptosis. 12 Percentage of lymphocytes and apoptotic cells OC9

Immune cell subsets with Bax and Bcl-2 measurements
The second cohort categorized PBMCs into four types of immune cells based on cell-surface markers, as follows: CD19 + B, CD4 + T, CD8 + T, and CD16 + CD56 +

NK. The mean fluorescence intensities (MFI) of Bax and
Bcl-2 were measured within these lymphocyte subsets.
One million PBMCs were washed with phosphate-

Percentage of lymphocytes and apoptotic PBMCs between OC patients and HC
Given the small sample of OC patients in the stages I, II, and III, we merged them into one group (OC stage I-III). The percentage of lymphocytes was higher in HC group, followed by OC stage I-III, and OC stage IV. The percentage of lymphocytes was significantly lower in OC stage IV than in HC samples (p<0.05; Figure 2A). However, the percentage of apoptotic cells gated from lymphocytes ( Figure 2B) gradually increased from HC, to OC stage I-III, and OC stage IV. We found a significant difference between patients with OC stage IV and HC (p<0.05; Figure 2C), but no significant correlations between the percentage of lymphocytes and apoptotic cells within OC samples (R 2 =0.03; p=0.43; Figure 2D). Figure 3A shows lymphocyte subsets. OC samples presented significantly higher CD4 + T cells (p<0.001) and significantly lower CD8 + T cells than HC. CD19 + B cells and CD16 + 56 + NK cells were significantly lower in OC than in HC samples (p<0.001 and p<0.01, respectively). Bax MFI of all four cell types was slightly higher in OC samples than in HC samples ( Figure 3B).

Percentage of lymphocyte subtypes and Bax/ Bcl-2 MFI and ratio between OC patients and HC
The two groups had similar Bcl-2 MFI levels ( Figure   3C). Bax/Bcl-2 ratio in CD19 + B, CD8 + T, and CD16 + 56 + NK cells were slightly higher in OC samples than in  Percentage of lymphocyte subtypes and Bax/ Bcl-2 MFI and ratio between OC patients in stage I-III and stage IV The percentage of CD16 + 56 + NK cells was significantly lower in OC stage IV than in stage I-III (p<0.05; Figure 4A). Bax MFI of all four cell types was slightly higher in stage IV than in stage I-III samples ( Figure 4B). Conversely, Bcl-2 MFI of all four cell types was significantly lower in stage IV (p<0.05; Figure 4C). Bax/Bcl-2 ratio in CD19 + B, CD4 + T, CD8 + T, and CD16 + 56 + NK cells had slightly higher levels in  2.12±0.92 (p=0.08) Figure 4D).

Percentage of lymphocytes subtypes and Bax/ Bcl-2 MFI and ratio according to tumor size and lymph node metastasis
Regarding tumor size, the percentage of CD16 + CD56 + NK cells was significantly lower in T4 than in T1, T2, and T3 of OC samples ( Figure 5A). Bax MFI of all four cell types gradually increased from T2 to T3 and T4 ( Figure 5B). Bcl-2 MFI of all four cell types -particularly CD19 + B cells and CD8 + T cells -was considerably lower in T4 tumors ( Figure 5C). Bax/Bcl-2 ratio was significantly higher in T4 than in T2 tumors for all four cell types, mainly for CD19 + B cells and CD4 + T cells (p<0.01) and CD8 + T and CD16 + 56 + NK cells (p<0.05) ( Figure 5D). Likewise, Bax/Bcl-2 ratio for CD8 + T and CD16 + 56 + NK cells was significantly higher in T4 than in T3 ( Figure 5D; p < 0.05).
As for the number of metastatic lymph nodes, the percentage of CD4 + T cells and CD16 + CD56 + NK cells was lower in N3, whereas for CD19 + B and CD8 + T-cells it was higher ( Figure 6A). Both Bax and Bcl-2 MFI were significantly different for all four cell types in N3 when compared with N0, N1, and N2 ( Figure   6B, 6C). Bax/Bcl-2 ratio in CD19 + B, CD4 + T, CD8 + T, and CD16 + 56 + NK cells had slightly higher levels in N3 than N0, N1, and N2 (data not shown), but without statically significant difference. N3 p-value, when compared with N0, N1, and N2 for all four cell types, ranged from 0.48 to 0.99 ( Figure 6D).

Discussion
The immune system is an important defense mechanism to eliminate tumor cells, and the decrease of its functional response is intrinsically associated with cancer's growth, metastasis, and its recurrence. 21 OC is an aggressive tumor, particularly in TNM stage IV -which corresponds to the metastatic stage. 3 Lymphocytes are an important type of immune cells that have been targeted for the treatment of OC 22 for being capable of recognizing cancer antigen and destroying cancer cells. In a recent study, we found that PBMCs in cancer patients may alter epigenetic regulation and gene expression as an effect of head and neck 23 and breast cancer. 24 Previous studies showed that lymphocyte apoptosis is related to weakness and tumor progression. 8,9 Based on these findings, we hypothesized that cancer cells can alter the levels of proteins associated with lymphocyte function, including apoptosis.
In this study, we found significantly lower CD19 + B and CD16 + 56 + NK cell counts in OC patients than Tumor size, lymph node involvement, and Bax/Bcl-2 ratio were also higher in advance-stage tumors in our study, which may suggest that Bax/Bcl-2 ratio levels are associated with OSCC aggressiveness. However, further studies with a larger cohort are necessary to clarify this correlation.
The limited number of patients with OC stage I-III poses a limitation for our study. We suggest further studies to be conducted with a larger number of patients to confirm our findings. In conclusion, our results show that lymphocyte apoptosis and Bax/Bcl-2 ratios were higher in patients with OC in stage 4, T4, and N3 tumors, indicating that they play an important role in cancer prognosis. These target molecules may prove useful for supportive treatment in the future.