Prognostic significance of tumor infiltrating immune cells in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a major cause of morbidity and mortality in patients with head and neck cancer. Even with multi-modality treatment, only modest improvement of patient survival has been reported. To date, the prognosis of OSCC patients remains unsatisfactory, as indicated by the poor 5-year survival rate of less than 20% in advanced patients [ 1– 3]. Such a poor survival in OSCC patients indicates not only the aggressiveness of this cancer, but also insufficient understanding of the disease, which hinders the development of effective treatments. Our recent understanding of the involvement of immune components in disease progression, prognostic or treatment stratification in other cancers revealed the significance of immuno-characterization of human malignancies [ 4, 5], which is lacking in OSCC. Our current TNM staging system for OSCC is informative for prognosis, however, it is likely that additional immuno-characterization of OSCC tumors in situ may further facilitate treatment stratification, especially for the new arrays of immune drugs for cancer [ 1].

Tumor infiltrating immune cells have been shown to provide prognostic values in several human malignancies [ 6– 10]. For adaptive immune cells, CD8 ⁺ cytotoxic lymphocytes (CTLs) were generally considered as the main force against cancer. Both intra- and peri-tumoral CD8 expression have been shown to predict better survival in colorectal and esophageal cancers [ 11– 13]. CD4 ⁺ cells consist of several subpopulations and its benefit was controversial [ 14– 17]. Among these subpopulations, Th1 is considered as a critical component of tumor surveillance. T-bet is commonly used as a specific marker of Th1 cells, its expression have been shown significantly associated with survival of patients with breast or gastric cancer [ 18, 19]. As the important components of innate immunity, functions of macrophages and natural killer (NK) cells in tumor microenvironment draw much attention in recent decades [ 20– 22]. CD68 has been widely used as a pan-macrophage marker, its expression was reported associated with poor prognosis in breast cancer and hepatocellular cancer [ 23, 24]. CD57 expression is most prominent in highly mature cytotoxic NK cells and terminally differentiated effector T-cells such as CTLs and Th cells [ 25]. NK cells were important effectors of both innate and adaptive immune response, their killing capacity against tumor cells enhances in absence of MHC class I molecules. Tumour CD57 expression has been reported independently predicting survival in patients with colorectal cancer, gastric cancer and prostate cancer [ 26– 28].

Yet, the precise role of immune cells in OSCC remained poorly defined and controversial, though primary OSCC tumors are known to be heavily infiltrated with lymphocytes [ 29]. Thus, in this study several representative immune subsets (CD8, CD4, T-bet, CD68 and CD57) in a cohort of surgically treated OSCC patients were determined. To keep the consistency and reproducibility, the methodology recommended by an international breast cancer TILs Working Group was tested [ 30]. This study will provide new strategies to select the most promising immune markers for clinic trials through an integrative scope.

A comprehensive detection and assessment of factors influencing prognosis is important for improving patient management of OSCC. This study performed on various immune parameters in OSCC patients has shown that infiltration levels of immune cells were important predictive factors for patient outcome. Particularly, among these, stroma CD8 and CD57 expression were found to be the most powerful prognostic indicators.

We found that patients with strong CD8 expression had a significantly better clinical outcome. CD8 ⁺ cell is a crucial component of cell-mediated immunity as it produces interferon-γ upon interaction with tumour targets. In agreement to our findings, strong tumour infiltration by CD8 ⁺ cells has been correlated with a favorable outcome in several tumor types [ 31– 36], including head and neck cancer [ 14– 17, 37]. Former study on colorectal cancer with a large cohort has shown that infiltrating CD8 ⁺ T cells had a prognostic value that superior to and independent of those of TNM classification [ 38]. In our study, CD8 expression was significantly higher in patients with no lymph node involvements, when stratified patients according to N stages, CD8 expression was still correlated with favorable prognosis regardless of N stages, which revealed the superior prognostic value of CD8 expression.

Detailed analysis of CD57 ⁺ inflammatory cell activity in the host immune systems and head and neck cancer development has not been well defined. Function of tumor infiltrated CD57 ⁺cells remain unclear. Former studies have shown that CD57 was not an independent factor associated with survival [ 39, 40]. A more recent study included 57 cases of OSCC indicated that high level of CD57 ⁺ cells correlated with longer OS in OSCC patients and CD57 expression could be considered as a powerful indicator of OS [ 41]. This result was accordant with our study. In the present study, high CD57 expression was significantly associated with early clinical stage and no lymph node metastasis. A strong CD57 expression was found to be an independent prognostic factor for longer survival, moreover, it represented better predictive value compared with other immune cells, including CD4 and CD8 positive cells, and TNM staging. As researchers have reported positive correlation between presence of CD57 ⁺ T cells and cancer progression [ 25], our results indicated the potential important role of CD57 ⁺ NK cells in anti-tumor immunity against OSCC, which remained to be further confirmed by co-staining for CD3.

CD4 ⁺ cells serve a variety of biological functions according to their subpopulations. Th1 assist cytotoxic function of CD8 ⁺ cells, while Th2, Th17 and regulatory T cells (Tregs) could negatively regulate the adaptive immunity. T-bet plays critical roles in the differentiation of Th1 and regulates the Th1/Th2 shift. In OSCC, the role of CD4 remains controversial as mixed findings have been reported. Balermpas P et al. found no correlation between CD4 expression and clinical outcome of patients with head and neck cancer [ 15], whereas Nguyen N et al. reported that higher CD4 levels predicted improved OS and disease-specific survival [ 17]. T-bet has been shown to be associated with better outcome in patients with renal cell cancer, breast cancer, gastric cancer and colorectal cancer [ 19, 42– 45]. We did not observe any correlation for either CD4 or T-bet expression and clinical outcome in our series. As CD4 ⁺ cells consist of various subpopulations, each of them could affect tumor behavior. The clinical significance according to each phenotype remains to be established.

Another important component of innate immunity, macrophages, is functionally differentiated into pro-inflammatory “M1” and alternative anti-inflammatory “M2” phenotypes. M1 type, commonly identified by staining the CD11c antigen, was conferred a significantly better prognosis. M2 type, expressing CD163 and MRC1, promoted tumor growth, invasion, angiogenesis, and metastasis [ 46]. Generally speaking, CD68 is the best established marker of tumor associated macrophages (TAMs), it is expressed on both M1 and M2 phenotypes. Ni YH et al. found that CD68 ⁺ TAMs infiltration in tumor stroma was correlated with high tumor grade and lymph node metastasis. More TAMs was correlated with short OS, but TAMs was not an independent predictive factor [ 46]. Our study showed that CD68 expression was not significantly associated with OSCC patient survival in both univariate and multivariate analysis, which was consistent with a more recent study including larger cohort of 278 patients by Nguyen N et al. [ 17] This result indicated the counterweight of M1 and M2 in tumor microenvironment of OSCC. Further study is required to differentiate M1 and M2 to determine their respective functions in OSCC.

In this study, special attention was paid for evaluating immune infiltrates. Regarding localization of the stained markers, stroma and tumor periphery not tumor nest were evaluated, because most current studies have shown that stroma immune cells were superior and more reproducible [ 47, 48]. We used full section over core biopsy (such as in the settings of tissue microarrays) because up till now, there was no published evidence that Tissue Microarrays (TMAs) can mirror or reflect the potential heterogeneity of immune cells in tumor, and the number and diameter of cores in TMAs vary, which will likely affect the accuracy needed for the determination of various immune components [ 30]. Regarding the scoring system, a quantitative parameter of cells count per HPF was applied. Machine scoring approaches, while promising, have not been published or validated yet, which needs to be explored in future studies.

There were some limitations in the current study. First was the limited size of our cohort with all patients being Chinese OSCC patients. It remains unclear if Chinese ethnic background is associated with particular immunological features. Second, a critical negatively regulated factor of immune response, Tregs, has not been separately stained. As a subpopulation of CD4 ⁺ cells, Tregs might contribute to the final negative results of association between CD4 and survival. Third, as CD68 expression cannot differentiate M1 and M2 subtypes of TAMs, further study should be designed including reliable markers of M1 and M2 TAMs for precise analysis. Fourth, without co-staining for CD3, the specific functions of CD57 ⁺ T cell and CD57 ⁺ NK cell in OSCC were not illuminated.

In conclusion, our study revealed that stroma CD57 and CD8 expression were independent prognostic markers for OS of OSCC patients. When compared with TNM staging, expression of CD8 and CD57 provided superior predictive function. The results from this small OSCC cohort suggest that tumor infiltrating immune cells can potentially predict patient survival, thus provide new clues to therapeutic strategies in OSCC based on utilizing host immune response.