In this well-defined, large population with incident HNSCC, we assessed the associations between biomarkers of HPV16 and overall survival of HNSCC patients. In agreement with previous findings [35
], our data show that all serological biomarkers that we examined were associated with improved overall survival and this improvement is particularly enhanced for oropharyngeal cancer [9
]. In our study the HPV16 E6 and E7 serology markers were superior to all other biomarkers that we examined, taken alone, in predicting the prognosis of HNSCC. Importantly, we observed that p16 overexpression by immunohistochemical staining or HPV16 DNA by PCR, as individual biomarkers, are not informative for predicting HNSCC prognosis in this population-based sample. Patients whose tumors were HPV16 DNA positive but E6 and/or E7 seronegative did not have any reduction in overall mortality, whereas patients with E6 and/or E7 seropositive demonstrated a substantial and significant reduction in overall mortality regardless of DNA status. P16 overexpression was only informative when serologic status for E6 and E7 was known. Importantly, E6 and/or E7 seropositive patients without p16 overexpression did not have enhanced survival. In contrast, the E6 and E7 seronegative patients with p16 overexpression were at a significantly increased hazard of death. This striking relationship is consistent with the known multiple mechanisms of inactivation of the retinoblastoma pathway, including the action of HPV16 or deletion, methylation or mutation of Rb or other genes in this pathway.
It has been suggested that the favorable prognosis associated with HPV is specific to DNA-positive HNSCC patients with transcriptionally active HPV genomes [37
]. Our finding of the clear importance of the combination of HPV16 DNA and seropositivity to early proteins for predicting survival strongly supports that view and thus the need to choose particular biomarkers that will be clinically useful. Our results also quite strongly suggest that the mere presence of HPV16 DNA in tumors (by PCR) is not an informative biomarker for HPV16 in predicting overall survival. Consistent with prior studies [38
], our data indicated patients with HPV16 serologic positivity tended to be younger, male, non-smokers and non-drinkers or light-drinkers. However, when we include HPV DNA status with E6/E7 serologic status, we found that patients with tumor DNA-positive but E6/E7 seronegative tended to be older (62.1 vs. 57.8), females (33.9% vs. 11.5%), smokers (78.0 vs. 71.2%), and heavy drinkers (64.4% vs. 53.9%) compared with DNA-positive and E6/E7 seropositive. Thus, our finding supports the hypothesis that cases whose tumors are HPV DNA positive, but without evidence of transcriptional activity (as evidenced by a lack of serologic response to the HPV early genes), are tumors that are not driven by HPV16.
Given our higher prevalence of HPV16 DNA positivity compared with other studies[40
], we assessed the impact of assay threshold on survival by increasing the cutoff of MFI level (see Supplementary Table 4
). There was no difference in the overall survival across different thresholds of DNA positivity (by the MPG method), although a slightly enhanced survival was observed in the cases of orophryngeal cancer when the cut off was set to 700 MFI (30% of cases DNA positive). The prevalence of HPV16 DNA (MPG) positive oropharyngeal cancer (72%) and the percentage of E6/E7 seropositivity among DNA positive oropharyngeal cases (71%) in our study are similar to those of D’Souza et al [43
] in that HPV16 DNA was detected in 72% of tumor specimens and 64% were seropositive for HPV16 E6/E7. Smith et al [35
], reported anti-E6 and/or E7 antibodies detectable in 75% of tumor HPV-16 positive cases and 5% of HPV-16 negative cases. We found E6/E7 seropositivity in 50% of HPV-16 DNA positive cases and 9% of negative cases. Smith et al also compared the HPV serology assays with tumor tissue findings in association with survival, indicating that E6/E7 seropositive cases have similar prognostic advantage as that based on tumor DNA status. This discrepancy is probably related to the concordance between HPV16 DNA and E6/E7 serology, which was 0.74 in their study and 0.36 in our study. This differing concordance could result from populations with different prevalence of HPV infection (our sample was population-based and Smith et al (35
) used a hospital-based design) or potentially with distinct immunologic status, as both studies used the same methods to measure serology and HPV DNA in the tumor.
Conflicting results exist regarding the prognostic significance of immunostaining for p16. P16 overexpression has been associated with improved outcome in head and neck cancer[17
] while other investigators have reported no association[44
]. Few studies have directly compared the prognostic significance in subgroups of HNSCC by p16 and HPV infection. Smith et al [45
] reported a worse overall survival (HR= 4.1, 1.7–9.9) and disease-specific survival (HR = 4.0, 1.5–10.7) for the group of p16−/p53+/HPV− compared with p16+/p53−/HPV+. However, we found that patients with p16+/E6−/E7− had an increased hazard of death while Smith et al [48
] reported no association with survival. Our data are consistent with that of Weinberger et al [46
] who reported that tumors that were HPV DNA negative with low p16 protein expression had a poor 5-year survival of 20%. Similar results were observed in tumors that were HPV DNA positive with low p16 protein expression [46
A major strength of this study is the fact that, in a single, population-based study, we examined the concordance between tumor DNA and serologic markers, compared the prognostic findings among the markers, and investigated their joint effects in predicting the overall survival of HNSCC cases. We were able to detect tumor DNA and L1 serology using different detection methods in order to determine whether the discrepancy of findings is due to the difference in detection methods. The results demonstrate remarkable consistency regardless of detection methods used. Because a serologic assay is not site-specific, it can be argued that infections outside the head and neck might influence the estimates (albeit, this is a bias to the null). Also, not all individuals exposed to HPV seroconvert or maintain detectable antibody levels over time[49
]. Serum antibodies elicited by distant, past infection may wane over time (and the antibody response for different proteins may wane in a different fashion) leading to potential exposure misclassification. In order to examine the expression levels of E6/E7 oncogenes, mRNA should be considered as a potential biomarker. Further, tumor DNA and p16 immunostaining were only measured in a subset of the entire sample. However, this subset was comparable with the entire sample in demographics and clinical characteristics (supplemental table 1
) and the HR estimates were similar when restricting to subjects with complete data ( and supplemental table 6
). In addition, we were not able to adjust for tumor stage for the estimates presented in given the paucity of available data for tumor stage. However, for each marker, hazard estimates were similar when comparing models adjusted for tumor stage with those that were not (Supplementary Table 5
). Further, we were not able to evaluate disease-specific survival because these data were not collected. It is possible that overall survival is subject to competing risk, especially tobacco use. Yet, we expect that this bias is small and non-differential with respect to HPV status and potentially results in a reduction of the observed estimates, as tobacco use has been shown to be independent of HPV in its association with outcome[20
]. Last but not least, the conclusions we draw were based on our data that were analyzed using specific detection methods. In particular, HPV DNA assays were PCR-based. Our results may not be generalized to the studies that used other detection methods (e.g. in situ hybridization, viral load) to assess HPV biomarkers.
In conclusion, our study strongly suggests that the combination of detection of HPV16 DNA in HNSCC tumors or p16 immunostaining with E6/E7 antibodies represents the most clinically valuable surrogate markers for the identification of HNSCC patients who have a better prognosis.