The influence of human papillomavirus on nasopharyngeal carcinoma in Japan

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The influence of human papillomavirus on nasopharyngeal carcinoma in Japan Makoto Kano a, Satoru Kondo a,*, Naohiro Wakisaka a, Makiko Moriyama-Kita a, Yosuke Nakanishi a, Kazuhira Endo a, Shigeyuki Murono a, Hiroyuki Nakamura b, Tomokazu Yoshizaki a a

Division of Otolaryngology-Head and Neck Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8640, Japan b Department of Environmental and Preventive Medicine, Graduate School of Advanced Preventive Medical Sciences, Kanazawa University, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 April 2016 Accepted 27 July 2016 Available online xxx

Objective: Although Japan is a non-endemic area with nasopharyngeal carcinoma (NPC), the proportion of WHO type I NPC in Japan are different from that in non-endemic areas such as North America and Europe. Recently, it is said that not only Epstein–Barr virus (EBV) but also human papillomavirus (HPV) has an influence on NPC in non-endemic areas. The aim of this study is to clarify the influence of HPV on NPC in Japan. Methods: Paraffin-embedded tumor specimens were available for 59 patients with NPC diagnosed between 1996 and 2015. We detected the virus status by p16 immunohistochemistry, HPV PCR, and in situ hybridization for Epstein–Barr virus (EBV)-encoded RNA. Kaplan–Meier curves were used to compare the overall survival by viral status. Results: Among the 59 patients, 49 (83%) were EBV-positive/HPV-negative, 2 (3%) were EBVpositive/HPV-positive, and 8 (16%) were EBV-negative/HPV-negative. All HPV-positive NPCs were co-infected with EBV. There were no significant differences between the overall survival in the three groups (p = 0.111). Conclusion: In Japan, HPV was detected in a few patients with NPC, and we suggest that HPV has no influence on NPC carcinogenesis in this population. ß 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Epstein–Barr virus Human papillomaviruses Japan Nasopharyngeal carcinoma Non-endemic diseases

1. Introduction Nasopharyngeal carcinoma (NPC) is an endemic disease in Southeast Asia and southern China. In contrast, it is relatively uncommon worldwide [1]. Although NPC is a relatively rare Abbreviations: ACRT, alternating chemo-radiotherapy; CT, computed tomography; EBV, Epstein–Barr virus; HPV, human papillomavirus; NPC, nasopharyngeal carcinoma; OS, overall survival; pRb, hypophosphorylated retinoblastoma protein; SCC, squamous cell carcinoma. * Corresponding author. Fax: +81 76 234 4265. E-mail address: [email protected] (S. Kondo).

disease in Japan, with an incidence of 0.2–0.3 per 100,000 people, in Southern China, the incidence is 19.5 per 100,000 people [2]. NPC has a variety of contributing etiologies, including environmental factors, genetic susceptibility, and infection with Epstein–Barr virus (EBV) [3]. EBV, a ubiquitous human herpesvirus, is the major etiologic agent in non-keratinizing NPC (WHO type II/III). In contrast, keratinizing carcinomas (WHO type I) lack a consistent association with EBV, suggesting differences in the pathogenesis of NPC [4]. Among non-endemic countries, specifically Japan, the proportion of WHO type I NPC is low, and almost all NPCs are linked to EBV

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Please cite this article in press as: Kano M, et al. The influence of human papillomavirus on nasopharyngeal carcinoma in Japan. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.07.015

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[5]. In contrast, there are a certain number of WHO type I NPCs in which no relationship with EBV has been identified in other non-endemic countries. Many studies have indicated that the high-risk type of human papillomavirus (HPV) is etiologically linked to oropharyngeal squamous cell carcinoma (SCC). Furthermore, patients with HPV-positive oropharyngeal SCC have better prognoses than HPV-negative counterparts [6]. Considering the resemblances between the epithelium and lymphoid tissue of the oropharynx and nasopharynx, the following question arises: what is the role of HPV in NPC carcinogenesis? Some studies have found the presence of HPV in NPC. One study reported a high HPV infection rate in a Caucasian population in non-endemic areas, and another study focusing on patients from endemic areas, reported a smaller HPV infection rate [7– 12]. Japan was considered a non-endemic area in these studies, but like endemic areas, it had only a few of WHO type I NPC. Consequently, Japan has characteristics of both endemic and non-endemic areas. To the best of our knowledge, this is the first study on the influence of HPV on NPC in Japan. 2. Materials and methods 2.1. Patients This study included Japanese patients with primary NPC who were diagnosed at Kanazawa University hospital and Toyama Prefectural Central Hospital between January 1996 and January 2015. Paraffin-embedded tumor specimens were available for 59 of these patients. All patients underwent clinical examinations, such as nasopharyngoscopy and contrast-enhanced MRI or computed tomography (CT) of the head and neck, to determine the tumor, node, and metastasis (TNM) classification. In this study, TNM classification was re-evaluated according to the 1997 UICC classification. All tumor specimens were obtained from the nasopharynx by biopsy. On the basis of hematoxylin and eosin staining, these samples were classified according to the WHO classification. 2.2. HPV detection and identification We detected HPV by PCR and p16 immunohistochemistry. Isolation of DNA. Genomic DNA was extracted from tumor specimens using the QIAamp DNA Mini Kit (Qiagen, Netherlands). p16 immunohistochemistry. Primary NPC paraffin-embedded specimens were used for the immunohistochemical analysis of p16 expression. Three-micrometer-thick sections were prepared from each block of tissue embedded in paraffin. Deparaffinized sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity. The sections were incubated with a protein blocker (Dako, Glostrup, Denmark) for 20 min and incubated at 4 8C overnight with a p16 (JC8) antibody (Santa Cruz Biotechnology, U.S.A.) as the primary antibody. The sections were washed three times with PBS (pH 7.2). After washing with PBS, the

sections were exposed to EnVision+ secondary antibody (Dako) for 30 min. The reaction products were developed by immersing the sections in a 30 ,3-diamidobenzidine tetrahydrochloride solution. The sections were counterstained with hematoxylin. We determined that of all carcinoma cells with strong and diffuse nuclear and cytoplasmic staining, >70% were positive [13]. HPV PCR. Samples (genomic DNA was extracted from tumor specimens) were subjected to PCR reactions using general primer GP5+/GP6+ sets and the TaKaRa Ex Taq PCR kit (TaKaRa, Japan). The final 10 ml PCR mixture contained 1 ml sample, 2 ml PCR Master Mix, and 1 ml of each primer. Cycling profile was performed with these amplification settings: incubation for 1 min at 94 8C, 36 cycles of denaturation for 20 s at 94 8C and annealing and elongation for 30 s at 48 8C and 72 8C, respectively, and final extension for 5 min at 72 8C. HPV thirteen. HPV thirteen is used for the identification and typing of HPVs by Nihon Gene Research Laboratories Inc. This method detects and identifies 13 high-risk HPV subtypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) from genomic DNA. Of the 59 samples, 10 samples were detected as HPVpositive and false positive using PCR. Then, we used HPV thirteen; HPV thirteen revealed that only two samples were HPV-positive. We adopted HPV thirteen’s result because HPV thirteen was more reliable than HPV PCR alone. 2.3. EBV detection EBV status was assessed by in situ hybridization for EBVencoded RNA (EBER-ISH) in paraffin embedded tissues, using the PNA ISH Detection kit and Epstein–Barr Virus (EBER) PNA Probe/Fluorescein (Dako, Denmark) according to the manufacturer’s protocol. EBER-ISH has been established as the most sensitive and practical method for detecting EBV [14]. We determined that of all carcinoma cells with an intense dark-blue staining limited to the nucleus, >90% showed positive. 2.4. Treatment Of the 59 patients, 36 patients received radiotherapy with cisplatin-based concurrent chemotherapy. According to the change in treatment protocol from 2005, 18 patients received alternating cisplatin and 5-FU chemo-radiotherapy, as previously described [15]. Four patients received radiotherapy alone due to increased age (>70 years) or renal dysfunction. The accumulated dose of radiation to the nasopharynx was 70– 77 Gy in all cases, and the dose to the neck was 40–70 Gy. One patient refused treatment. 2.5. Statistical methods We excluded the one patient who refused treatment from the statistical analysis. In total, all 58 patients were linked to a mortality registry until December 25, 2015. The primary endpoint was death following NPC diagnosis. Overall survival (OS) was calculated using the duration from diagnosis to the

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date of death caused by NPC, date of censoring due to other causes of death, loss to follow-up, or end of the follow-up period (December 25, 2015). In addition, Fisher’s exact test was used to determine prognosis. The patients were classified depending on whether they were dead or alive during a period of 5 years postdiagnosis. Thus, the final study population included 42 patients, of which 25 were alive and 17 were dead. 3. Results 3.1. EBV status We evaluated the correlation between EBV and NPC by EBER-ISH. The expression of EBER was detected as dark brown staining (Fig. 1). Among the 59 specimens, 51 (86%) cases were categorized as EBER-positive, and 8 (14%) were categorized as EBERnegative (Table 1).

Fig. 1. Immunohistochemistry for p16 is shown. Brown staining indicates nuclear and cytoplasmic expression of p16 in patients with NPC. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 Characteristics of study cases according to viral status. Characteristics

All patients (n = 59)

EBV+/HPV (n = 49)

EBV+/HPV+ (n = 2)

EBV/HPV (n = 8)

p-value

Age Gender Male Female WHO type I II III EBER Positive Negative HPV-DNA Positive Negative p16 Positive Negative Tumor classification T1 T2 T3 T4 Nodal classification N0 N1 N2 N3 AJCC stage I II III IV Treatment RT CCRT ACRT Reject treatment

59.6  12.7

58.9  13.3

60.0  1.0

63.3  9.4

0.688

51 (86.4) 8 (13.6)

41 8

2 0

8 0

0.395

1 50 8

0 42 7

0 2 0

1 6 1

0.593

51 8

49 0

2 0

0 8

0

2 57

0 49

2 0

0 8

0

3 56

3 46

0 2

0 8

0.728

18 14 8 19

14 12 7 16

0 1 0 1

4 1 1 2

0.543

11 13 28 7

9 9 24 7

0 0 2 0

2 4 2 0

0.143

4 9 20 26

2 7 17 23

0 0 1 1

2 2 2 2

0.194

4 36 18 1

2 31 15 1

0 1 1 0

2 4 2 0

0.516

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Table 2 Relationship between viral status and prognosis. Status

EBV+/HPV (n = 35)

Five-year post-diagnosis prognosis Alive 20 Dead 15 57.1 Survival rate (%)

EBV+/HPV+ (n = 2)

EBV/HPV (n = 5)

1 1 50.0

4 1 80.00

3.4. Patients’ status

Fig. 2. In situ hybridization for EBER. Positive reaction is observed in almost all tumor cells. Blue staining indicates expression of EBER. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. p16 status We used p16 immunohistochemistry as a surrogate marker for HPV infection. We determined that of all carcinoma cells with staining, >70% were positive (Fig. 2). Among the 59 specimens, 3 (5%) cases were categorized as p16-positive, and 56 (95%) were p16-negative (Table 1).

Among the 59 patients, 51 (86%) had EBV-positive tumors, and 2 of these (3%) had EBV and HPV co-infection present in the tumor tissues; 8 (14%) patients had both EBV- and HPVnegative tumors. Of the two HPV-positive tumors, one case was HPV type 18, and the other contained types 16 and 18; however, there were three p16-overexpressing tumors in the 59 patients. p16-overexpressing tumors and HPV-positive tumors were mutually exclusive. Based on the result of the viral status, the samples were classified into three groups: EBV-positive/HPV-negative, EBVpositive/HPV-positive and EBV-negative/HPV-negative. There were no significant differences between the three groups in terms of TNM classification and the method of treatment (Table 1). 3.5. Survival analysis

3.3. HPV status We detected HPV using PCR. Three samples provided a clear band, but the other seven samples provided uncertain bands that may have been positive. Finally, HPV thirteen of these 10 samples was conducted, and only 2 samples were detected as HPV-positive; one case was HPV type 18, and the other was type 16 and 18 (Table 1).

The median follow-up time was 58 months. There were no significant differences between the OS in the three groups (p = 0.111; Fig. 3) due to the low number of medical cases. EBV-negative/HPV-negative patients with NPC tended to have a better OS rate than EBV-positive/HPV-negative patients with NPC. Furthermore, there were no significant differences in the three groups (p = 0.819; Table 2) using Fisher’s exact test (Table 2). 4. Discussion

Fig. 3. Overall survival by viral status (EBV-positive/HPV-negative, EBVpositive/HPV-positive, and EBV-negative/HPV-negative).

The current study investigated the relationship between EBV and HPV in NPC. According to previous studies in nonendemic areas, HPV was detected in 11 of 67 (16.4%) and 18 of 61 (30%) cases [8,9]. In contrast, HPV was detected at a lower rate [2 of 70 (3%) and 2 of 20 (10%)] in endemic areas [12,16]. As a general trend, in non-endemic areas, WHO type I NPCs exists to some extent, a proportion of HPV-positive NPC is relatively high, and EBV and HPV were mutually exclusive in NPC. In contrast, in endemic areas, WHO type II/III NPCs occurred in majority and strongly correlated with EBV infection. Then, a proportion of HPV-positive NPC is low, but cases in which EBV and HPV are co-infected in NPC sometimes occurred. The epidemiological features of NPC in Japan differed from those in the countries described above. Japan is not an endemic area of NPC, although EBV has been linked to most NPC cases. To the best of our knowledge, this is the first study to investigate

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the prevalence of HPV in NPC in Japan. Using PCR, our study revealed that only 2 of the 59 patients had HPV DNA, specifically described as HPV-18 and HPV-16/18 subtypes. Moreover, both HPV-positive cases were WHO type II and showed EBER positivity. Furthermore, there is no clinical difference between HPV-positive NPC and HPV-negative NPC. This result is similar to the statistics observed in endemic areas [12,16]. Although the incidence of HPV positivity in NPC is rare in Japan, there are several subsequent questions. First, how does HPV infect the nasopharynx? For latent infection, HPV, also known as a major etiologic agent for cervical cancer, has to infect epithelial stem cells that are located in the basal epithelial cell layer and have the ability to differentiate to multiple cell types. The squamocolumnar junction (SCJ) of the cervix is a transitional area between the squamous epithelium of the vagina and the columnar epithelium of the endocervix. This region is a suitable site for HPV to infect epithelial stem cells, because the basal epithelial cell layer is exposed in SCJ [17]. In the oropharynx, HPV infects epithelial stem cells within the tonsillar crypt epithelium of the palatine tonsils. Mucosal epithelium in the nasopharynx, which has small crypt epithelium similar to the oropharynx, is composed of the columnar epithelium which is typically observed in the respiratory tract and stratified squamous epithelium [18]. This transitional epithelium between the two different epithelia may function like a SCJ. It appears that HPV reaches the pharyngeal mucosa by the direct contact infection with the perineum or indirect contact infection through the saliva and infects the nasopharyngeal epithelial stem cells through a small crypt epithelium and transitional epithelium. Second, dose HPV positivity affects the development of NPC? In general, most HPVs following infection are excluded by the natural immune response in oropharynx. However, a few HPV survives and infects persistently with the assistance of two viral genes, E6 and E7. These genes are expressed in HPVpositive oropharyngeal carcinoma cells. Their gene products inactivate the major tumor suppressors p53 and pRb, respectively, and this leads to generate carcinoma [19]. In contrast, as there are no studies that have investigated the relationship between HPV and NPC carcinogenesis, the mechanism of carcinogenesis is still unknown. The lower number of HPV-positive patients with NPC in our study suggests that HPVs following infection might subsequently be excluded by not only the natural immune response but also EBV. This might be why oncogenic viruses such as EBV and HPV are mutually exclusive in non-endemic areas. It is possible that HPV which is not excluded by EBV affects the carcinogenesis of WHO type I NPCs. Although there were only a small number of HPV cases, this finding suggests that HPV is not etiologically relevant to NPC carcinogenesis in Japan. Further studies need to define the influence of HPV on NPC. Third, what is the discrepancy between the overexpression of p16 and HPV-positive NPC. p16 is a tumor suppressor gene that inhibits cyclin-dependent kinase 4A. In the presence of

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transcriptionally active HPV, hypophosphorylated retinoblastoma protein (pRb) binds to the HPV oncoprotein E7, enabling the transcriptional activator E2F to be constitutionally active while effectively stopping the negative feedback of free pRb on p16. Overexpression of p16 then occurs. Thus, the immunohistochemical detection of p16 is often used as a surrogate marker for HPV infection and as a marker for activity of viral oncoproteins in HPV-related oropharyngeal carcinoma [20]. Multiple genetic abnormalities were detected in NPC, particularly deletions in the p16 gene. The p16 gene is inactivated in 62–86% of NPC cases [21]. The above may explain the discrepancy between HPVpositive rates by PCR and p16 overexpression in our study; if a p16 gene deletion existed in NPC at the beginning of oncogenesis, p16 overexpression would not occur if HPV infection influenced NPC oncogenesis. Other studies investigating HPV infection in NPC have reported that almost all HPV-positivity by PCR corresponded to p16 overexpression in immunohistochemistry. Thus, p16 was used as a proof of HPV infection; however, we regarded only HPV DNA detection by PCR as a proof of HPV infection, with or without concomitant p16 overexpression. In Japan it is difficult to estimate the prognosis of HPVrelated NPC because of the small number of identified HPVpositive cases identified. Our study could not reveal this estimation, although Fisher’s exact test was used to estimate the prognosis. Additional research is necessary to reveal the influence of HPV on NPC in the future. In general, high-level EBER expression, namely EBV-positive NPC, correlated with a good prognosis in patients with NPC [22]; however, in our study, EBV-negative patients with NPC tended to have better OS than EBV-positive patients with NPC. Kaplan–Meier curves show that among 8 EBV-negative/HPV-negative patients with NPC, 4 patients who lived longer had a smaller tumor (3 patients with T1 tumor and 1 patient with T2 tumor) or little or no lymph node metastasis (2 patients with N0 and 2 patients with N1; Fig. 3). According to some studies reporting that primary tumor volume had an impact on prognosis [23,24], we suggest that small primary tumor volume is one of the reasons why EBV-negative patients with NPC had a good prognosis in our study. In summary, we propose that HPV is not etiologically linked to NPC in Japan. However, EBV is a major etiologic agent, particularly in non-keratinizing NPC (WHO type II/III). Conflict of interest The authors have no conflict of interest. Acknowledgement The authors would like to thank Enago (www.enago.jp) for the English language review. References [1] Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 2006;15:1765–77.

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