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. Author manuscript; available in PMC: 2012 May 3.
Published in final edited form as: Arch Otolaryngol Head Neck Surg. 2010 Dec;136(12):1267–1273. doi: 10.1001/archoto.2010.211

Correlation of Cellular Immunity with Human Papillomavirus 16 Status and Outcome in Patients with Advanced Oropharyngeal Cancer

Derrick Wansom 1, Emily Light 5, Frank Worden 3, Mark Prince 1, Susan Urba 3, Douglas B Chepeha 1, Kitrina Cordell 2, Avraham Eisbruch 4, Jeremy Taylor 5, Nisha D’Silva 2, Jeffrey Moyer 1, Carol R Bradford 1, David Kurnit 6, Bhavna Kumar 1, Thomas E Carey 1, Gregory T Wolf 1
PMCID: PMC3342998  NIHMSID: NIHMS370931  PMID: 21173378

Abstract

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.

Introduction

The observation that patients with HPV-16-associated oropharyngeal squamous carcinoma have a better prognosis may define these patients as a distinct subset of head and neck cancers1,2. These findings have provided an opportunity to investigate how the biologic differences of HPV-positive and -negative patient cohorts influence disease progression and outcomes.

The role of adaptive immunity in the development and progression of HPV-16 related oropharyngeal cancer is largely unknown. Deficits in the cellular and humoral immune system are commonly identified in patients with head and neck cancer and have been shown to be correlated with prognosis3,4. In particular, our group previously reported that increased peripheral blood levels of CD8 T lymphocytes predict improved overall survival5. Recently, HPV-16 E7-specific CD8 T cells were identified in a small number of patients with oropharyngeal cancer. Although higher levels were noted in patients with HPV-16 positive cancers, correlations with outcome were not reported6. Others have shown significant decreases in CD8 T cells reactive to wild-type p53 after surgical removal of HPV-16 positive cancers, suggesting that an immune-mediated process might be involved in these patients7. In an animal model of HPV-16-associated cancer, an intact immune response was essential for successful tumor clearance with chemotherapy or radiotherapy8.

To better understand the clinical importance of adaptive immunity in patients with HPV-16-associated oropharyngeal cancer, our group prospectively measured pretreatment peripheral blood levels of T-lymphocyte subsets in patients with advanced oropharyngeal cancer as part of a prospective Phase 2 clinical trial of therapeutic chemoradiotherapy. That trial included patients with HPV-16-positive and -negative cancers treated in a uniform fashion. Trial details and clinical outcomes have been reported previously9.

In the present study, levels of T-cell subsets were correlated with HPV-16 status, overall and disease free survival and response to induction chemotherapy.

Methods

Patient Population

The phase 2 clinical trial included 66 patients with stage III (n=20) or IV (n=46) oropharyngeal squamous cell carcinoma. Of these, 36 cancers were located in the base of tongue, and 30 were located in the tonsil or lateral pharynx. Pretreatment tumor tissue was available from 50 patients for creation of a tissue microarray (TMA). Sufficient tumor for extraction of DNA for p53 mutational analysis was available from 42 patients. 9 There was adequate tumor biopsy tissue to determine HPV status in 41 patients using a highly sensitive and specific quantitative method that combines polymerase chain reaction and mass spectroscopy. 10 There were 27 HPV-positive patients and 14 HPV-negative patients. Clinical tumor response and survival analyses were reported for the patients with biomarker data. There were no statistically significant differences in clinical outcomes among patients with and those without tissue available for bioanalysis. Median clinical follow up of these patients was 6.6 years.

Treatment Regimen

Patients received induction chemotherapy as one cycle of cisplatin (100 mg/m2/d for 1 day) and fluorouracil (1000 mg/m2/d for 5 days). Carboplatin (area under the curve) was used in place of cisplatin in patients with renal insufficiency or hearing loss. Response to the initial chemotherapy was evaluated by surface measurements at direct laryngoscopy, supplemented by radiographic imaging (computed tomography or magnetic resonance imaging) for deeply infiltrative tumors. Patients who demonstrated at least a 50% tumor reduction of the primary tumor underwent definitive concurrent chemotherapy (cisplatin 100 mg/m2, days 1, 22, 43) and radiation therapy (70 Gy; daily 2-Gy fractions for 7 weeks). Following radiotherapy, 2 cycles of adjuvant paclitaxel (175 mg/m2), every 21 days were administered to complete responders. Nonresponders to induction chemotherapy received salvage surgery and radiotherapy.

Lymphocyte Subpopulations

In a total of 47 patients, pretreatment peripheral blood samples were analyzed by routine automated flow cytometry for T, B, and NK cells and subpopulations of CD3, CD4, and CD8 T lymphocytes. Detailed methods have been described previously. 5 Determinations were made using commercially available monoclonal antibody reagents by an indirect immunofluorescent technique and were performed in the clinical laboratories of the University of Michigan Department of Pathology. Correlations of percentages and absolute numbers of the various subpopulations with HPV status, primary tumor response, and overall survival were determined.

HPV-16 Status, Epidermal Growth Factor Receptor Expression and p53 Mutation

Pretreatment biopsies were retrieved from 50 of 66 patients for construction of a TMA. Blocks were not available from 15 patients who had undergone biopsy outside our institution. One patient had no tumor left on biopsy specimen and so was not included. In 42 of 50 biopsy specimens, sufficient tumor was present for DNA isolation and HPV analysis, although 1 patient had insufficient DNA quality for HPV analysis. There was no residual tumor after TMA construction in 8 cases.

HPV type and copy number were assessed with a sensitive, specific, quantitative method that involved real-time competitive PCR and MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) mass spectroscopy separation of products on a matrix-loaded silicon chip array10. Internal standards allowed quantification of HPV copies per cellular genome copy. Primers designed to amplify the E6 region distinguished 13 discrete high risk HPV types (HPV -16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 and -68) 11. We previously measured and reported levels of epidermal growth factor receptor (EGFR) expression and p53 mutation status in this patient cohort using methods previously described12. Briefly, TMA slides created from biopsy specimens from the patients in this trial were deparaffinized, rehydrated, and peroxidase quenched (DakoCytomation, Glostrup, Denmark). For antigen retrieval, slides were incubated with pepsin (EGFR;10 minutes at 37°C) or with citrate buffer (p16, p53; 30 minutes at 92°C) and were blocked with horse serum (30 minutes at 25°C). Primary antibody, EGFR/31G7 (Zymed Laboratories, South San Francisco, California), p16/16P04, p53/DO1, (Laboratory-Vision, Fremont, CA) were added for 1 hour and were probed with avidin-biotin peroxidase (ABC Kit; Vector Laboratories, Burlingame, California). Antibody binding was scored by a pathologist (K.C.) who was blinded to the clinical outcome using a continuous scale (10%, 30%, 90%, etc) for the proportion of EGFR-positive tumor cells in each core. For p16, and p53, a scale of 1 to 4 was used: 1 was less than 5%; 2, 5% to 20%; 3, 21% to 50%; and 4, 51% to 100% tumor staining. Intensity was scored as 1 (no staining), 2 (low intensity), 3 (moderate), and 4 (high intensity). Scores for multiple cores from each patient were averaged. For p53 mutation, p53 exons 4 to 9 were amplified by using specific primers. Products from 2 polymerase chain reactions were sequenced in both directions and were analyzed by using Mutation Surveyor version 2.61 (Soft Genetics, State College, Pennsylvania) and by manual review. Although limiting the mutational analysis to exons 4 to 9 may be inadequate, data were also analyzed by p53 expression and obtained similar results. However, the study was not powered to determine the impact of p53 or EGFZR on response to therapy. Correlations of lymphocyte subpopulations with EGFR and p53 status were determined.

Statistical analysis

Pretreatment levels (percentages and absolute counts) of CD3, CD4, CD8, natural killer, B cells, and overall white blood cell (WBC) counts were treated as continuous variables for the assessment of univariate associations with survival in Cox proportional hazard models. To illustrate survival rates, quartiles were plotted using the Kaplan-Meier method. Log-rank tests were employed to test the homogeneity of survival across groups. Cutoffs explored in separate tests included quartiles, the median, and previously determined cutoff points from healthy individuals to classify “high” and “low” levels of CD3, CD4, CD8, and CD4:CD8 ratios 5.

Rank-based statistical methods were employed for the assessment of univariate associations between continuous lymphocyte levels and covariates of interest. Patient-level averages for EGFR and the titer of HPV-16 (log-transformed) were used in this analysis. Patients were categorized as current, past (quit> 6 months ago), or never smokers. Spearman rank correlation was used to assess the correlation of subsets with performance status, response to therapy, smoking status, stage, T class, N class, and EGFR expression. The Wilcoxon rank sum test was used to compare the distributions of subsets by HPV-16 status, tumor subsite, and p53 mutation type.

Overall survival was defined as time to death from any cause. The Kaplan-Meier method and the log-rank test were used to compare the homogeneity of overall survival and disease specific survival across quartiles using the median as a cutoff point and using cutoff points from healthy patients established in previous work5. Disease-specific survival was defined as the time to death from oropharynx cancer. Patients who were alive at last follow-up or who had died as a result of reasons other than oropharyngeal cancer were censored. Cox proportional hazards models were used to assess time-to-event outcomes. For each time-to-event outcome, 3 models were constructed: a model with subset levels (treated as a continuous variable), a model with clinical variables, and a model with both clinical variables and subset levels. Likelihood ratio tests were used to compare models.

All statistical analyses were done in SAS version 9.2 (SAS Institute, Carey, North Carolina). A 2-tailed P ≤ .05 was considered statistically significant.

Results

Lymphocyte Subsets and HPV-16 Status

Of 66 patients entered in the prospective clinical trial, pretreatment lymphocyte subsets were available in 47. The mean percentage of CD8 cells were significantly higher (P=.04), and the CD4:CD8 ratio lower (P=.02) in patients with HPV-16-positive cancers (Figure 1). There were no significant differences in other T-cell or B-cell levels among HPV-16-positive or HPV-16-negative patients. Overall WBC count was higher in HPV-16-negative patients and in smokers (P=.02 and .051, respectively) and hemoglobin levels were lower in smokers compared with past or never smokers. All of the HPV-16-negative patients studied were smokers. Percentages of CD8 cells were significantly and similarly elevated in both HPV-16-positive smokers and nonsmokers compared to HPV-16-negative smokers (P=.04, Figure 2). There were no significant differences in the proportions of lymphocyte subset levels among smokers or nonsmokers despite overall lower WBC count among HPV-16-positive patients suggesting that differences in CD8 levels were more closely associated with HPV status than with smoking history.

Figure 1.

Figure 1

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Levels of T lymphocytes (A) and CD4:CD8 ratios (B) according to human papillomavirus (HPV) status. The mean (SEM) percentage of CD8 cells was significantly elevated (P=.04), and the CD4:CD8 ratio was significantly lower (P=.02) in HPV-16-positive patients compared with HPV-16-negative patients. Significance was determined by the Wilcoxon rank sum test.

Figure 2.

Figure 2

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Levels of T Lymphocyte levels by human papillomavirus (HPV) 16 and smoking status. The mean (SEM) percentage of CD8 cells was significantly elevated in HPV-16-positive smokers and nonsmokers compared with HPV-16-negative smokers (P=.04, Spearman rank correlation)

Lymphocyte Subsets and Response to Chemotherapy

All patients received a single cycle of induction chemotherapy, and clinical tumor response was assessed by direct laryngoscopy and radiologic imaging in order to allow bioselection for subsequent treatment according to the protocol. An elevated percentage of CD8 cells (P=.02) and low CD4:CD8 ratio (P=.02) were significantly associated with complete and partial clinical tumor response to neoadjuvant chemotherapy (Figure 3). This was true when we used both mean levels and our previously established cutoff points for normal or elevated levels.5 In fact, although HPV-16 status was also a predictor of response to neoadjuvant chemotherapy (P=.01, Fisher Exact test), the CD8 levels were more predictive than HPV-16 status alone (P=.04). Higher percentages of CD8 levels were also significantly associated with ultimate tumor response after chemoradiotherapy (P=.045, Kruskal-Wallis, and P=.04, Fisher Exact Test). Smoking status was not found to have a significant correlation with response to neoadjuvant chemotherapy or to overall response after chemoradiotherapy.

Figure 3.

Figure 3

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Peripheral blood levels of T lymphocytes (A) and CD4:CD8 ratios (B) according to tumor response to induction chemotherapy. The mean (SEM) percentage of CD8 cells was significantly higher (P=.02) and the CD4:CD8 ratio was significantly lower (P=.02) in responders to induction chemotherapy. Significance was determined by the Wilcoxon rank sum test.

Lymphocyte Subsets, p53 mutation and EGFR expression

Among the other biomarkers examined, HPV status, EGFR expression, WBC and smoking were significantly associated with survival as single variables. Both HPV status (P=.02) and intensity of EGFR over-expression (P=.002) were significantly associated with disease-free and overall survival consistent with our previous analysis in the larger cohort of patients 12. Intensity of EGFR was a strong predictor of survival even after controlling for HPV-16 (P=.01) and smoking (P=.02). However, there were no significant correlations of any lymphocyte subset with EGFR overexpression. Likewise, p53 mutations were found in only 5 of our 33 patients (15%) and were not significantly predictive of survival (P=.13). The study was not powered to adequately determine the impact of p53 status on patient outcome. The correlation of p53 status and EGFR expression with clinical outcomes was previously reported12. In all, 21 of 22 HPV-16-positive cancers had wild type p53 (21 of 22) and 64% of HPV-16-negative cancers had wild type p53. Because WBC count tended to be higher in patients with HPV-16-negative tumors, WBC count was also higher in patients with mutated p53, but we did not find any correlation of individual lymphocyte subset levels with p53 mutation status.

Lymphocyte Subsets, HPV-16 Status and Survival

In the subset of patients included in this study, HPV-16 status was a significant prognostic factor, consistent with our previous analysis of the entire study population (P=.005, Figure 4). Although mean levels of the various lymphocyte subsets did not differ significantly among patients by survival status, the mean percentage of CD8 cells tended to be higher (P=.13) and CD4:CD8 ratio lower (P=.06) in surviving patients assuming unequal variances in mean levels. Using previously established cut-points5 for elevated CD8 levels (>24%) and low CD4:CD8 ratio (<2.0), we noted a trend for improved overall survival in patients with high percentage of CD8 cells and low CD4:CD8 ratio (P=.14 and P=.109, respectively, Figure 5). In assessing other traditional prognostic factors, there were no significant correlations of T-lymphocyte subsets with Karnofsky performance status, sex, T class, N class, tumor stage (III vs IV), or tumor subsite. Specifically, there were no significant differences in CD8 or CD4 levels among patients with tonsil or base of tongue primary tumors, although CD8 cell levels tended to be higher (P=.054) and CD4:CD8 ratio lower (P=.08) in patients with stage III cancer.

Figure 4.

Figure 4

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Overall survival of patients positive for human papillomavirus (HPV) 16 is improved compared to patients with HPV-16-negative cancers (P=.005).

Figure 5.

Figure 5

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Overall survival of patients using previously established cut points for the percentage of CD8 cells (A) and for CD4:CD8 ratios (B). Improved overall survival in patients tended to be associated with high perectange of CD8 cells and a low CD4:CD8 ratio (P=.14 and P=.11, respectively)

Discussion

In this retrospective analysis of a prospective observational study, we evaluated the relationship of pretreatment peripheral blood lymphocytes with HPV-16 status, tumor response to chemoradiotherapy, and overall survival. The most significant finding in the present study was that elevated percentages of CD8 cells and low CD4:CD8 ratios were associated with HPV-16 status, improved disease-free interval and overall survival. Improved survival was also significantly associated with low EGFR expression, positive HPV-16 status, and nonsmoking history. These various factors, except for HPV-16 status, appeared to be independent of lymphocyte subset levels.

Our data suggest that alterations in cellular immune response in patients with HPV-16-positive cancers may be partially responsible for the better prognosis associated with HPV-16-positive cancers. T cells appear to be important in the pathogenesis of HPV-16-associated infections13 because there is an increased incidence in T-cell-immunosuppressed patients14. Although memory cytotoxic T-cells directed against an HPV-16 E7-encoded epitope have been demonstrated in patients with HPV-16-positive cervical lesions,15 little is known about T-cell levels in HPV-16-associated oropharyngeal cancer. Previously, CD8 T-cells specific for HPV-16 E7 epitopes and for wild-type p53 peptide sequences have been described in a small number of patients with oropharyngeal cancers, but correlations with outcome have been lacking.6,7 Our results in this larger cohort of patients with advanced disease support these findings and suggest that circulating CD8 cell levels may be an important prognostic factor.

The measurement of peripheral blood lymphocyte subpopulations represents a reflection of immune homeostasis and is not a direct functional measure of overall immune competence. Many studies, however, have demonstrated the functional relevance of peripheral CD8 T cell frequency to tumor immunity and autoimmunity.16, 17 Mice with mutated or depleted CD8 cells challenged with HPV-16-positive tumor cells showed greatly decreased tumor regression compared to controls, demonstrating that antitumor effects are CD8 dependent.18 The mechanism and significance of increased T lymphocytes in the peripheral blood of HPV-16-positive patients has not been elucidated. One of many possible explanations is that HPV-16-positive tumors have increased antigenicity through the E7 antigen, causing enhanced stimulation of the immune response. The expression of E7 in dysplastic tumor cells may allow the immune system to more readily identify tumor cells as foreign. This is supported by DNA vaccine evidence from a mouse model using HPV16 E7/heat shock protein 70 genes which induced a stronger E7-specific CD8 T-cell immune response and resulted in a more significant therapeutic effect against E7-expressing tumor cells 19. In addition, in an animal model of HPV-16-transformed murine tumors, intralymphatic immunization with E7 peptide resulted in expansion of E7-specific CD8 cells responsible for tumor regression. In that study, the effect could be enhanced by removal of immunosuppressive T-regulatory cells 20. Taken together, these findings demonstrate that clearance of HPV-16-positive cells is antigen dependent and immune mediated and offers support for novel approaches to immunotherapy of HPV-16-related disease.

Many patients with HPV-16 positive cancers are nonsmokers. Smoking was an important and independent prognostic factor in this study. Interestingly, there were no observed effects of smoking on the relative frequency of CD4 or CD8 cells. Smoking was only significantly associated with an increase in mean WBC. This increase is possibly a result of non-specific inflammation related to smoking.21 Our group previously reported the profound negative impact of smoking status on overall survival in head and neck cancer22 and in HPV-16-related oropharyngeal cancer 12. A smoking habit has many potential detrimental effects, including causing gene mutations, epigenetic silencing of tumor suppressor genes, and increased mucosal inflammation resulting in production of immunosuppressive inflammatory cytokines. In the present study, WBC levels were higher in smokers compared to nonsmokers but were not directly related to alterations in percentage of cells among the T-lymphocyte subsets. Surprisingly, levels of CD8 cells were similarly elevated in HPV-16-positive smokers and nonsmokers. However, the study population, had few never smokers and all of the HPV-16-negative patients were smokers. Because smokers with HPV-16-positive cancers tend to have a poorer prognosis compared to nonsmokers, CD8 level is likely one of several related factors associated with better outcomes in HPV-16-associated cancers.

In this cohort of patients, HPV-16 status and lymphocyte subsets were independent markers of response to chemotherapy and radiotherapy. Elevated percentage of CD8 cells and low CD4:CD8 ratios were more predictive of chemotherapy response than HPV-16 status alone. Similarly, percentages of CD8 cells and CD4:CD8 ratios were significantly associated with overall response to chemoradiotherapy. These observations are consistent with in vivo results in mice, in which HPV-16-positive tumors had complete responses while HPV-16-negative tumors showed only partial responses to cisplatin therapy. 8 Furthermore, the same treatment was administered to immunodeficient mice that were found to be unable to clear HPV-16-positive tumor cells as effectively as in immunocompetent mice 8, 18. These combined findings are consistent with the hypothesis that a robust immune response can augment the antitumor activity of therapeutic chemotherapy and radiotherapy.

Lymphocyte subset alterations were independent of other important biomarkers such as EGFR expression and p53 mutation. Overexpression of EGFR has been associated with poor prognosis independent of HPV-16 status in oropharyngeal cancer and also directly associated with CD3 T-cell infiltration of oropharyngeal tumors. 23 However, CD3 infiltration was only found to be of prognostic significance in patients with HPV-16-negative cancers. Typically, patients with head and neck cancers and p53 mutations tend to have a poorer prognosis. We found no significant relationship of lymphocyte subsets with EGFR expression or p53 mutation. The failure to find a significant prognostic effect of p53 or correlations of p53 mutation with lymphocyte subsets was not surprising because most of the patients studied had cancers with wild-type p53. Only 5 patients had p53 mutations and these patients did tend to have a poorer prognosis (P=.13).

Although there is no proven causal relationship between HPV-16 status and T-cell subsets, our data suggest that measures of adaptive immunity could be useful in the clinical setting. Cutoff points for percentage of CD8 cells and CD4:CD8 ratios previously established from a large group of age-matched smoking control subjects could be applied in clinical scenarios to predict prognosis. In this study, levels of lymphocyte subsets were measured by automated flow cytometry in routine pathology department clinical laboratories. Patients with percentage of CD8 levels greater than 24% and CD4:CD8 ratios less than 2.0 tended to have a higher tumor response rates and improved survival compared with patients with low percentages of CD8 cells and high CD4:CD8 ratio. This is in agreement with our previous data in surgically treated patients who also exhibited significantly improved outcomes using these same clinical laboratory cutoff points.

Our overall findings of significant alterations in peripheral T-lymphocyte subsets that correlate with HPV-16 status and prognosis in patients with oropharyngeal cancer help to better characterize and confirm the importance of adaptive immunity in these patients. Functional analyses of these lymphocyte subsets are clearly needed to better understand the mechanistic role of these cells in the biology of HPV-16-related cancer. Longitudinal studies of changes in subset levels after therapy may provide insight into the design and monitoring of secondary cancer-prevention strategies for these patients. Detailed analysis of tumor-infiltrating lymphocytes in HPV-16-positive and -negative patients may also help provide a better understanding of the relationship between increased levels of circulating T cells, tumor infiltrating lymphocytes, host immune reactivity and outcome in the unique setting of HPV-16 related oropharyngeal cancer.

Acknowledgments

This study would not have been possible without the generosity and participation of our patients and the dedicated assistance and teamwork of the members of the Head and Neck Oncology Program at the University of Michigan who enrolled and cared for the patients in this study. The EGFR expression and p53 mutation analyses were performed in the laboratory of Drs. Carey and Bradford. The HPV-16 analyses were performed by the late Dr. David Kurnit who carried out the HPV assays using blinded samples with no knowledge of outcome or clinical course using a novel multiplex PCR, mass spectroscopy HPV detection method developed by him and subsequently licensed to SensiGen LLC and then to Sequenom.

Research support was provided by NIH NCI P50 CA097248; NIDCR R01 DE13346; NIDCD P30 DC 05188 and T32 DC005356 grants awarded to the University of Michigan Comprehensive Cancer Center and the Department of Otolaryngology, respectively. The research was also supported in part by the Sinabaldo and Diane Tozzi Research Fund.

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