Human Papillomavirus-16 (HPV-16) associated squamous carcinoma of the oropharynx has a favorable prognosis. Patients with HPV-16 positive cancers have elevated peripheral blood CD8+ T lymphocyte levels that correlate with response to chemotherapy and survival. Tumor infiltrating lymphocyte subpopulations (TIL) were assessed in pretreatment biopsies from a prospective patient cohort to determine if TIL subsets differed by HPV status, clinical factors, patient outcome or correlated with peripheral blood T cell levels. Methods Measured were CD8, CD4, CD68 and Treg (FoxP3) lymphocytes by immunohistochemistry in a tissue microarray created from patients (n=46) with advanced oropharynx cancer. Correlations with peripheral blood levels, HPV status, expression of EGFR, clinical tumor and patient characteristics and outcome were determined. Patients were treated with a single course of neoadjuvant chemotherapy (cisplatin, 5-fluorouracil) followed by either surgery (non-responders) or chemoradiation (cisplatin 100 mg/m2 every 3 weeks × 3; 70 Gy, 2 Gy daily × 7 wks) for responders. Median follow up was 6.6 years. Results HPV-16 positive patients had improved survival (p=0.016). Degree of T cell infiltration did not differ by HPV status but was significantly related to disease specific (DSS) and overall survival (OS). Higher infiltration by CD8, CD4 and FoxP3 subsets was significantly associated with lower T stage and survival. Even after adjusting for HPV status, CD8, FoxP3 and total T cells were significantly associated with DSS (p=0.0236; 0.0040; 0.0197) and OS (p=0.0137; 0.0158; 0.0115, respectively). Less T cell infiltration (p=0.0130) and CD4 cells in particular (p=0.0792) were associated with higher EGFR expression. FoxP3 infiltration correlated significantly and directly with CD4 and CD8 infiltration but not with peripheral blood levels. Conclusions Improved outcomes are associated with increased TILs independent of HPV status and suggest the local immune response to HPV-16 may be related in part to factors such as tumor size, EGFR expression, smoking history, performance status or innate immunity. Assessment of TILs in tissue microarrays is difficult due to small core sample size and variation in tumor representation in tissue cores. Further study of larger numbers of patients and infiltrates in whole tumor sections combined with functional analysis of individual subsets may be necessary to detect differences in local immunity in HPV-16 related cancers.

Objective To determine whether the favorable outcome associated with human papillomavirus (HPV) 16-positive cancer is related to a patient’s adaptive immunity. Setting Academic medical center. Methods Forty-seven of 66 previously untreated patients (6 of 20 patients with stage II and 41 of 46 with stage IV cancer) in a prospective clinical trial of chemoradiotherapy. Intervention All patients were treated with a single course of neoadjuvant chemotherapy followed by either surgery (for non-responders) or concurrent chemotherapy and conventional radiation. Main Outcome Measures Pretreatment levels (percentages and absolute counts) of CD3, CD4, CD8, NK, and B cells and overall WBC were measured by flow cytometry. Correlations of subsets with HPV-16 status, tumor subsite, stage, T class, N class, smoking status, performance status, gender, response to chemoradiotherapy, p53 mutation type, epidermal growth factor receptor expression, and disease specific and overall survival were determined. Results After median follow up of 6.6 years, improved survival was associated with elevated percentage of CD8 levels (P=.04), a low CD4:CD8 ratio (P=.01), low epidermal growth factor receptor expression (P=.002), and HPV status (P=.02). The percentage of CD8 cell levels were significantly higher (P=.04) in HPV-16-positive patients. A higher percentage of CD8 cells was associated with response to induction chemotherapy (P=.02) and complete tumor response after chemoradiation (P=.045) Conclusions These findings confirm previous correlations of outcome with circulating CD8 cell levels and support the conjecture that improved adaptive immunity may play a role in the favorable prognosis of patients with HPV-16-positive cancers.