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Journal of Clinical Oncology, Vol 24, No 36 (December 20), 2006: pp. 5630-5636 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.04.6136 High-Risk Human Papillomavirus Affects Prognosis in Patients With Surgically Treated Oropharyngeal Squamous Cell Carcinoma
From the Head and Neck Cancer Medical Oncology Unit, Unit of Experimental Molecular Pathology, Medical Statistics and Biometry, Department of Head and Neck Surgery, Department of Pathology, Radiotherapy Department, and Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori; and Fondazione Italiana per la Ricerca sul Cancro (FIRC) Institute of Molecular Oncology, Milan, Italy Address reprint requests to Lisa Licitra, MD, Head and Neck Cancer Medical Oncology Unit, Cancer Medicine Department Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy; e-mail: lisa.licitra{at}istitutotumori.mi.it
PURPOSE: Human papillomavirus (HPV) DNA tumors actively integrating the E6 and E7 oncogenes have a distinct biologic behavior resulting in a more favorable prognosis. To which extent the viral integration by itself, and/or the associated wild-type (wt) TP53 status, and/or a functional p16 contribute to prognosis is unclear. PATIENTS AND METHODS: To clarify how the presence of high-risk (HR) -HPV, TP53, and p16INK4a status interact with clinical outcome, we considered a retrospective series of 90 consecutive oropharyngeal cancer patients treated primarily with surgery. RESULTS: Seventeen (19%) patients showed integrated HPV 16 DNA (HPV positive), wt TP53 in all but two patients, normal p16INK4a in 15 assessable patients, and p16 expression in all 17 patients. Thirty-five patients (39%), two of whom were HPV positive, harbored TP53 mutations. p16INK4a deletion and p16 null immunophenotype occurred in 28 and 58 patients, respectively, and was similarly distributed in both patients with mutated TP53 (48% and 82%, respectively) and in patients with wt TP53 (46% and 77%, respectively). Statistical analysis showed that HPV-positive status significantly affects all investigated end points: overall survival (P = .0018), incidence of tumor relapse (P = .0371), and second tumor (P = .0152), whereas TP53 and p16INK4a status and p16 expression were not prognostic by themselves. CONCLUSION: Our molecular and clinical results are in agreement with previous findings but provide additional information into the biologic mechanisms involved in HR-HPV oropharyngeal cancer in comparison to HPV-negative tumors. According to the reduced risk of relapse and second tumors associated with HR-HPV positivity of oropharyngeal cancer, the therapeutic strategy and follow-up procedures should be reviewed.
Recent studies showed an etiologic role of infection with high-risk human papillomavirus (HR-HPV) in a subset of oropharyngeal squamous cell carcinomas (SCCs) and a distinct biologic behavior of tumors integrating E6 and E7 oncogenes, resulting in a more favorable prognosis.1-4 In some studies, HPV-positive tumors were more prevalent in younger patients,4,5 and they were associated with lower exposure to alcohol or tobacco6,7 and advanced disease.8,9 These factors are implicated potentially in prognosis, regardless of HPV positivity. However, not all studies confirmed these associations,10,11 due to an insufficient sample size to adjust for these and other important prognostic factors (such as sex and treatment),9,12-14 so that some reports failed to detect a survival advantage of HPV-positive patients bearing tumors.15,16 Oropharyngeal cancer is commonly treated with surgery and/or radiotherapy and, in advanced disease, also with concomitant chemoradiotherapy. In retrospective studies detecting the favorable prognostic effect of HPV status, the correlation between HPV status and the type of treatment was not specifically addressed.12,14,17,18 At the molecular level, in head and neck cancer, the presence of E6 oncoprotein, which mediates p53 degradation, generally is coupled with wild-type (wt) TP53,13,19 although it was suggested that p53 may partly retain its functional role, despite coexpression of viral E6 oncoprotein.20 Moreover, the presence of E7 oncoprotein, which binds and functionally inactivates retinoblastoma protein, parallels an upregulation of p16 expression suggestive of a functional p16 protein.21 To which extent the viral integration by itself, and/or the specific associated TP53 status, and/or p16INK4a status contribute to prognosis, and whether this is dependent on the delivered treatment, is unclear. This study investigated the prognostic effect of HR-HPV, TP53, and p16INK4a status and expression in oropharyngeal SCCs, based on a series of 90 consecutive patients treated at our institution with surgery, followed by radiotherapy in patients with high-risk disease.
Between 1990 and 1999, 100 consecutive patients with oropharyngeal SCC were treated surgically at the National Cancer Institute of Milan (Milan, Italy). Ninety patients were fully assessable in terms of availability of pathologic specimen and follow-up information. Fifty-eight patients (64%) received postoperative radiotherapy according to commonly accepted postoperative pathologic risk features. Radiation doses ranged from 45 to 66 Gy. Two HPV-negative patients did not complete irradiation. During a median follow-up of 5.8 years (range, 12 to 129 months), patients were assessed for the occurrence of SCC relapse, second tumor (ST), and eventually death. ST was defined clinically as a tumor occurring more than 2 cm away from and/or more than 3 years after the treatment of the index tumor.22 Reliable information about smoking and alcohol habits was not available consistently. Analyses of pathology specimen were performed on formalin-fixed, paraffin-embedded tissue samples. HR-HPV presence was investigated through real-time polymerase chain reaction (PCR), TP53 mutational status through double-gradient–denaturing gradient gel electrophoresis (dg-DGGE) analysis followed by automated sequencing, p16INK4a homozygous deletion through comparative duplex PCR, and by p16 immunohistochemical analysis.
High-Risk HPV DNA Detection and Assessment of HPV Physical Status DNA extracted from CaSki and HeLa cell lines was used as positive control for HPV 16 and HPV 18, respectively. To assess the physical status of HPV, each HPV DNA-positive sample was quantified absolutely for E2 and E6 by means of a real-time PCR TaqMan assay, as described previously.25 Given that the integration of viral DNA into the host cell usually disrupts the viral E2 open reading frame, whereas the E6 generally remains intact, equivalent copy numbers of E2 and E6 should be detected by real-time PCR when only the episomal form is present. In contrast, an E2/E6 of more or less than 1 indicates the presence of both integrated and episomal forms, and the absence of E2 amplification indicates the presence of only the integrated form. Plasmid HPV 16 DNAs from known commercially available standards (cloning) were analyzed in parallel with the samples. Standard curves were created automatically by plotting the threshold cycle values against the logarithm of the copy numbers of plasmid DNA standards (serially diluted 10-fold from 107 to 101 copies of HPV DNA). The copy number in the unknown samples was evaluated by means of integrated software using regression analysis. All of the experiments were performed in triplicate.
Detection of TP53 Gene Mutations
The detected mutations were confirmed at least twice by independent amplifications and sequence reactions. Moreover, in all samples showing the wt TP53 gene from exons 5 to 8, but showing a p53 immunoreactivity  50% of the nuclei, molecular analysis was extended to exons 4, 9, and 10. The primers used to amplify DNA from exons 4 to 10 are listed in Table 2. For exon 4, we performed two internal PCRs on the same external PCR.
Homozygous Deletions of the p16INK4a Gene To investigate homozygous deletion (HD) of p16INK4a exons 1  and 2, a comparative duplex PCR was performed in which each exon was coamplified with a fragment of the human ß-globin gene. The primers used for p16INK4a amplification for exon 1 were forward: 5'-GCC CAA CGC ACC GAA TAG T-3'; reverse: 5'-TAC CTG ATT CCA ATT CCC CTG C-3'. The primers used for p16INK4a amplification for exon 2 were forward: 5'-CTT CCT GGA CAC GCT GGT-3'; reverse: 5'-GCA GGT ACC GTG CGA CAT-3'. The occurrence of HD was evaluated on the basis of previously described criteria.23 The K562 cell line, which has an HD at the INK4A locus, was used as a negative control.
Immunohistochemistry
Statistical Analysis The end points of interest were overall survival, tumor relapse, and second primaries. Time to occurrence of any of these events was computed from the date of surgery to the date when the event was recorded, or censored at the latest follow-up date available in event-free patients. To investigate the pattern of occurrence of any of the aforementioned end points over time, descriptive analyses were carried out by estimating Kaplan-Meier overall survival curves and crude cumulative incidence curves of tumor relapse or second primaries,28 whereas inferential analyses relied on cumulative hazards. In particular, unadjusted P values for testing the prognostic effect of HPV/TP53 status were obtained from the log-rank test, and adjusted P values were obtained from the likelihood ratio test in a multivariable Cox regression model. The covariates entered into the latter for the purpose of adjustment (treatment for overall survival; patient age at surgery and tumor stage for tumor relapse) were chosen from a number of predictors as those that maximized the Cox model fit, as estimated by the Akaike information criterion. For second primaries, because of the limited number of events, only unadjusted P values are given. P values below the conventional 5% threshold were regarded as significant. All of the analyses were carried out using SAS software (SAS Institute Inc, Cary, NC).
High-Risk HPV DNA Detection and Assessment of HPV Physical Status Out of the 90 investigated patients, 17 (19%) showed integrated HPV 16 DNA (HPV positive) and none were positive for HPV 18 DNA. We observed an E2-to-E6 transcript ratio consistent with full viral integration in two HPV-positive patients and the presence of both episomal and integrated viral forms in the remaining patients.
TP53 Mutation
HDs of the p16INK4a Gene
p16 Immunohistochemistry
Statistical Analysis During a median follow-up of 5.8 years, 49 deaths, 43 locoregional relapses (of which seven also occurred with distant metastasis), and 10 STs were recorded. STs were diagnosed mainly in the lung (five patients), in the head and neck area (four patients), and in the urinary bladder (one patient; later, this patient developed a third primary in the lung). Overall survival, crude cumulative incidence of tumor relapse, and STs were 63.9%, 41.6%, and 2.2% at 3 years, and 50.2%, 47.6%, and 10.2% at 5 years.
By testing the prognostic role of HPV/TP53 status, using either univariable or multivariable analyses (Table 2), significant results were obtained for each investigated end point, namely overall survival (P = .0018 at the multivariable analysis), occurrence of tumor relapse (P = .0371), and ST (P = .0152). Figures 1 to
A similar investigation of the joint prognostic effect of HPV and p16 expression (or p16INK4a deletion) yielded similar results (not shown in detail): HPV status retains its favorable prognostic effect, whereas it did not influence the clinical outcome of HPV-negative patients.
The presence of HR-HPV together with the analysis of TP53 and p16INK4a status and p16 immunophenotyping was assessed in a series of 90 oropharyngeal SCCs, all of which were primary tumors treated homogeneously with surgery with or without radiation. The results showed that 17 (19%) of 90 patients harbored integrated HPV 16 DNA, mostly coupled with wt TP53, normal p16INK4a gene, and a p16-positive immunophenotype. Thirty-five (39%) of 90 patients harbored TP53 mutations and not surprisingly, HR-HPV and TP53 mutation coexisted in only two patients, who retained both normal p16INK4a and expression of its encoded protein. p16INK4a homozygous deletion occurred in 28 patients and p16 null immunophenotype occurred in 58 HPV-negative patients, both similarly distributed among mutated TP53 (48% and 82%, respectively) and wt TP53 (46% and 77%, respectively) groups. The correlation between p16 immunophenotyping and HR-HPV as well as p16INK4a molecular analyses confirmed that immunohistochemistry is a good surrogate for HR-HPV infection, with a p16 overexpression in all HPV-positive patients, and for p16INK4a inactivation, with evidence of loss of p16 nuclear staining in all p16INK4a-deleted patients. However, p16 immunostaining may be misleading because it does not entirely mirror molecular findings. Indeed, in our series, despite the significant correlation between p16 expression and presence of HR-HPV (P < .0001), 12 p16-positive patients were HR-HPV negative. Conversely, p16 null patients outnumber the molecular deleted patients, suggesting the existence of other inactivating p16INK4a mechanisms such as promoter methylation. The outcome analysis of the different biomarker association resulted in the definition of three independent prognostic groups among which patients harboring HR-HPV showed a significant improvement in terms of survival, occurrence of relapse, and ST incidence. This apparently was independent from tumor stage as well as the delivered treatment, confirming the observation that HPV-positive tumors represent a distinct disease among oropharyngeal cancers. In contrast, TP53 mutations seem not to be significant in prognostication of oropharyngeal SCCs primarily undergoing surgery, a notion consistent with the fact that TP53 mutations act as a predictive factor under particular treatment conditions such as chemotherapy and radiotherapy, rather than as a prognostic factor.29-34 In our study, deregulation of the p16INK4a gene and its encoded protein also seem to lack prognostic significance (in the absence of HPV infection). Indeed, the role of this deregulation in head and neck tumors is not yet well defined.35,36 Few studies concluded that the reason for a better prognosis would be explained by an enhanced radiosensitivity of HPV-positive patients in comparison to HPV-negative patients.3,29,37-39 It was suggested that the interaction between E6 and p53 does not result in its full functional abrogation as compared with mutated TP53 patients.20,40 This would justify an improved radiosensitivity sustained by a functioning p53 protein20,40 to which we can add a functional p16 protein. To our knowledge, this is the first clinical series in which p16INK4a molecular analysis was performed to complement p16 immunophenotypic data. Conversely, it has been shown that E6 and E7 oncogenes can modulate abrogation of response to DNA damage, such as radiation, using both a p53-dependent and p53-independent pathways,41-43 indicating that the interpretation of an enhanced radiosensitivity is oversimplified. Our results would confirm the prognostic role of HPV also for patients primarily treated with surgery, and a better prognosis was seen in HPV-positive patients independent of whether they received postoperative radiation. This suggests that mechanisms other than or in addition to TP53 and p16INK4a status are involved in conferring a favorable prognosis, and that to the peculiar features of HPV-positive oropharyngeal SCC might contribute to its gene profile. In presence of transcriptionally active HR-HPV, there is a substantial difference in terms of lower rate of loss of heterozygosity, in particular at chromosomal regions 3p, 9p, and 17p, compared with HPV-inactive or HPV-negative tumors.19 These findings are consistent with our observation of the mutual exclusion between HPV 16 DNA and both TP53 mutations and p16INK4a deletion, indicating the presence of two functional proteins. In addition to the possible role of genetic instability, the presence of a tumor-causative virus could contribute (by itself) in reducing tumor aggressiveness by inducing a more efficient immune response. In fact, there is strong evidence that integrated E6 and E7 proteins are particularly immunogenic in humans, with the production of both humoral and cell-mediated responses,44 which might prevail on the protective role of all oncoproteins in the infected cells from the host immune response.45 A peculiar finding of HR-HPV–related oropharyngeal SCC in comparison to HPV-negative tumors, which again speaks in favor of a distinct entity, is represented by the reduced tendency of developing second tumors. One may speculate about the reasons involved in the uneventful behavior of HPV-related cancers within the framework of the head and neck cancer genetic progression model46,47 and the most recent proposed interpretations of the genetic mechanisms at the basis of the Slaughter's cancerization concept.48 Head and neck tumors have long been recognized to be associated with carcinogen exposure and to carry TP53 mutations and/or p16INK4a deregulation. Long-term exposure of the aerodigestive mucosa would lead to the development of multifocal genetically unrelated carcinoma. This concept has been revised recently in the light of the presence of a genetically altered mucosa field in the tissue surrounding oral and oropharyngeal tumors, which has been shown to share common alterations with the index tumor.22,49 This field is capable of superficial progressive expansion and would be responsible for the development of clonally related second tumors (the so-called second field tumors in the patch field carcinoma model), as opposed to the tumors arising de novo (so-called second primary tumors), lacking a common genetic alteration with the index tumor. At present, these mechanisms are accepted to coexist.50 The absence or reduced exposure to carcinogens has been observed in HR-HPV–related tumors,17,51 and our results seem to corroborate this observation indirectly. The virtual absence of second tumors in HPV-positive patients would call into question the role of the relatively preserved 3p, 9p, and 17p chromosome regions in HPV-positive tumors,19 in contrast to what happens at the molecular level of an expanding field. Expanding field is genetically characterized by loss of heterozygosity at all of these loci along with the presence of TP53 mutation47 and p16INK4a alterations.52 Consistently, our HPV-positive patients mostly harbored wt TP53 and all carried normal p16INK4a gene coupled with p16 overexpression. On the basis of its favorable prognosis, including the reduced incidence of second tumors in HR-HPV SCCs, the reconsideration of therapeutic attitudes, as well as follow-up procedures tailored according to the risk of relapse and second primaries, should take place. Similar considerations should be made for the inclusion of these patients into prospective trials in which HPV positivity should be regarded at least as a stratification factor.53
The authors indicated no potential conflicts of interest.
This article is dedicated to our late friend and colleague, Alberto, whose personal suffering prompted our investigations into this disease.
Supported in part by grants from the Italian Association for Cancer Research (AIRC) Grant (S.P.) and Consiglio Nazionale delle Ricerche/Ministero dell'Universitá e della Ricerca (CNR/MIUR). L.L. and S.P. contributed equally to this work. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Semin Oncol 31:744-754, 2004[CrossRef][Medline] Submitted October 17, 2005; accepted April 27, 2006. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 tests. 
b, –0.27; P = .0125). Consequently, HPV and TP53 could not be investigated reliably as two independent factors. Rather, they were investigated after splitting the whole series into three subsets: HPV positive/TP53 wt (15 patients), HPV positive or negative/TP53 mutation (35 patients), and HPV negative/TP53 wt (40 patients). Patients, disease, and treatment characteristics according to HPV/TP53 and are listed in 
