Tumor infiltrating CD8+ and Foxp3+ lymphocytes correlate to clinical outcome and human papillomavirus (HPV) status in tonsillar cancer

Abstract

Background: Human papillomavirus (HPV) is a causative factor for tonsillar squamous cell carcinoma (TSCC) and patients with HPV positive (HPV(+)) TSCC have a better clinical outcome than those with HPV negative (HPV(-)) TSCC. However, since not all patients with HPV(+) TSCC respond to treatment, additional biomarkers are needed together with HPV status to better predict response to therapy and to individualize treatment. For this purpose, we examined whether the number of tumor infiltrating cytotoxic and regulatory T-cells in TSCC correlated to HPV status and to clinical outcome.

Methods: Formalin fixed paraffin embedded TSCC, previously analysed for HPV DNA, derived from 83 patients, were divided into four groups depending on the HPV status of the tumor and clinical outcome. Tumors were stained by immunohistochemistry and evaluated for the number of infiltrating cytotoxic (CD8(+)) and regulatory (Foxp3(+)) T-cells.

Results: A high CD8(+) T-cell infiltration was significantly positively correlated to a good clinical outcome in both patients with HPV(+) and HPV(-) TSCC patients. Similarly, a high CD8(+)/Foxp3(+) TIL ratio was correlated to a 3-year disease free survival. Furthermore, HPV(+) TSCC had in comparison to HPV(-) TSCC, higher numbers of infiltrating CD8(+) and Foxp3(+) T-cells.

Conclusions: In conclusion, a positive correlation between a high number of infiltrating CD8(+) cells and clinical outcome indicates that CD8(+) cells may contribute to a beneficial clinical outcome in TSCC patients, and may potentially serve as a biomarker. Likewise, the CD8(+)/Foxp3(+)cell ratio can potentially be used for the same purpose.

Figure 1. Examples of TSCC stained for tumor infiltrating CD8⁺ and Foxp3⁺ cells.

TSCC with (A) low and (B) high CD8⁺ infiltration and (C) low and (D) high Foxp3⁺ infiltration.

Figure 2. Box plots presenting tumor infiltrating CD8⁺ and Foxp3⁺ cells for different groups of TSCC.

A) number of CD8⁺ TILs, B) number Foxp3⁺ TILs and C) the CD8⁺/Foxp3⁺ cell ratio. “Good” and “poor” denotes clinical outcome. In addition to the four TSCC groups defined in the Methods section, different combinations of groups have also been compared. Thus, “HPV⁺ good” corresponds to group A; “HPV⁺ poor” to group B; “HPV⁻ good” to group C; “HPV⁻ poor” to group D; HPV⁺ to groups A+B, HPV⁻ to groups C+D; “good” to groups A+C; and “poor” to groups B+D. In order to better visualize the details in the lower part of Figure 1C the upper part of the diagram including the upper whisker (at a ratio of 80) was omitted.

Figure 3. Kaplan-Meier curves showing disease free patient survival depending on TSCC HPV status and TILs, and n denotes the number of patients in each stratified group.

A) HPV⁺TSCC and B) HPV⁻TSCC patients stratified by the number of CD8⁺ TILs. High and low denotes CD8⁺ TIL count above and below the median values of 33 for HPV⁺TSCC and 4.4 HPV⁻TSCC. C) HPV⁺TSCC and D) HPV⁻TSCC patients stratified by the ratio of CD8⁺/FoxP3⁺ cell ratio. High and low denotes CD8⁺/FoxP3⁺ cell ratio above and below 1.