A total of 132 consecutive patients in this cohort were diagnosed with HPSCC and the clinical characteristics of these patients are summarized in Table 1. The samples included 131 males and 1 female with a median age of 60 years (range: 40-76 years). A higher pathological grading (Grade III) group numbered 29 (22%), and the lower gradings (Grades I and II) group accounted for 103 (78%) patients. As described above, patients were divided into 2 groups depending on HPSCC clinical stage according to the AJCC 7th edition cancer staging system: namely Stage III (35, patients, 26.5%) and Stage IVA or IVB (97 patients, 73.5%). Patients smoked at least 20 packs of cigarettes per year as many as 115 (87.1%) and smoking less than 20 packs group was 17 (12.9%). Most tumors were located in the pyriform sinus (PS).
With a median follow-up of 28.4 months (Interquartile Range 20.9-39.1 months), the 3-year OS, DFS, DMFS and LRFS for the entire cohort were 68.2% (95% confidence interval [CI], 57.8% to 78.6%), 62.1% (95%CI, 52.1% to 72.1%), 72.6% (95%CI, 62.2% to 83.0%) and 79.7% (95%CI, 72.4% to 87.0%), respectively. During the follow-up period, treatment failed in 42 (31.8%) patients. Local-regional recurrence was observed in 25 (18.9%) patients, and distant metastasis occurred in 26 (19.7%) patients.
Expression of CD8, CD4 and Foxp3
Representative images for immunohistochemical detection of tumor-infiltrating T lymphocytes are shown (Fig. 1A-F). Two independent pathologist who were blinded to the patient data reviewed the slides. Median values were used for cut-offs and the patient cohort was separated into high and low groups, as described in our previous study23. The medians were 80 for CD8 (range 1 to 900), 30 for Foxp3 (range 2 to 300) and 30 for CD4 (range 1to 400), respectively. Areas of the tumor with hemorrhage or necrosis were avoided. We also investigated the ratios of CD8/Foxp3 and CD4/CD8, calculating them for each individual tumor. Similarly, the optimal cut-off points were calculated, along with their medians: the values were 2.50 (range 0.1 to 33.33) for CD8/Foxp3 and 0.33 (range 0.01 to 6.00) for CD4/CD8.
Association among different variables
Regarding the correlation of the immune markers with the clinicopathologic characteristics, high levels of TILs (CD8, Foxp3 and CD4) showed evidently correlations with lower histopathological grade and the ratios of CD8/Foxp3 and CD4/CD8 were correlated with the expression of each subtype (CD8, Foxp3 and CD4, CD8, respectively) (P < 0.05). Increased Foxp3+ TIL also exhibited a significant association with both stage and T stage (P =0.048 and P= 0.046, respectively). We also found markedly correlations among CD8, CD4 and FoxP3, using Pearson’s correlation coefficient (P<0.001, Fig. 2A-C). Other relationships between immune markers expression and clinicopathologic parameters are summarized in Table1.
Correlation with prognosis
The Kaplan-Meier curves of 3-year OS, DFS, DMFS, LRFS for patients with TILs and the ratios (low or high) are shown (Figs. 3-4). The 3-year OS, DFS, DMFS and LRFS rates, according to high and low CD8+TIL density, were 80.9% vs 56.3%, 73.2% vs 51.4%, 80.4% vs 64.5% and 77.8% vs 82.1%, respectively. Significant differences were found between the high and low CD8+TIL groups in 3-year OS, DFS and DMFS, but not in the LRFS (Fig. 3A-D). Similarly, a higher FOXP3+TIL was also strongly correlated with better OS, DFS and DMFS (P=0.001, P= 0.028 and P=0.009, respectively, Fig. 3E-H). Further analysis revealed that patients with a high CD8/FoxP3 ratio had significantly better DFS and DMFS (P=0.013 and P= 0.029, respectively) (Fig. 4B-C), while higher ratio of CD4/CD8 had evidently improved 3-year DFS and LRFS compared with lower CD4/CD8 ratio (P = 0.029 and P = 0.033, respectively) (Fig. 4F, H). By contrast, no association between the status of CD8/FoxP3 or CD4/CD8 ratio and OS was observed (Fig. 4A, E). Both UVA and MVA were performed to determine the associations between prognosis and clinic- pathological variables (Table 2-3). The multivariate variables were adopted from their prognostic significance in UVA (P < 0.05). The results did reveal that high ratio of CD8/FoxP3 remained an independent favorable prognostic factor for DFS (HR=2.613; 95% CI, 1.203-5.673; P =0.015) and DMFS (HR=3.606; 95% CI, 1.334-9.748; P = 0.011). Meanwhile, the ratio of CD4/CD8 was also an independent prognostic factor for LRFS (HR=0.414; 95% CI,0.178-0.959; P = 0.040) in the MVA. In addition, FoxP3+ TIL, T stage and site were found to be independent prognosis factors associated with DMFS, DFS and LRFS, respectively (Table 3).