HIGH-RISK HUMAN PAPILLOMAVIRUS IN NASOPHARYNGEAL CARCINOMA

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1 ORIGINAL ARTICLE HIGH-RISK HUMAN PAPILLOMAVIRUS IN NASOPHARYNGEAL CARCINOMA Aatur D. Singhi, MD, PhD, 1 Joseph Califano, MD, 2 William H. Westra, MD 1,2 1 Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland. wwestra@jhmi.edu 2 Department of Otolaryngology Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland Accepted 11 November 2010 Published online 11 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: /hed Abstract: Background. Human papillomavirus (HPV), a cause of oropharyngeal carcinoma, has also been implicated as an etiologic agent in nasopharyngeal carcinomas. Methods. We performed p16 immunohistochemistry and in situ hybridization for Epstein Barr virus (EBV) and HPV on 45 carcinomas of the nasopharynx. Results. Thirty-four (76%) carcinomas were EBV-positive/ HPV-negative, 7 (16%) were EBV-negative/HPV-negative, and 4 (9%) were EBV-negative/HPV-positive. HPV was more likely to be detected in carcinomas from white patients than nonwhite patients (16% vs 0%; p ¼.03). Of the 3 patients with HPV-positive carcinomas and available staging information, all were found to have extension into the oropharynx. All HPV-positive carcinomas were p16 positive, but none of the HPV-negative carcinomas were p16 positive (p <.001). Conclusion. HPV can be detected in a subset of carcinomas involving the nasopharynx, but many of these may represent extension from an oropharyngeal primary. P16 immunohistochemistry is a reliable marker for separating EBV-related and HPV-related carcinomas of Waldheyer s ring. VC 2011 Wiley Periodicals, Inc. Head Neck 34: , 2012 Keywords: Epstein Barr virus; lymphoepithelioma; oropharyngeal carcinoma; head and neck squamous cell carcinoma; p16 immunohistochemistry Dr. Califano is the Director of Research of the Milton J. Dance Head and Neck Endowment. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies. Correspondence to: W. H. Westra VC 2011 Wiley Periodicals, Inc. Human papillomavirus (HPV), particularly the tumorigenic 16 type, has been confirmed as a causative agent in the development of a subset of head and neck squamous cell carcinomas. 1 3 Most of these HPV-positive carcinomas localize to the oropharynx, 1,4 7 but detection of HPV in carcinomas of the nasopharynx has, to some extent, undermined the concept that HPV targets highly specific anatomic subsites of the head and neck Indeed, the recent detection of HPV in 4 of 4 nonkeratinizing nasopharyngeal carcinomas from white patients has implicated HPV, not Epstein Barr virus (EBV), as the primary etiologic agent in this particular patient population. 13 The purpose of the present study was to further clarify the incidence and implications of HPV detection in carcinomas of the nasopharynx using direct in situ hybridization (ISH) and p16 immunohistochemistry. PATIENTS AND METHODS Cases. Study approval, including a waiver of written consent, was obtained from the Johns Hopkins Hospital Medical Institutions Internal Review Board. The surgical pathology files of the Johns Hopkins Hospital were reviewed for all nonkeratinizing nasopharyngeal carcinomas biopsied or resected between 1985 and The slides were reviewed to confirm the diagnosis, and a tumor block was selected for HPV analysis. The study cohort consisted of 45 cases. Medical records were reviewed to document patient and tumor parameters including age, sex, race, stage, site of tumor origin, and patterns of local tumor extension specifically, relationship of the tumor in the nasopharynx to the oropharynx. In Situ Hybridization. Formalin-fixed, paraffin-embedded tumor specimens were evaluated for high-risk HPV DNA using the protocol for the automated Ventana HR HPV III probe set (Ventana Medical Systems, Tucson, AZ). This DNA probe cocktail has affinity to HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. For any equivocal cases, the tumors were also evaluated for HPV-16 DNA using the ISH-catalyzed signal amplification method for biotinylated probes (Dako GenPoint, Carpinteria, CA). 14 A positive hybridization was defined as detection of punctate signals localized to tumor cell nuclei. HPVpositive controls included HPV-16 positive squamous cell carcinoma cell lines (SiHa and CaSki). The SiHa cell line is known to harbor 1 to 2 copies of integrated HPV-16 per cell, and the Caski cell line is known to harbor 60 to 600 copies of integrated HPV-16 per cell. An HPV-16 negative head and neck squamous cell carcinoma served as a negative control. ISH for EBV was performed on 4-lm deparaffinized sections using a digoxigenin labeled 30-mer Human Papillomavirus in Nasopharyngeal Carcinoma HEAD & NECK DOI /hed February

2 Table 1. Characteristics of study cases grouped by ethnicity. Patient/tumor characteristic White (n ¼ 20) Asian (n ¼ 11) African American (n ¼ 11) Middle Eastern (n ¼ 2) Hispanic (n ¼ 1) Sex Male 17 (85) 6 (55) 10 (91) 2 (100) 1 (100) Female 3 (15) 5 (45) 1 (9) 0 (0) 0 (0) Age <60 17 (85) 9 (82) 11 (100) 2 (100) 0 (0) 60 3 (15) 2 (18) 0 (0) 0 (0) 1 (100) Lymph node metastasis 17 (85) 9 (82) 10 (91) 1 (50) 1 (100) TNM staging I 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) II 5 (25) 4 (36) 3 (27) 2 (100) 0 (0) III 8 (40) 3 (27) 2 (18) 0 (0) 0 (0) IV 7 (35) 4 (36) 6 (55) 0 (0) 1 (100) IVA 4 (20) 2 (18) 3 (27) 0 (0) 0 (0) IVB 0 (0) 1 (9) 0 (0) 0 (0) 0 (0) IVC 3 (15) 1 (9) 3 (27) 0 (0) 1 (100) EBVþ/HPV 10 (50) 11 (100) 10 (91) 2 (100) 1 (100) EBV /HPVþ 4 (20) 0 (0) 0 (0) 0 (0) 0 (0) EBVþ/HPVþ 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) EBV /HPV 6 (30) 0 (0) 1 (9) 0 (0) 0 (0) P16 positive 4 (20) 0 (0) 0 (0) 0 (0) 0 (0) Abbreviations: EBV, Epstein Barr virus; HPV, human papillomavirus. oligonucleotide probe (EBER kit, Ventana Medical Systems) complimentary to small nuclear EBV encoded-rna1. A positive hybridization was defined as punctuate or diffuse signals in the nucleus of nearly all of the tumor cells. An undifferentiated nasopharyngeal carcinoma known to be EBV positive served as a positive control. P16 Immunohistochemistry. Formalin-fixed and paraffin-embedded tissues of 5-um thick sections were deparaffinized and subjected to antigen retrieval using 10 mm citrate buffer (92 C for 30 minutes). Tissue sections were incubated with a mouse monoclonal antibody against p16 (MTM Laboratories, Heidelberg, Germany) at a 1:500 dilution. The p16 antibody was visualized using the avidin-biotin-peroxidase technique (Dako LSAB Kit, Dako Cytomation). Staining was scored as positive if it was strong and diffuse (>70% of the tumor cells) or negative if absent or focal. For squamous cell carcinomas of the head and neck, only strong and diffuse staining of both the nuclei and cytoplasm, and not focal or weak staining, is regarded as positive. 7 Immunohistochemical interpretation was performed without knowledge of HPV or EBV status. Statistical Analysis. Test results (EBV and HPV ISH, p16 immunohistochemistry) were recorded as dichotomous variables (positive or negative) and analyzed by use of the Fisher exact test. All p values are 2-sided. RESULTS A total of 45 patients with nonkeratinizing nasopharyngeal carcinomas were identified. The clinical and pathologic features are summarized in Table 1. Patients ranged in age from 15 to 83 years (mean, 42 years; median, 44 years); and 36 (80%) were men. Twenty patients (44%) were white, 11 (24%) Asian, 11 (24%) were African American, 2 (4%) were Middle Eastern, and 1 (2%) was Hispanic. Fourteen patients (31%) had TNM classification II tumors, 13 (29%) had TNM classification III tumors, and 18 (40%) had TNM classification IV tumors. Overall, 34 of 45 carcinomas (76%) were EBV positive by ISH, 4 (9%) were HPV positive, and 7 (16%) were neither EBV nor HPV positive. None of the carcinomas harbored both EBV and HPV. The type and frequency of oncovirus varied among ethnic groups. EBV was more likely to be detected in carcinomas from nonwhite than from white patients (96% vs 50%; p <.001). Conversely, HPV was more likely to be detected in nasopharyngeal carcinomas from white than from nonwhite patients (16% vs 0%; p ¼.03). There was no statistically significant association between viral status and patient age, sex, tumor stage, or lymph node metastases. The 4 HPV-positive carcinomas were diagnosed after 2002 (2003, 2005, 2009, and 2010), but the number of HPV-positive cases was too small to discern statistically significant differences in incidence over time. All4ofthecarcinomasthatwereHPV-positivebyISH were p16-positive by immunohistochemistry (Figure 1), but none of the carcinomas that were negative for HPV were p16 positive (100% vs 0%; p <.001). Of the 4 carcinomas in the nasopharynx that were HPV positive, 3 had available detailed staging information to help discern the relationship of the tumor in the nasopharynx to the oropharynx. All 3 were found to have co-involvement of the oropharynx. Patient 1 presented with a necrotic neck mass that was diagnosed as metastatic squamous cell carcinoma by fine-needle aspiration. Positron emission tomography coupled with CT showed irregular uptake in an area of fullness involving 214 Human Papillomavirus in Nasopharyngeal Carcinoma HEAD & NECK DOI /hed February 2012

3 FIGURE 1. Human papillomavirus (HPV)-related nonkeratinizing carcinoma involving the nasopharynx. (A) The tumor infiltrates the subepithelial lymphoid stroma as nests of undifferentiated slightly spindled cells without evidence of keratinization. (B) The tumor cells are p16 positive by immunohistochemistry. The presence of dot-like hybridization signals within the tumor nuclei (arrow) indicates the presence of HPV-16 at low viral copy numbers (inset, HPV-16 in situ hybridization). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] the right fossa of Rosenmueller. By intraoperative palpation, there was an area of firmness in the right nasopharynx that extended in continuity to the right tonsillar fossa. Biopsies taken from both of these anatomic sites showed syncytial nests of poorly differentiated nonkeratinizing squamous cell carcinoma in a lymphoid background; and both specimens were HPV positive (Figure 2). Patient 2 presented with a sore throat, bilateral tinnitus, and difficulty breathing. Endoscopic examination revealed an exophytic lesion of the nasopharynx. The mucosa lining the rest of the upper aerodigestive tract, including the oropharynx, was unremarkable. The biopsy specimen of the nasopharyngeal lesion showed a poorly differentiated nonkeratinizing carcinoma. A staging positron emission tomography coupled with CT showed intense fluorodeoxyglucose uptake in the inferior right soft palate with extension into the right oropharynx, and focal uptake in the midline of the nasopharynx. The soft palate and right tonsillar mass was resected revealing syncytial nests of poorly differentiated nonkeratinizing squamous cell carcinoma in a lymphoid background. The carcinomas in the nasopharynx and oropharynx were both HPV positive. Patient 3 presented with otitis media. Physical examination revealed a 2-cm nasopharyngeal mass. The oropharynx was unremarkable. A CT scan showed a 3-cm mass centered in the left fossa of Rosenmueller with inferior extension into the upper pole of the right tonsil. A biopsy of the nasopharyngeal mass revealed syncytial nests of poorly differentiated nonkeratinizing carcinoma in a lymphoid background. The tonsil was not biopsied. Of the 41 patients with HPV-negative carcinomas, 34 (83%) had sufficient clinical and radiographic information to discern the relationship of the tumor in the nasopharynx to the oropharynx. Although oropharyngeal involvement was a consistent finding with the HPV-positive cases, this was not the case with the HPV-negative carcinomas. Only 4 of the HPV-negative carcinomas involved the oropharynx (100% vs 12%; p ¼.005). The strong association of oropharyngeal involvement and HPV positivity argues for origin from the oropharynx. DISCUSSION Although EBV is recognized as a major etiologic agent for nonkeratinizing carcinomas of the nasopharynx, Human Papillomavirus in Nasopharyngeal Carcinoma HEAD & NECK DOI /hed February

4 FIGURE 2. Simultaneous involvement of the nasopharynx and oropharynx by an human papillomavirus (HPV)-related carcinoma. The carcinoma in the nasopharynx (A) and oropharynx (C) seem histologically identical; and they both harbor high risk HPV by in situ hybridization (B) nasopharynx (D) oropharynx. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] EBV is not detected in all nasopharyngeal carcinomas. In particular, the constant association between EBV and nonkeratinizing nasopharyngeal carcinomas noted in Asian patients is not maintained in white patients. 13 Our analysis of a large number of nonkeratinizing nasopharyngeal carcinomas, including a sizeable number of white North American patients, confirmed a substantial variation in EBV detection as a function of ethnicity. EBV was detected in all 11 tumors from Asian patients but in only 10 of 20 tumors from white patients. The nonobligatory presence of EBV has suggested the presence of other causative agents working in cooperation with or jointly of EBV in tumorigenesis of the nasopharynx. High-risk HPV has emerged as a leading candidate. Several studies have detected HPV in nasopharyngeal carcinomas, either alone or as a co-infectant in EBVpositive tumors This association has been particularly strong for keratinizing nasopharyngeal carcinomas 15 ; but in their limited analysis of nasopharyngeal carcinomas from white patients, Maxwell et al 13 identified high-risk HPV in 4 of 4 carcinomas of the nonkeratinizing type. To date, those polymerase chain reaction (PCR)- based studies that have implicated a causative role for HPV in the development of nasopharyngeal carcinomas have not been confirmed by ISH methods of detection. Unlike PCR-based detection methods, ISH permits direct visualization of viral tissue distribution, allowing for distinction between viral infections that are biologically relevant from those that are not. Our HPV ISH assay has single viral-copy sensitivity and is strongly correlated with HPV E6 and E7 oncogene expression the gold standard for defining a tumor as HPV-associated. 16,17 A pattern of hybridization signals localized to the nuclei of a clonally expanded population of neoplastic cells supports a viral role that is causal rather than incidental. This ability to discern oncologically relevant HPV infection helps clarify certain unsettled issues raised by more ambiguous detection assays. First, EBV 216 Human Papillomavirus in Nasopharyngeal Carcinoma HEAD & NECK DOI /hed February 2012

5 and HPV infections seem to be mutually exclusive for carcinomas involving the nasopharynx. Eighty-four percent of the carcinomas harbored either EBV or HPV, but none harbored both. The absence of co-infection challenges the notion that the pathogenesis of nasopharyngeal carcinomas requires the combined effects of both EBV and HPV. 10,11,18 Second, the frequency and relevance of HPV infection in carcinomas of the nasopharynx may be overestimated by PCR-based detection methods. Although we were indeed able to detect HPV in a small subset of nasopharyngeal carcinomas using an ISH approach, we could not confirm the finding of others that HPV is present in the majority of nasopharyngeal carcinomas, 10,11 even among a highly selected group of white patients where the association with EBV is weak. 8,13 Third, many if not all HPV-related carcinomas involving the nasopharynx may actually represent oropharyngeal carcinomas secondarily involving the nasopharynx by direct extension. In all 3 of the HPV-positive carcinomas in which detailed staging information was available for review, the carcinomas in the nasopharynx had clinical, radiologic, and/or histopathologic evidence of synchronous involvement of the oropharynx. Certain features of HPV-positive oropharyngeal carcinomas may enhance their ability to masquerade as primary nasopharyngeal carcinomas. HPV-related oropharyngeal carcinomas tend to arise from deep within the tonsillar crypts in the absence of surface dysplasia 2,4 where they frequently propagate beneath the surface epithelium without inducing stromal desmoplasia or surface ulceration. As a result, inspection of the pharyngeal membranes often fails to recognize the full extent of tumor spread. Moreover, this pattern of subepithelial spread is anatomically unconstrained. The oropharynx and nasopharynx form part of a contiguous and noncompartmentalized wreath of lymphoepithelial tissues known as Waldheyer s ring, and the propensity for unrestricted migration along this lymphoid sheath from 1 site to the next has long frustrated attempts to discern precise tumor origin. 19 Indeed, clonal migration of neoplastic cells along this track of lymphoepithelial tissue has recently been proposed as a mechanism to account for tumor multifocality in patients with synchronous HPV-related squamous cell carcinomas of Waldheyer s ring including dual involvement of the oropharynx and nasopharynx. 20 Finally, HPV-positive oropharyngeal carcinomas resemble EBV-positive nasopharyngeal carcinomas at the histologic level. They are typically poorly differentiated and nonkeratinizing, and they even may have well developed lymphoepithelial features (eg, syncytial cytoplasm, prominent nucleoli, or a rich lymphoid stroma) that render them histologically indistinguishable from the undifferentiated subtype of nasopharyngeal carcinoma. 7,21 The site selectivity of HPV has provided a basis for using HPV detection as a means of tumor localization in patients presenting with lymph node metastases. In effect, the identification of HPV in a cervical lymph node metastasis has been used to implicate the oropharynx and exclude the nasopharynx and other non-oropharyngeal locations as the site of tumor origin in that troubling group of patients with occult primary cancers This rationale has been recently undermined by the detection of HPV in a sizable percentage of carcinomas of presumed nasopharyngeal origin. 13 Our findings suggest that when HPV-related carcinomas are discovered in a site adjacent to the oropharynx such as the nasopharynx, involvement likely represents secondary extension. Confirmation of the site specificity of HPV-driven tumorigenesis to the oropharynx helps restore confidence in HPV detection as a useful staging tactic. In HPV-positive carcinomas, transcription of the viral oncoprotein E7 functionally inactivates the retinoblastoma (Rb) gene product, causing a perturbation of key components of the Rb pathway including overexpression of the p16 tumor suppressor gene to levels that can be detected immunohistochemically Due to the strength of this association, p16 staining is commonly used to discern squamous cell carcinomas that are HPV-related from those that are not. As a discriminator of a biologically distinct tumor entity, p16 immunohistochemistry has taken on various practical applications in the clinical arena. HPV detection in squamous cell carcinoma of the head and neck has been correlated with improved survival such that p16 immunohistochemistry may serve as a useful marker of prognoses. 28 Patients with HPV-induced squamous cell carcinoma of the head and neck may benefit from HPV-targeted treatment strategies (eg, therapeutic HPV vaccines) such that p16 immunohistochemistry may help tailor therapy. 29,30 P16 immunohistochemistry is used to help pinpoint the site of tumor origin for patients with metastatic head and neck squamous cell carcinoma. 22 The discriminating power of simple p16 immunohistochemistry might also have a role in differentiating HPV-related from EBV-related carcinomas as both tumors can simultaneously involve the oropharynx and the nasopharynx, metastasize to cervical lymph nodes in the absence of a clinically apparent primary, and exhibit overlapping morphologic features. Toward this end, we found that p16 immunostaining is very reliable in making this distinction: p16 overexpression was noted in all 4 of the HPV-positive carcinomas but in none of the 34 EBV-positive carcinomas. HPV-related squamous cell carcinoma is now regarded as a distinct subtype of head and neck carcinoma. Its propensity to specifically target the unique microenvironment of the oropharynx represents 1 of its more biologically distinct and clinically relevant features. 2 Our study supports the site specificity of HPV-related carcinoma of the head and neck. Carcinomas of the nasopharynx do not commonly harbor HPV, and its detection at this site should direct attention to the oropharynx as a possible site of tumor origin. Human Papillomavirus in Nasopharyngeal Carcinoma HEAD & NECK DOI /hed February

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