Journal of Medical Virology
Low Prevalence of Human Papillomavirus in Head and Neck Squamous Cell Carcinoma in Chinese Patients Yanan Xu,1,2,3 Suru Liu,1,3 Hongliang Yi,1,3 Jiadong Wang,2,3 Yanli Luo,4 and Shankai Yin1,3* 1
Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China 2 Department of Head and Neck Surgery, Renji Hospital of Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Otolaryngological Institute of Shanghai Jiaotong University, Shanghai, China 4 Department of Pathology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
The aim of this study was to ascertain the prevalence and prognostic value of human papillomavirus (HPV) infection status in head and neck squamous cell carcinoma. Immunohistochemistry and GenoArray HPV genotyping assays were used to evaluate the HPV infection status of 256 Chinese patients with head and neck squamous cell carcinoma. Long-term survival rates were calculated using the Kaplan– Meier method. Immunostaining for p16 was prevalent in 6.6% (17/256) of patients, whereas HPV infection was detected in 9 of 256 (3.5%) patients using the HPV genotyping test. None of the p16-negative subjects were found to have HPV infection according to the HPV genotyping test. P16 positivity was more common among nonsmokers than smokers (16.3% vs. 4.7%, respectively; P ¼ 0.02). Among nine HPV-positive cases, seven were infected with HPV-16, one with HPV-33, and one with both HPV-16 and HPV-11. The 3-year overall survival rate was 87.5% for p16-positive/HPV-positive patients, 75.0% for p16-positive/HPV-negative patients, and 76.9% for p16-negative/HPV-negative patients (P ¼ 0.30). A very low rate of HPV infection was found among the study patients. HPV infection status may not be a useful prognostic marker for head and neck cancer patients. J. Med. Virol. # 2014 Wiley Periodicals, Inc.
KEY WORDS:
head and neck squamous cell carcinoma; human papillomavirus; prevalence; prognosis; survival
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION Head and neck cancer is the sixth most common cancer worldwide [Siegel et al., 2012], and its treatment can result in cosmetic deformity and functional impairment of vital functions, including breathing, swallowing, speech, phonation, taste, hearing, and smell. Head and neck cancer derives from mucosa lining the oral cavity, oropharynx, nasopharynx, larynx, and hypopharynx. The most well-established risk factors for head and neck cancer are tobacco and alcohol abuse. High-risk human papillomavirus (HPV) infection, whose role in carcinogenesis of the uterine cervix has been extensively studied, is now an emerging risk factor for head and neck cancer [Bouvard et al., 2009; Leemans et al., 2011]. In the last decade, the incidence of oropharyngeal squamous cell cancer has increased sharply, in contrast to other head and neck cancers. Many attribute this to a rise in HPV-related oropharyngeal squamous cell cancer cases. Little has been mentioned about the association of HPV and head and neck cancer, excluding the oropharynx. Prevalence rates of HPV infection in head and neck cancer, excluding the oropharynx, are reported to be lower than those for oropharyngeal cancer [Li et al., 2013]. The reported prevalence of HPV DNA in patients with head and neck cancer, excluding the oropharynx, varies widely, ranging from 0% to 86% [Ribeiro et al., 2011; Li et al., 2013]. This disparity is
Correspondence to: Shankai Yin, MD PhD, Affiliated Sixth People’s Hospital of Shanghai Jiaotong University, 200233 Shanghai, China. E-mail: [email protected] Accepted 27 July 2014
DOI 10.1002/jmv.24052 Published online in Wiley Online Library (wileyonlinelibrary.com).
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primarily due to geographical differences among the studies conducted and the inadequate distinction of patients with oropharyngeal carcinoma from those with other cancers of the head and neck region. Additionally, differences in the analytical sensitivity and specificity of HPV genotyping methods, the limited spectrum of HPV types analyzed, and differences among HPV diagnostic criteria [D’Souza et al., 2007; Wei et al., 2012] have all contributed to inconsistent results regarding HPV infection in patients with head and neck cancer. Over 200 types of HPV have been identified. Similar to cervical, vaginal, and vulvar cancers, head and neck cancer is associated predominantly with high-risk HPV, particularly HPV-16 [Snow and Laudadio, 2010]. The International Agency for Research on Cancer reported in 2012 that HPV-16 causes cancer of the oropharynx. HPV-positive head and neck cancer seems to be a distinct clinical entity with a better prognosis than HPV-negative tumors [Liang et al., 2012], which may be attributable to enhanced sensitivity to treatment due to wild-type TP53, allowing an apoptotic response to radiation and chemoradiation in cancercells [Guihard et al., 2012]. Marur et al., 2010 emphasized that HPV-associated head and neck cancer is distinct, characterized by p53 degradation, retinoblastoma protein (pRb) pathway inactivation, and p16 upregulation while tobacco-associated head and neck cancers are characterized by TP53 mutation and downregulation of CDKN2A encoding p16, which are responsible for poor prognosis. The survival benefit was most predominant or restricted in patients with oropharyngeal cancer [O’Rorke et al., 2012], but limited data were obtained in those with head and neck cancer, excluding the oropharynx, particularly with regard to p16-positive/HPV-negative cases. The current study focused exclusively on head and neck cancer, excluding the oropharynx. The aim was to evaluate the HPV infection rate in study patients with head and neck cancer and assess its prognostic value.
treated at Shanghai Sixth People’s Hospital (Shanghai, China) were enrolled in this study. Among them, 22 patients were excluded because of poor extracted DNA quality. The clinical charts of the remaining 256 patients (229 men and 27 women; ages, 21–93 years; median age, 60.5 years) were reviewed. The anatomic locations of the tissue samples were as follows: nasopharynx (n ¼ 77), larynx (n ¼ 159), and hypopharynx (n ¼ 20). Data on clinical pathological characteristics were obtained from the electronic medical record system used in Shanghai Sixth People’s Hospital. Tumor Node Metastasis (TNM) staging was used to classify tumors in accordance with the American Joint Committee on Cancer classification system. Patient distribution according to TNM classification is provided in Table I. Note that no patients had distant metastasis. Smoking and drinking history was also available in the hospital’s electronic medical record system and thus, patients were classified as smokers/nonsmokers and drinkers/nondrinkers. One representative formalin-fixed, paraffin-embedded (FFPE) block was retrieved for each case. Histological diagnosis of head and neck cancer was confirmed by two independent pathologists. Approval for the study was obtained from the Research Ethics Committee of Shanghai Sixth People’s Hospital. Treatment Patients with nasopharyngeal cancer received radiotherapy, while those with cancers in other locations received surgery therapy. Postoperative radiotherapy followed surgery if a patient was T3 or N2 stage according to the pathologic diagnosis and/or clinical data. Additionally, patients with T4 or a positive incisal margin received cisplatin and Taxotere during postoperative radiotherapy. None of the additional postoperative treatments were performed in patients of N0, N1, T1, or T2 stage. None of the treatments were based on HPV status because these markers are not examined routinely before treatment.
MATERIALS AND METHODS
HPV Detection
Study Patients From April 2004 to April 2013, 278 patients with head and neck cancer who were diagnosed and
P16 immunohistochemistry. Immunostaining for p16 was performed on representative 4-mm sections cut from FFPE tissue blocks obtained before the
TABLE I. Distribution of Patients According to TNM Stage (n ¼ 256) No. Patients (%) Tumor classification T1 T2 T3 T4 Total
N0
N1
N2a
N2b
N2c
N3
46 62 22 8 138 (53.9)
5 9 7 4 25 (9.8)
5 11 9 3 28 (10.9)
6 15 9 11 41 (16.0)
0 3 6 7 16 (6.3)
0 1 2 5 8 (3.1)
J. Med. Virol. DOI 10.1002/jmv
Total 62 101 55 38 256
(24.2) (39.5) (21.5) (14.8) (100)
HPV in Head and Neck Cancer
patient underwent chemotherapy or radiotherapy. P16 immunohistochemistry was performed using a proprietary kit (CINtec1 p16 Histology; Roche, mtm laboratories, Heidelberg, Germany) on a Roche Autostainer (Benchmark XT; Roche, Switzerland). Laryngeal cancer with high p16 expression was used as a positive control. The primary antibody was omitted from the negative controls. P16 immunohistochemistry was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of the malignant cells [Schache et al., 2011]. All other staining patterns were scored as negative. All samples were scored independently by two senior head and neck pathologists. DNA Extraction and Assessment of Sample Adequacy. Sectioning was performed using the appropriate precautions to prevent inter-block DNA contamination. DNA was extracted from 2- 10-mm sections of FFPE biopsies using the Qiagen FFPE Kit (Hilden, Germany) according to the manufacturer’s instructions. A NanoDrop was used to measure the concentration and purity of DNA. The resulting DNA preparations were stored at 20˚C. HPV GenoArray and Genotyping Assay. The GenoArray test by HybriBio (Guangzhou, China) is an L1 consensus primer-based PCR assay capable of amplifying 21 HPV genotypes, including 13 high risk (HR) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), two probable HR types (53 and 66), and six low risk (LR) and unknown-risk types (6, 11, 42, 43, 44, and CP8304 [HPV-81]). The assay was performed according to the manufacturer’s protocol [Liu et al., 2010]. Tumors were classified as HPV-positive if they contained HPV DNA (according to HPV genotyping) and over-expressed p16 (according to immunohistochemistry) [Marur et al., 2010]. Statistical Analysis. Categorical variables were compared using the chi-square test and Fisher’s exact test. Differences were considered significant at P < 0.05 in two-tailed tests. Overall survival (OS) analysis was determined as the time from the end of treatment to death; survivors were censored at their last follow-up. The Kaplan–Meier method was used for survival analysis. Data were analyzed using SAS 9.3. RESULTS Clinical Characteristics and HPV Prevalence Table II provides data on HPV from the pretreatment specimens as well as clinical data from 256 patients with head and neck cancer. Immunostaining for p16 was found in 6.6% (17/256) of patients, whereas HPV infection was detected in 9 of 256 (3.5%) patients by HPV genotyping. None of the p16negative subjects were found to have HPV infection via HPV genotyping. Eight cases were p16-positive/ HPV-negative.
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P16 positivity was more common among nonsmokers than smokers (16.3% vs. 4.7%, respectively; P ¼ 0.02) (Table II). Of the 256 specimens analyzed, the prevalence of HPV was 6.5% (5/77) in nasopharyngeal cancer, 5.0% (1/20) in hypopharyngeal cancer, and 1.9% (3/159) in laryngeal cancer specimens. Among eight p16-positive/HPV-negative cases, seven were laryngeal cancer and one was nasopharyngeal cancer. There was no significant correlation between positive HPV status and tumor localization (P ¼ 0.22). Prevalence of Different HPV Genotypes in HPVPositive Patients A total of three HPV genotypes were found among the nine HPV-positive cases. Seven patients were infected with HPV-16 (five with nasopharyngeal cancer, one with laryngeal cancer, and one with hypopharyngeal cancer). One patient with laryngeal cancer was infected with HPV-33 and one patient with laryngeal cancer was infected with both HPV-16 and HPV-11. HPV Status and Patient Survival For survival analysis, the mean follow-up was 35.3 months (range, 2–113 months). For the surviving patients (n ¼ 195), the mean follow-up was 35.9 months (range, 6–113 months), and for the nonsurviving patients (n ¼ 61), the mean follow-up was 33.5 months (range, 2–80 months). The 3-year overall survival rate was 77.4% (Fig. 1). Figure 2 shows 3-year overall survival rates of 87.5% for p16-positive/HPV-positive cases (n ¼ 9), 75.0% for p16-positive/HPV-negative cases (n ¼ 8), and 76.9% for p16-negative/HPV-negative cases (n ¼ 239) (P ¼ 0.30). Figure 3 shows that the 3-year overall survival rate was 83.9% for p16-positive cases (n ¼ 17) and 76.9% for p16-negative cases (n ¼ 239) (P ¼ 0.13). DISCUSSION In the current study, the sensitivity of p16 immunostaining was higher than that of the genoarray test (6.6% vs. 3.5%, respectively) for detecting HPV infection. Using E6 and E7 mRNA levels as conclusive evidence of HPV involvement, Smeets et al., 2007 found that p16 immunostaining in head and neck cancer has a 100% sensitivity and 79% specificity as a surrogate marker of HPV infection. With regard to sensitivity, p16 immunostaining is a good first-line assay for elimination of HPV-negative cases from any further analysis. Since the p16 assay is not specific for HPV-16, the higher rate of positivity that observed with this assay likely indicates detection of HPV genotypes beyond the 21 tested here. Additionally, p16 overexpression may indicate pRB pathway disturbances unrelated to HPV (e.g., mutational inactivation of pRB). J. Med. Virol. DOI 10.1002/jmv
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Xu et al. TABLE II. Clinical Parameters of Patients With Head and Neck Cancer No. Patients (%)
Characteristic Gender Males Females Age, median [range], years Tobacco Nonsmokers Smokers Alcohol Nondrinkers Drinkers Tumor localization Nasopharynx Larynx Hypopharynx Clinical tumor classification cT1 cT2 cT3 cT4 Clinical lymph node classification cN0 cN1 cN2 cN3 Clinical stage I II III IV
p16þ/Genotypingþ
p16þ/Genotyping-
p16-/Genotyping-
P
8 (3.5) 1 (3.7) 62.6 [51–71]
7 (3.1) 1 (3.7) 64.3 [46–75]
214 (93.5) 25 (92.6) 60.3 [21–84]
0.84
4 (9.3) 5 (2.3)
3 (7.0) 5 (2.3)
36 (83.7) 203 (95.3)
0.02
6 (4.7) 3 (2.4)
3 (2.3) 5 (3.9)
120 (93.0) 119 (93.7)
0.55
5 (6.5) 3 (1.9) 1 (5.0)
1 (1.3) 7 (4.4) 0 (0)
71 (92.2) 149 (93.7) 19 (95)
0.22
2 5 2 0
(3.2) (5.0) (3.6) (0)
2 2 3 1
(3.2) (2.0) (5.5) (2.6)
58 94 50 37
(93.6) (93.1) (90.9) (97.4)
0.79
4 3 2 0
(2.9) (12.0) (2.4) (0)
4 0 3 1
(2.9) (0) (3.5) (12.5)
130 22 80 7
(94.2) (88.0) (94.1) (87.5)
0.21
2 4 3 0
(1.8) (6.2) (7.3) (0)
2 3 2 1
(1.8) (4.6) (4.9) (2.6)
108 58 36 37
(96.4) (89.2) (87.8) (97.4)
0.22
Fig. 1. Kaplan–Meier survival curves for overall survival.
J. Med. Virol. DOI 10.1002/jmv
0.34
Fig. 2. Kaplan–Meier curves for overall survival rate by HPV status. The survival rates are shown for p16þ/HPVþ, p16þ/ HPV-, and p16-/HPV- patients (P ¼ 0.30).
HPV in Head and Neck Cancer
Figure 3. Kaplan–Meier curves for the overall survival rate according to p16 status. The survival rates for p16þ and p16patients are shown (P ¼ 0.13).
Since the sensitivity of p16 immunohistochemistry was almost 100%, a positive finding by HPV genotyping reduces the number of false-positive cases detected by p16 staining alone. Therefore, immunohistochemical analysis of p16 assay is a useful first assay to eliminate HPV-negative head and neck cancer, and HPV genotyping is a useful second assay to identify p16-positive cases. This strategy will help to reduce the false-positive detection rate of HPV-positive head and neck cancers. Variability in HPV positivity rates in head and neck cancer, excluding the oropharynx, has ranged from 0% to 86% across published surveys [Hobbs et al., 2006; Ribeiro et al., 2011; Li et al., 2013]. HPV-16 should overwhelmingly be the most common papillomavirus found in head and neck cancer; however, the present study suggests that the levels of HPV-16 circulating in the study population may be relatively low. Indirect evidence supporting this presumption includes the lower incidence of cervical cancer (for which HPV-16 is the major risk factor) reported in Eastern Asia compared with Central America (9.6 vs. 22.2 per 100,000 in 2008) [Jemal et al., 2011]. Li et al., 2003 found that none of the 16 tonsil cancer specimens from Chinese patients were HPV DNA-positive, whereas those from Australia had a positivity rate of 46% using the same test method. Therefore, our findings imply that the involvement of HPV in head and neck cancer may vary with ethnicity.
5
Traditionally, tobacco smoking and excessive alcohol consumption have been considered the main risk factors for head and neck cancer. Individuals with HPV-positive head and neck cancer tend to be white, male, nonsmokers, and nondrinkers when compared with those with HPV-negative tumors [Gillison et al., 2008]. In the present study, most of the HPVpositive cases were also nonsmokers (P ¼ 0.02). The percentage of nonsmokers among subjects with head and neck cancer has varied across different published studies: 16.8% in the current study, 29.5% in Saito’s study [Saito et al., 2013], and 44% reported by D’Souza et al. [D’Souza et al., 2007]. One interpretation of these findings is that the proportion of HPVpositive head and neck cancers is smaller in countries where the burden of the classical risk factor, tobacco, is high. Two recent large-scale exome sequencing projects in head and neck cancer [Agrawal et al., 2011; Stransky et al., 2011] revealed that HPV-negative tumors accumulate at least two times as many mutations as HPV-positive tumors. This may indicate that carcinogenesis in HPV-negative head and neck cancer is a result of the acquisition of a large number of mutations in many different signaling pathways. In contrast, carcinogenesis in HPV-positive tumors is modulated by the activities of E6/E7 viral oncoproteins. Leemans et al., 2011 considered that the survival benefit observed in HPV-associated head and neck cancer may not be the result of HPV positivity per se, but rather the absence of TP53 gene mutations or CDKN2A deletion in HPV-positive tumors, which are responsible for poor prognoses in HPVnegative patients. An association between HPV-positive, p16-positive oropharyngeal tumors and survival outcomes was reported by another retrospective analysis in a large phase 3 trial of chemoradiation, which concluded that oropharyngeal squamous cell cancer patients with HPV-positive/p16-positive tumors had better prognoses than those with HPV-negative/p16negative cancers [Rischin et al., 2009]. Ragin and Taioli, [2007] showed that there was no difference in overall survival between HPV-positive and -negative head and neck cancer, excluding the oropharynx patients. No data were obtained regarding overall survival in p16-positive/HPV-negative head and neck cancer, excluding the oropharynx patients. In the current study, there was no significant difference in overall survival among patients with p16-positive/ HPV-positive, p16-positive/HPV-negative, and p16negative/HPV-negative head and neck cancers, excluding the oropharynx. The HPV infection rate in the study patients with head and neck cancer, excluding the oropharynx, was 3.5%, suggesting that HPV was not a major causal factor of head and neck cancer in these patients. HPV infection did not affect the overall survival in this study.
J. Med. Virol. DOI 10.1002/jmv
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O’Rorke MA, Ellison MV, Murray LJ, Moran M, James J, Anderson LA. 2012. Human papillomavirus related head and neck cancer survival: A systematic review and meta-analysis. Oral Oncol 48:1191–1201. Ragin CC, Taioli E. 2007. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection: Review and meta-analysis. Int J Cancer 121:1813– 1820. Ribeiro KB, Levi JE, Pawlita M, Koifman S, Matos E, Eluf-Neto J, Wunsch-Filho V, Curado MP, Shangina O, Zaridze D, SzeszeniaDabrowska N, Lissowska J, Daudt A, Menezes A, Bencko V, Mates D, Fernandez L, Fabianova E, Gheit T, Tommasino M, Boffetta P, Brennan P, Waterboer T. 2011. Low human papillomavirus prevalence in head and neck cancer: Results from two large case-control studies in high-incidence regions. Int J Epidemiol 40:489–502. Rischin D, Young RJ, Fisher R, Fox SB, Le QT, Peters LJ, Solomon B, Choi J, O’Sullivan B, Kenny LM, McArthur GA. 2009. Prognostic significance of HPV and p16 status in patients with oropharyngeal cancer treated on a large international phase III trial. 2009 ASCO annual meeting; Orlando, FL, USA; May 29– June2, 2009 Abstr 6004. Saito Y, Yoshida M, Ushiku T, Omura G, Ebihara Y, Shimono T, Fukayama M, Yamasoba T, Asakage T. 2013. Prognostic value of p16 expression and alcohol consumption in Japanese patients with oropharyngeal squamous cell carcinoma. Cancer 119:2005–2011. Schache AG, Liloglou T, Risk JM, Filia A, Jones TM, Sheard J, Woolgar JA, Helliwell TR, Triantafyllou A, Robinson M, Sloan P, Harvey-Woodworth C, Sisson D, Shaw RJ. 2011. Evaluation of human papilloma virus diagnostic testing in oropharyngeal squamous cell carcinoma: Sensitivity, specificity, and prognostic discrimination. Clin Cancer Res 17:6262–6271. Siegel R, Naishadham D, Jemal A. 2012. Cancer statistics, 2012. Ca-Cancer J Clin 62:10–29. Smeets SJ, Hesselink AT, Speel EJ, Haesevoets A, Snijders PJ, Pawlita M, Meijer CJ, Braakhuis BJ, Leemans CR, Brakenhoff RH. 2007. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer 121:2465–2472. Snow AN, Laudadio J. 2010. Human papillomavirus detection in head and neck squamous cell carcinomas. Adv Anat Pathol 17: 394–403. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, Shefler E, Ramos AH, Stojanov P, Carter SL, Voet D, Cortes ML, Auclair D, Berger MF, Saksena G, Guiducci C, Onofrio RC, Parkin M, Romkes M, Weissfeld JL, Seethala RR, Wang L, Rangel-Escareno C, Fernandez-Lopez JC, Hidalgo-Miranda A, Melendez-Zajgla J, Winckler W, Ardlie K, Gabriel SB, Meyerson M, Lander ES, Getz G, Golub TR, Garraway LA, Grandis JR. 2011. The mutational landscape of head and neck squamous cell carcinoma. Science 333:1157–1160. Wei W, Shi Q, Guo F, Zhang BY, Chen C, Zhang NS, Dong XP. 2012. The distribution of human papillomavirus in tissues from patients with head and neck squamous cell carcinoma. Oncol Rep 28:1750–1756.
Low Prevalence of Human Papillomavirus in Head and Neck Squamous Cell Carcinoma in Chinese Patients Yanan Xu,1,2,3 Suru Liu,1,3 Hongliang Yi,1,3 Jiadong Wang,2,3 Yanli Luo,4 and Shankai Yin1,3* 1
Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China 2 Department of Head and Neck Surgery, Renji Hospital of Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Otolaryngological Institute of Shanghai Jiaotong University, Shanghai, China 4 Department of Pathology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
The aim of this study was to ascertain the prevalence and prognostic value of human papillomavirus (HPV) infection status in head and neck squamous cell carcinoma. Immunohistochemistry and GenoArray HPV genotyping assays were used to evaluate the HPV infection status of 256 Chinese patients with head and neck squamous cell carcinoma. Long-term survival rates were calculated using the Kaplan– Meier method. Immunostaining for p16 was prevalent in 6.6% (17/256) of patients, whereas HPV infection was detected in 9 of 256 (3.5%) patients using the HPV genotyping test. None of the p16-negative subjects were found to have HPV infection according to the HPV genotyping test. P16 positivity was more common among nonsmokers than smokers (16.3% vs. 4.7%, respectively; P ¼ 0.02). Among nine HPV-positive cases, seven were infected with HPV-16, one with HPV-33, and one with both HPV-16 and HPV-11. The 3-year overall survival rate was 87.5% for p16-positive/HPV-positive patients, 75.0% for p16-positive/HPV-negative patients, and 76.9% for p16-negative/HPV-negative patients (P ¼ 0.30). A very low rate of HPV infection was found among the study patients. HPV infection status may not be a useful prognostic marker for head and neck cancer patients. J. Med. Virol. # 2014 Wiley Periodicals, Inc.
KEY WORDS:
head and neck squamous cell carcinoma; human papillomavirus; prevalence; prognosis; survival
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION Head and neck cancer is the sixth most common cancer worldwide [Siegel et al., 2012], and its treatment can result in cosmetic deformity and functional impairment of vital functions, including breathing, swallowing, speech, phonation, taste, hearing, and smell. Head and neck cancer derives from mucosa lining the oral cavity, oropharynx, nasopharynx, larynx, and hypopharynx. The most well-established risk factors for head and neck cancer are tobacco and alcohol abuse. High-risk human papillomavirus (HPV) infection, whose role in carcinogenesis of the uterine cervix has been extensively studied, is now an emerging risk factor for head and neck cancer [Bouvard et al., 2009; Leemans et al., 2011]. In the last decade, the incidence of oropharyngeal squamous cell cancer has increased sharply, in contrast to other head and neck cancers. Many attribute this to a rise in HPV-related oropharyngeal squamous cell cancer cases. Little has been mentioned about the association of HPV and head and neck cancer, excluding the oropharynx. Prevalence rates of HPV infection in head and neck cancer, excluding the oropharynx, are reported to be lower than those for oropharyngeal cancer [Li et al., 2013]. The reported prevalence of HPV DNA in patients with head and neck cancer, excluding the oropharynx, varies widely, ranging from 0% to 86% [Ribeiro et al., 2011; Li et al., 2013]. This disparity is
Correspondence to: Shankai Yin, MD PhD, Affiliated Sixth People’s Hospital of Shanghai Jiaotong University, 200233 Shanghai, China. E-mail: [email protected] Accepted 27 July 2014
DOI 10.1002/jmv.24052 Published online in Wiley Online Library (wileyonlinelibrary.com).
2
Xu et al.
primarily due to geographical differences among the studies conducted and the inadequate distinction of patients with oropharyngeal carcinoma from those with other cancers of the head and neck region. Additionally, differences in the analytical sensitivity and specificity of HPV genotyping methods, the limited spectrum of HPV types analyzed, and differences among HPV diagnostic criteria [D’Souza et al., 2007; Wei et al., 2012] have all contributed to inconsistent results regarding HPV infection in patients with head and neck cancer. Over 200 types of HPV have been identified. Similar to cervical, vaginal, and vulvar cancers, head and neck cancer is associated predominantly with high-risk HPV, particularly HPV-16 [Snow and Laudadio, 2010]. The International Agency for Research on Cancer reported in 2012 that HPV-16 causes cancer of the oropharynx. HPV-positive head and neck cancer seems to be a distinct clinical entity with a better prognosis than HPV-negative tumors [Liang et al., 2012], which may be attributable to enhanced sensitivity to treatment due to wild-type TP53, allowing an apoptotic response to radiation and chemoradiation in cancercells [Guihard et al., 2012]. Marur et al., 2010 emphasized that HPV-associated head and neck cancer is distinct, characterized by p53 degradation, retinoblastoma protein (pRb) pathway inactivation, and p16 upregulation while tobacco-associated head and neck cancers are characterized by TP53 mutation and downregulation of CDKN2A encoding p16, which are responsible for poor prognosis. The survival benefit was most predominant or restricted in patients with oropharyngeal cancer [O’Rorke et al., 2012], but limited data were obtained in those with head and neck cancer, excluding the oropharynx, particularly with regard to p16-positive/HPV-negative cases. The current study focused exclusively on head and neck cancer, excluding the oropharynx. The aim was to evaluate the HPV infection rate in study patients with head and neck cancer and assess its prognostic value.
treated at Shanghai Sixth People’s Hospital (Shanghai, China) were enrolled in this study. Among them, 22 patients were excluded because of poor extracted DNA quality. The clinical charts of the remaining 256 patients (229 men and 27 women; ages, 21–93 years; median age, 60.5 years) were reviewed. The anatomic locations of the tissue samples were as follows: nasopharynx (n ¼ 77), larynx (n ¼ 159), and hypopharynx (n ¼ 20). Data on clinical pathological characteristics were obtained from the electronic medical record system used in Shanghai Sixth People’s Hospital. Tumor Node Metastasis (TNM) staging was used to classify tumors in accordance with the American Joint Committee on Cancer classification system. Patient distribution according to TNM classification is provided in Table I. Note that no patients had distant metastasis. Smoking and drinking history was also available in the hospital’s electronic medical record system and thus, patients were classified as smokers/nonsmokers and drinkers/nondrinkers. One representative formalin-fixed, paraffin-embedded (FFPE) block was retrieved for each case. Histological diagnosis of head and neck cancer was confirmed by two independent pathologists. Approval for the study was obtained from the Research Ethics Committee of Shanghai Sixth People’s Hospital. Treatment Patients with nasopharyngeal cancer received radiotherapy, while those with cancers in other locations received surgery therapy. Postoperative radiotherapy followed surgery if a patient was T3 or N2 stage according to the pathologic diagnosis and/or clinical data. Additionally, patients with T4 or a positive incisal margin received cisplatin and Taxotere during postoperative radiotherapy. None of the additional postoperative treatments were performed in patients of N0, N1, T1, or T2 stage. None of the treatments were based on HPV status because these markers are not examined routinely before treatment.
MATERIALS AND METHODS
HPV Detection
Study Patients From April 2004 to April 2013, 278 patients with head and neck cancer who were diagnosed and
P16 immunohistochemistry. Immunostaining for p16 was performed on representative 4-mm sections cut from FFPE tissue blocks obtained before the
TABLE I. Distribution of Patients According to TNM Stage (n ¼ 256) No. Patients (%) Tumor classification T1 T2 T3 T4 Total
N0
N1
N2a
N2b
N2c
N3
46 62 22 8 138 (53.9)
5 9 7 4 25 (9.8)
5 11 9 3 28 (10.9)
6 15 9 11 41 (16.0)
0 3 6 7 16 (6.3)
0 1 2 5 8 (3.1)
J. Med. Virol. DOI 10.1002/jmv
Total 62 101 55 38 256
(24.2) (39.5) (21.5) (14.8) (100)
HPV in Head and Neck Cancer
patient underwent chemotherapy or radiotherapy. P16 immunohistochemistry was performed using a proprietary kit (CINtec1 p16 Histology; Roche, mtm laboratories, Heidelberg, Germany) on a Roche Autostainer (Benchmark XT; Roche, Switzerland). Laryngeal cancer with high p16 expression was used as a positive control. The primary antibody was omitted from the negative controls. P16 immunohistochemistry was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of the malignant cells [Schache et al., 2011]. All other staining patterns were scored as negative. All samples were scored independently by two senior head and neck pathologists. DNA Extraction and Assessment of Sample Adequacy. Sectioning was performed using the appropriate precautions to prevent inter-block DNA contamination. DNA was extracted from 2- 10-mm sections of FFPE biopsies using the Qiagen FFPE Kit (Hilden, Germany) according to the manufacturer’s instructions. A NanoDrop was used to measure the concentration and purity of DNA. The resulting DNA preparations were stored at 20˚C. HPV GenoArray and Genotyping Assay. The GenoArray test by HybriBio (Guangzhou, China) is an L1 consensus primer-based PCR assay capable of amplifying 21 HPV genotypes, including 13 high risk (HR) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), two probable HR types (53 and 66), and six low risk (LR) and unknown-risk types (6, 11, 42, 43, 44, and CP8304 [HPV-81]). The assay was performed according to the manufacturer’s protocol [Liu et al., 2010]. Tumors were classified as HPV-positive if they contained HPV DNA (according to HPV genotyping) and over-expressed p16 (according to immunohistochemistry) [Marur et al., 2010]. Statistical Analysis. Categorical variables were compared using the chi-square test and Fisher’s exact test. Differences were considered significant at P < 0.05 in two-tailed tests. Overall survival (OS) analysis was determined as the time from the end of treatment to death; survivors were censored at their last follow-up. The Kaplan–Meier method was used for survival analysis. Data were analyzed using SAS 9.3. RESULTS Clinical Characteristics and HPV Prevalence Table II provides data on HPV from the pretreatment specimens as well as clinical data from 256 patients with head and neck cancer. Immunostaining for p16 was found in 6.6% (17/256) of patients, whereas HPV infection was detected in 9 of 256 (3.5%) patients by HPV genotyping. None of the p16negative subjects were found to have HPV infection via HPV genotyping. Eight cases were p16-positive/ HPV-negative.
3
P16 positivity was more common among nonsmokers than smokers (16.3% vs. 4.7%, respectively; P ¼ 0.02) (Table II). Of the 256 specimens analyzed, the prevalence of HPV was 6.5% (5/77) in nasopharyngeal cancer, 5.0% (1/20) in hypopharyngeal cancer, and 1.9% (3/159) in laryngeal cancer specimens. Among eight p16-positive/HPV-negative cases, seven were laryngeal cancer and one was nasopharyngeal cancer. There was no significant correlation between positive HPV status and tumor localization (P ¼ 0.22). Prevalence of Different HPV Genotypes in HPVPositive Patients A total of three HPV genotypes were found among the nine HPV-positive cases. Seven patients were infected with HPV-16 (five with nasopharyngeal cancer, one with laryngeal cancer, and one with hypopharyngeal cancer). One patient with laryngeal cancer was infected with HPV-33 and one patient with laryngeal cancer was infected with both HPV-16 and HPV-11. HPV Status and Patient Survival For survival analysis, the mean follow-up was 35.3 months (range, 2–113 months). For the surviving patients (n ¼ 195), the mean follow-up was 35.9 months (range, 6–113 months), and for the nonsurviving patients (n ¼ 61), the mean follow-up was 33.5 months (range, 2–80 months). The 3-year overall survival rate was 77.4% (Fig. 1). Figure 2 shows 3-year overall survival rates of 87.5% for p16-positive/HPV-positive cases (n ¼ 9), 75.0% for p16-positive/HPV-negative cases (n ¼ 8), and 76.9% for p16-negative/HPV-negative cases (n ¼ 239) (P ¼ 0.30). Figure 3 shows that the 3-year overall survival rate was 83.9% for p16-positive cases (n ¼ 17) and 76.9% for p16-negative cases (n ¼ 239) (P ¼ 0.13). DISCUSSION In the current study, the sensitivity of p16 immunostaining was higher than that of the genoarray test (6.6% vs. 3.5%, respectively) for detecting HPV infection. Using E6 and E7 mRNA levels as conclusive evidence of HPV involvement, Smeets et al., 2007 found that p16 immunostaining in head and neck cancer has a 100% sensitivity and 79% specificity as a surrogate marker of HPV infection. With regard to sensitivity, p16 immunostaining is a good first-line assay for elimination of HPV-negative cases from any further analysis. Since the p16 assay is not specific for HPV-16, the higher rate of positivity that observed with this assay likely indicates detection of HPV genotypes beyond the 21 tested here. Additionally, p16 overexpression may indicate pRB pathway disturbances unrelated to HPV (e.g., mutational inactivation of pRB). J. Med. Virol. DOI 10.1002/jmv
4
Xu et al. TABLE II. Clinical Parameters of Patients With Head and Neck Cancer No. Patients (%)
Characteristic Gender Males Females Age, median [range], years Tobacco Nonsmokers Smokers Alcohol Nondrinkers Drinkers Tumor localization Nasopharynx Larynx Hypopharynx Clinical tumor classification cT1 cT2 cT3 cT4 Clinical lymph node classification cN0 cN1 cN2 cN3 Clinical stage I II III IV
p16þ/Genotypingþ
p16þ/Genotyping-
p16-/Genotyping-
P
8 (3.5) 1 (3.7) 62.6 [51–71]
7 (3.1) 1 (3.7) 64.3 [46–75]
214 (93.5) 25 (92.6) 60.3 [21–84]
0.84
4 (9.3) 5 (2.3)
3 (7.0) 5 (2.3)
36 (83.7) 203 (95.3)
0.02
6 (4.7) 3 (2.4)
3 (2.3) 5 (3.9)
120 (93.0) 119 (93.7)
0.55
5 (6.5) 3 (1.9) 1 (5.0)
1 (1.3) 7 (4.4) 0 (0)
71 (92.2) 149 (93.7) 19 (95)
0.22
2 5 2 0
(3.2) (5.0) (3.6) (0)
2 2 3 1
(3.2) (2.0) (5.5) (2.6)
58 94 50 37
(93.6) (93.1) (90.9) (97.4)
0.79
4 3 2 0
(2.9) (12.0) (2.4) (0)
4 0 3 1
(2.9) (0) (3.5) (12.5)
130 22 80 7
(94.2) (88.0) (94.1) (87.5)
0.21
2 4 3 0
(1.8) (6.2) (7.3) (0)
2 3 2 1
(1.8) (4.6) (4.9) (2.6)
108 58 36 37
(96.4) (89.2) (87.8) (97.4)
0.22
Fig. 1. Kaplan–Meier survival curves for overall survival.
J. Med. Virol. DOI 10.1002/jmv
0.34
Fig. 2. Kaplan–Meier curves for overall survival rate by HPV status. The survival rates are shown for p16þ/HPVþ, p16þ/ HPV-, and p16-/HPV- patients (P ¼ 0.30).
HPV in Head and Neck Cancer
Figure 3. Kaplan–Meier curves for the overall survival rate according to p16 status. The survival rates for p16þ and p16patients are shown (P ¼ 0.13).
Since the sensitivity of p16 immunohistochemistry was almost 100%, a positive finding by HPV genotyping reduces the number of false-positive cases detected by p16 staining alone. Therefore, immunohistochemical analysis of p16 assay is a useful first assay to eliminate HPV-negative head and neck cancer, and HPV genotyping is a useful second assay to identify p16-positive cases. This strategy will help to reduce the false-positive detection rate of HPV-positive head and neck cancers. Variability in HPV positivity rates in head and neck cancer, excluding the oropharynx, has ranged from 0% to 86% across published surveys [Hobbs et al., 2006; Ribeiro et al., 2011; Li et al., 2013]. HPV-16 should overwhelmingly be the most common papillomavirus found in head and neck cancer; however, the present study suggests that the levels of HPV-16 circulating in the study population may be relatively low. Indirect evidence supporting this presumption includes the lower incidence of cervical cancer (for which HPV-16 is the major risk factor) reported in Eastern Asia compared with Central America (9.6 vs. 22.2 per 100,000 in 2008) [Jemal et al., 2011]. Li et al., 2003 found that none of the 16 tonsil cancer specimens from Chinese patients were HPV DNA-positive, whereas those from Australia had a positivity rate of 46% using the same test method. Therefore, our findings imply that the involvement of HPV in head and neck cancer may vary with ethnicity.
5
Traditionally, tobacco smoking and excessive alcohol consumption have been considered the main risk factors for head and neck cancer. Individuals with HPV-positive head and neck cancer tend to be white, male, nonsmokers, and nondrinkers when compared with those with HPV-negative tumors [Gillison et al., 2008]. In the present study, most of the HPVpositive cases were also nonsmokers (P ¼ 0.02). The percentage of nonsmokers among subjects with head and neck cancer has varied across different published studies: 16.8% in the current study, 29.5% in Saito’s study [Saito et al., 2013], and 44% reported by D’Souza et al. [D’Souza et al., 2007]. One interpretation of these findings is that the proportion of HPVpositive head and neck cancers is smaller in countries where the burden of the classical risk factor, tobacco, is high. Two recent large-scale exome sequencing projects in head and neck cancer [Agrawal et al., 2011; Stransky et al., 2011] revealed that HPV-negative tumors accumulate at least two times as many mutations as HPV-positive tumors. This may indicate that carcinogenesis in HPV-negative head and neck cancer is a result of the acquisition of a large number of mutations in many different signaling pathways. In contrast, carcinogenesis in HPV-positive tumors is modulated by the activities of E6/E7 viral oncoproteins. Leemans et al., 2011 considered that the survival benefit observed in HPV-associated head and neck cancer may not be the result of HPV positivity per se, but rather the absence of TP53 gene mutations or CDKN2A deletion in HPV-positive tumors, which are responsible for poor prognoses in HPVnegative patients. An association between HPV-positive, p16-positive oropharyngeal tumors and survival outcomes was reported by another retrospective analysis in a large phase 3 trial of chemoradiation, which concluded that oropharyngeal squamous cell cancer patients with HPV-positive/p16-positive tumors had better prognoses than those with HPV-negative/p16negative cancers [Rischin et al., 2009]. Ragin and Taioli, [2007] showed that there was no difference in overall survival between HPV-positive and -negative head and neck cancer, excluding the oropharynx patients. No data were obtained regarding overall survival in p16-positive/HPV-negative head and neck cancer, excluding the oropharynx patients. In the current study, there was no significant difference in overall survival among patients with p16-positive/ HPV-positive, p16-positive/HPV-negative, and p16negative/HPV-negative head and neck cancers, excluding the oropharynx. The HPV infection rate in the study patients with head and neck cancer, excluding the oropharynx, was 3.5%, suggesting that HPV was not a major causal factor of head and neck cancer in these patients. HPV infection did not affect the overall survival in this study.
J. Med. Virol. DOI 10.1002/jmv
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Xu et al.
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