A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16INK4a expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16INK4a reliably indicates HPV infection. Here, to evaluate the accuracy of p16INK4a expression for determining HPV infection and the prognostic value of HPV infection and p16INK4a expression for HNSCC survival, especially oropharyngeal squamous cell carcinoma (OPSCC) survival, 150 fresh-frozen HNSCC samples were analyzed for HPV DNA, E6/E7 mRNA and p16INK4a expression by polymerase chain reaction and immunohistochemistry. p16INK4a expression was scored from 0 to 4 according to the percentage of p16INK4a-positive cells, with overexpression defined as >40% positive cells. Of the 150 tumor samples tested, 10 tumors were nasopharyngeal, 53 oropharyngeal, 39 hypopharyngeal, 24 laryngeal and 24 were located in the oral cavity. HPV DNA was detected in 47 (31.3%) samples, but only 21 also exhibited HPV mRNA expression. Inter-rater agreement was low between p16INK4a expression and HPV DNA presence and between p16INK4a expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16INK4a overexpression and HPV mRNA expression. Three-year recurrence-free survival was significantly higher for OPSCC patients who were HPV DNA-positive than for OPSCC patients who were HPV DNA-negative (P=0.008) and for OPSCC patients over-expressing p16INK4a than for without overexpressing p16INK4a (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16INK4a overexpression predicted significantly better recurrence-free survival. This combination is a more accurate marker for active HPV infection in HNSCC than HPV DNA status or general p16INK4a-positive status alone and offers a useful and reliable method for detecting and determining the prognosis of HPV-related HNSCC.


Introduction
Each year, 600,000 new cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed worldwide (1). Common risk factors for most forms of HNSCC include heavy consumption of tobacco and/or alcohol (2), although the oropharyngeal squamous cell carcinoma (OPSCC) is less likely to be associated with tobacco and alcohol exposure and more often correlated with human papillomavirus (HPV) infection (3). The incidence of OPSCC associated with HPV infection is increasing; for example, among cases of tonsillar cancer in Stockholm, HPV-positive cases rose from 23% in the 1970s to 57% in the 1990s and 79% from 2000 to 2007 (4). Moreover, alongside tobacco and alcohol, high-risk HPV variants (HR-HPVs) have emerged as risk factor for HNSCC, including OPSCC (5).
HNSCC patients who are HPV positive have substantially better prognosis than those who are HPV negative (6)(7)(8)(9)(10). Although the detection of E6/E7 mRNA transcripts is regarded as the gold standard for the presence of clinically relevant (active) HPV (11), the requirement of unfixed (fresh frozen) tissue and the cost of polymerase chain reaction (PCR) make direct detec-A comprehensive evaluation of human papillomavirus positive status and p16 INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma tion of E6/E7 impractical for cancer diagnostics at present. Accordingly, many studies have attempted to identify an easily measured surrogate maker for the diagnosis of HPV-associated HNSCC.
Expression of the tumor suppressor p16 INK4a has been proposed as a surrogate marker for HPV infection: its overexpression is thought to reflect the presence of biologically active HPV infection given that functional inactivation of pRb by viral E7 induces p16 INK4a upregulation. Detection of p16 INK4a expression can also be performed using formalinfixed, paraffin-embedded (FFPE) samples (11)(12)(13). However, there is controversy as to whether p16 INK4a expression reliably indicates HPV infection (11,12).
Klaes et al classified p16 INK4a staining as negative (<1% of cells positive), sporadic (<5% of cells positive), focal (<25% of cells positive) or diffuse (>25% of cells positive) (14). Other studies have defined p16 INK4a expression in tumors as strong and diffuse when ≥70% of cells (cytoplasm and nuclei) are stained (15)(16)(17), while Fischer et al assessed tumors as p16 INK4a positive when ≥5% of cells were immunopositive (18). These diverse scoring systems may lead to significant discrepancies across studies in the relationship between HPV infection and p16 INK4a expression. Furthermore, p16 INK4a expression has been observed in tumor-free tonsillar tissue without HPV infection, implicating other mechanisms in p16 INK4a upregulation (19). Bussu et al concluded that it is unnecessary to measure a surrogate, such as p16 INK4a expression, for objective, reliable, and direct diagnosis because HPV nucleic acids can be detected by PCR without requiring subjective assessments by histopathologists (17).
In this study, we evaluated the relationship between HPV infection and p16 INK4a expression and the value of both HPV-positive status and p16 INK4a expression levels for HNSCC prognosis using tissue samples from a well-characterized cohort of Japanese patients with HNSCC receiving curative treatment. We measured the presence of HPV DNA, HPV E6/E7 mRNA expression using fresh-frozen samples and measured p16 INK4a expression using FFPE samples.

Materials and methods
Subjects and study design. The eligibility criteria for this study were as follows: the presence of previously untreated, pathologically confirmed primary HNSCC without distant metastasis (M0); receiving curative treatment of surgery alone, surgery combined with radiation therapy (RT) or chemoradiotherapy (CRT), concurrent chemoradiotherapy (CCRT) or RT alone with >66 Gy of total dosage; and complete remission after primary treatment. The treatment modalities were determined according to tumor location, tumor stage, response to induction chemotherapy, and general physical condition, not by the results of HPV status and p16 INK4a in tumor tissue. Based on these criteria, 150 patients treated by the Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Japan were recruited between October 2006 and June 2013. In contrast to our previous study on HPV status and squamous cell carcinoma antigen (SCCA), this study involved a greater total number of cases who met the inclusion criteria and we also updated the prognostic information of some of the patients reported previously (8). Each patient gave written informed consent before enrolment. The research protocol was approved by the Ethics Committee of the University of the Ryukyus. Tissue samples were snap-frozen in liquid nitrogen during biopsy or surgical excision and stored in liquid nitrogen until further analysis. Demographic and clinicopathologic parameters for each patient were collected at scheduled intervals during the follow-up period.
Cell lines and culture. The cervical cancer cell lines CaSki (harboring ~600 copies of integrated HPV-16 DNA/genome) and SiHa (1-2 copies of integrated HPV-16 DNA/genome) were purchased from the European Collection of Animal Cell Cultures (Salisbury, UK) and the American Type Culture Collection (Tokyo, Japan), respectively, and cultured according to the suppliers' instructions.
DNA and RNA extraction. Genomic DNA and total RNA were extracted from frozen tumor samples, SiHa cells and CaSki cells using the Gentra Purification Tissue kit (Qiagen, Germantown, MD) and the ToTALLY RNA™ kit (Ambion, Austin, TX), respectively, according to the manufacturers' protocols. The extracted RNA was suspended in 50 µl ultrahigh quality diethyl pyrocarbonate-treated water.
PCR for detection of HPV DNA. The presence and integrity of the DNA in all samples was verified by PCR β-globin gene amplification using the primers PC04 and GH20 (20). Water (negative control) and DNA from HPV-16-positive CaSki cells (positive control) were included in each amplification series. The presence of HPV DNA was analyzed by PCR using the general consensus primer sets GP5 + /GP6 + and MY09/11 (21,22). DNA samples that were negative for HPV using GP5 + /GP6 + or MY09/11 PCR were re-amplified by (auto-) nested PCR using the GP5 + /GP6 + primer pair as previously described (23). PCR products were purified and directly sequenced with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Obtained sequences were aligned and compared with those of known HPV types in the GenBank database using the BLAST program.

Detection of HPV E6/E7 mRNA by reverse transcription PCR.
Before cDNA synthesis, any residual DNA was removed by incubation with 1 U DNase I (Ambion) at room temperature for 25 min. cDNA was then synthesized from DNA-free total RNA using the RETROscript ® kit (Ambion) according to the manufacturer's instructions. To examine the presence of contaminating DNA in RNA samples, all the assays were performed both with and without reverse transcriptase.
To detect high-risk E6/E7 mRNA transcripts, PCR was performed with the cDNA from HPV DNA-positive samples using the Takara PCR Human Papillomavirus Typing Set (Takara, Bio Inc., Otsu, Shiga, Japan), which can identify high-risk HPV types 16,18,31,33,35,52 and 58. To verify the HPV-16 E6/E7 mRNA transcripts, the HPV-16 DNA-positive samples were also examined using a half-nested PCR approach with cDNA as previously described by Wiest et al (24). Positive PCR products were purified and directly sequenced using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).
Immunohistochemistry for p16 INK4a and scoring of results. Serial 4-µm-thick sections from FFPE tumor samples were deparaffinized in a graded alcohol series. Epitope retrieval was performed by heating at 95-99˚C for 10 min in Tris/EDTA buffer (pH 9.0). Endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide plus 15 mM sodium azide for 5 min. The sections were subsequently incubated overnight at 4˚C with primary monoclonal mouse anti-p16 INK4a antibody (MTM Laboratories AG, Heidelberg, Germany). After extensive washing in phosphate-buffered saline, the slides were incubated for 30 min at room temperature with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (MTM Laboratories). Immunolabeling was visualized by incubation in 3-3'-diaminobenzidine for 10 min. Stained slides were counterstained with hematoxylin.
Survival analysis. Descriptive statistics were used to characterize patient baseline characteristics. The Mann-Whitney U-test or Kruskal-Wallis test was used for continuous variables, and Pearson's χ 2 test or Fisher's exact test was used for categorical variables. The inter-rater agreements between HPV-DNA presence and p16 INK4a expression and between HPV mRNA expression and p16 INK4a expression were measured by calculating Cohen's κ coefficient. A κ-value <0.20 was considered slight agreement, 0.21-0.40 as fair, 0.41-0.60 as moderate, 0.61-0.80 as good and 0.81-0.99 as excellent agreement (17,(25)(26)(27).
Locoregional control was defined as complete and persistent disappearance of disease at the primary tumor (T site) and regional lymph nodes (N site) after treatment. Recurrence-free survival was defined as the time from the end of treatment to cancer recurrence or last follow-up. Disease-specific survival was defined as the time from the end of treatment to subsidence of disease or last follow-up. Survival curves were evaluated by the Kaplan-Meier method, and survival distributions were compared using the log-rank test. Multivariate Cox proportional hazard analysis was used to identify prognostic parameters and treatments associated with risk of recurrence and disease-specific death. P-values <0.05 were considered significant. All statistical analyses were performed using the SPSS statistical package (SPSS for Windows version 12.0; SPSS, Inc., Chicago, IL).
As two of these HPV-positive samples were insufficient for RNA assay, E6/E7 mRNA expression by HPV-16, HPV-33, HPV-35, HPV-58 and HPV-56 was examined by reverse transcription PCR in 45 samples. The E6 and E7 mRNA transcripts were detected in 21 of 45 (46.7%) specimens, the majority from OPSCC cases (18/25 HPV-positive cases), as shown in Table II.
Between the HPV DNA-positive and -negative groups, there were significant differences in the distribution of histological differentiation and tumor location; for example, the HPV DNA-positive group showed poor differentiation in histology and has a higher occurrence of oropharyngeal carcinoma compared with HPV DNA-negative group. The p16 INK4a overexpression group showed similar clinical characteristics (Table I).
p16 INK4a expression and correlation with HPV status. In this study, the p16 INK4a expression scoring system (0-4) was based on the percentage of p16 INK4a -positive cells (Fig. 1)        combination of HPV DNA status and p16 INK4a overexpression had both high sensitivity (94.4%) and specificity (100%) for detecting HPV E6/E7 mRNA expression in OPSCC (Table V).  (Fig. 2). The recurrence-free survival after 3 years was 72.7% for patients with HPV DNA-negative OPSCC and 100% for patients with HPV DNA-positive OPSCC. On the contrary, there were no significant differences in recurrencefree survival and disease-specific survival between HPV DNA-positive and -negative patients with non-OPSCC (P=0.139 and 0.144, respectively; Kaplan-Meier curves not shown).
HPV mRNA-positive OPSCC patients exhibited a trend toward improved recurrence-free survival compared with HPV mRNA-negative patients with OPSCC (P=0.051) (Fig. 2). The recurrence-free survival after 3 years was 78.3% for patients Table III. Scoring of p16 INK4a overexpression and its association with HPV DNA in HNSCC and OPSCC.     with HPV mRNA-negative OPSCC and 100% for patients with HPV mRNA-positive OPSCC. OPSCC patients with p16 INK4a overexpression showed significantly improved recurrence-free survival compared with OPSCC patients without p16 INK4a overexpression (P=0.034) (Fig. 2). The recurrence-free survival after 3 years was 100 and 77.1%, respectively.
iii) Multivariate analysis using Cox proportional-hazard model in HNSCC. To assess the independent predictive value of all these factors for recurrence-free survival in HNSCC, multivariate analysis using Cox proportional-hazards models was performed. Both HPV DNA presence and p16 INK4a overexpression was modeled as one factor. Since no patients with positive HPV E6/E7 mRNA expression had any recurrent lesion, the influence of HPV mRNA expression on recurrencefree survival could not be evaluated.

Discussion
Over the past two decades, HR-HPV has been firmly established as a common etiologic factor in OPSCC and is now widely used as a prognostic marker for OPSCC. Although there are a number of studies on the epidemiologic role and prognostic value of HPV in OPSCC, some did not distinguish between patients receiving curative treatment from those receiving palliative care (28)(29)(30)(31). Moreover, there are few studies on the prognostic value of HPV infection in non-oropharyngeal SCC. In this study, we analyzed the prognostic value of HPV infection in a retrospectively selected cohort of HNSCC patients receiving curative treatment. While the oropharynx was the site of highest prevalence (47.2%), this cohort also included patients with nasopharyngeal, hypopharyngeal, laryngeal and oral cavity tumors. The relatively high prevalence of HPV in  cases of nasopharynx and oral cavity SCC suggests that HPV may play an important role in these non-oropharynx HNSCCs as well as in OPSCC.
A significant correlation was found between the presence of HPV DNA and improved recurrence-free survival in OPSCC, with HPV DNA-negative patients demonstrating an apparent greater risk of recurrence compared with HPV DNA-positive patients. OPSCC patients with HPV mRNA expression (active infection) also displayed improved recurrence-free survival compared with OPSCC patients without HPV mRNA expression (P=0.051), in line with previous studies (6,9,(32)(33)(34)(35). Although HPV DNA-positive patients with nonoropharyngeal SCCs also showed better recurrence-free and disease-free survival, the prognostic value did not reach statistical significance. Since OPSCC patients accounted for 34% of subjects in our series, the fair prognostic significance of HPV status for non-OPSCC HNSCC patients may be influenced by the generally excellent prognosis of patients with OPSCC. Indeed, Isayeva et al systematically reviewed the published data regarding the prognostic significance of HPV in SCCs of the oral cavity, larynx, sinonasal tract and nasopharynx and found no association between HPV status and treatment outcome (36). Further studies are needed to clarify the influence of HPV infection on prognosis in non-OPSCC cases.
Several studies have suggested that p16 INK4a expression can be used as a surrogate marker for HPV infection in OPSCC (14,37 (40).
In the present study, the sensitivity of general p16 INK4a expression for detecting HPV DNA in HNSCC was also low, and there was generally low rate of agreement between p16 INK4a -positive status and HR-HPV E6/E7 mRNA expression in both HNSCC (κ=0.56) and OPSCC (κ=0.57). These results indicate that p16 INK4a expression alone is not suitable for identi fying HPV-related tumors. However, p16 INK4a overexpression (p16 INK4a expression score ≥3) was a sensitive and specific marker for detecting HR-HPV mRNA expression in both HNSCC and OPSCC. This result also underscores the potential of our scoring system for evaluating p16 INK4a expression and determining prognosis. Recent studies have demonstrated a significant correlation between p16 INK4a expression as a surrogate marker of HPV infection and fair prognosis in OPSCC. Lassen et al reported that p16 INK4a -positive HNSCC showed a better response to conventional radiotherapy than p16 INK4a -negative HNSCC, and ascribed this survival benefit to a better locoregional control rate (13). In a study by Fischer et al (18), p16 INK4a -negative OPSCC patients demonstrated a more than 2-fold greater risk of death compared with p16 INK4a -positive patients. Although locoregional OPSCC relapse was independent of p16 INK4a expression, multivariate survival analysis indicated that p16 INK4a expression was an independent prognostic indicator for OPSCC, but not HNSCC, after adjustment for clinical T classification, clinical N classification, and treatment modality (18). In the present study, the combined evaluation of HPV DNA status and p16 INK4a overexpression could predict HPV E6/E7 mRNA expression in OPSCC with both high specificity (100%) and sensitivity (94.4%). Furthermore, the combined evaluation of HPV DNA status and p16 INK4a overexpression was an independent prognostic indicator for HNSCC in multivariate analysis. Given the time and expense of E6/E7 mRNA analysis, this combination may be particularly useful for larger scale clinical studies.
The combined evaluation of HPV DNA status and p16 INK4a overexpression was strongly correlated with HPV mRNA expression in OPSCC. Since the combination of HPV DNA-positive status and p16 INK4a overexpression showed a close relation with fair recurrence-free survival in HNSCC in multivariate analysis, the combination can serve as an accurate surrogate marker for biologically active HPV infection. This combined evaluation appears to be a useful and reliable method for detecting HPV-related HNSCC and determining its prognosis.