Detection of Human Papillomavirus in Cervical Lymph Nodes: a Highly Effective Strategy for Localizing Site of Tumor Origin

Vol. 9, 6469–6475, December 15, 2003 Clinical Cancer Research 6469

Detection of Human Papillomavirus in Cervical Lymph Nodes: A Highly Effective Strategy for Localizing Site of Tumor Origin

Shahnaz Begum,1 Maura L. Gillison,2 the oropharynx, either directly by ISH or indirectly by M. Ali Ansari-Lari,1 Keerti Shah,3 and immunohistochemistry for p16 overexpression. William H. Westra1,4 Departments of 1Pathology, 2Oncology, 3Molecular Microbiology and INTRODUCTION Immunology, and 4Otolaryngology-Head and Neck Surgery, The Nearly 40,000 patients in the United States (1) and over Johns Hopkins Medical Institutions, Baltimore, Maryland 500,000 patients worldwide (2) are diagnosed with head and neck squamous cell carcinoma (HNSCC) each year. Despite ABSTRACT vast improvements in the diagnostic and therapeutic armament over the past several decades, mortality rates have not changed Purpose: Patients with head and neck squamous cell significantly. Patients tend to present with advanced disease carcinoma (HNSCC) often come to clinical attention with a when opportunities for cure are not optimal. Indeed, most pa- neck mass due to metastatic spread to lymph nodes. Treat- tients already have metastatic spread to regional lymph nodes at ment is dictated by the subsequent determination of primary the time of presentation (3); 13% of patients present with a neck tumor site and stage. However, the primary site remains mass as the first and only clinical manifestation (4), and 3% to elusive in some patients even after an exhaustive examina- 9% of the primary tumors continue to elude detection even after tion. Human papillomavirus type 16 (HPV-16) is an impor- clinical, radiographic, endoscopic, and histopathologic evalua- tant etiologic agent for HNSCCs that arise within the oro- tion (5). The treatment of patients with occult primary HNSCCs pharynx but less so for tumors from nonoropharyngeal sites. is challenging and controversial. One option is to blanket the Detection of HPV-16 or a surrogate marker may be useful in mucosa judged most at risk with wide-field radiation, but such localizing tumor origin in patients who present with meta- prophylactic treatment is associated with the risk of xerstomia, static HNSCC. mucositis, and other complications of radiation. Experimental Design: We performed in situ hybridiza- Human papillomavirus (HPV), particularly the tumorigenic tion (ISH) for HPV-16 on lymph node metastases from 68 16 subtype, has been confirmed recently as a causative agent in patients with HNSCC. P16 immunohistochemistry was also the development of a subset of HNSCCs (6). In particular, performed because HPV-16 integration disrupts the retino- HPV-16 is present in the majority of tumors arising in the blastoma pathway and induces an overexpression of p16. oropharynx, but in only a small percentage of HNSCCs arising Results: HPV-16 was detected in 22 of the 68 (32%) in nonoropharyngeal sites (6–8). In HPV-positive oropharyn- cases by ISH. When stratified by site of origin, HPV-16 was geal carcinomas, transcription of the viral oncoprotein E7 is detected in 22 of 31 (71%) metastases from the oropharynx, known to functionally inactivate the Retinoblastoma (Rb) gene but in none of the 37 (0%) metastases from other sites (P < product, causing a perturbation of other key components of the 0.001; Fisher’s exact). P16 expression was associated with Rb pathway (7, 9–11). As one example, functional inactivation the presence of HPV-16 by ISH: 21 of 22 HPV-16 positive of Rb by E7 is known to induce an up-regulation of p16 tumors exhibited p16 expression, whereas only 4 of the 46 expression (12, 13), reaching levels that can be readily detected HPV-16-negative tumors were p16 positive (95% versus 9%; by routine immunohistochemistry (IHC). Although p16 IHC has P < 0.001; Fisher’s exact). P16 expression in the node shown promise as an ancillary diagnostic tool in the recognition metastases also correlated with site of tumor origin: 24 of 31 of HPV-induced neoplasia of the female genital tract (14–16), oropharyngeal tumors were p16 positive, whereas only 1 of potential applications to tumors of the upper aerodigestive tract 37 nonoropharyngeal tumors was p16 positive (77% versus have yet to be defined. The purpose of the present study was to 3%; P < 0.001; Fisher’s exact). determine whether detection of HPV, by direct in situ hybrid- Conclusions: For patients with metastatic HNSCC, de- ization (ISH) or by immunohistochemical detection of p16 as a tection of HPV-16 is a reliable way to establish origin from surrogate marker, is helpful in pinpointing the site of tumor origin in those patients with cervical lymph node metastases.

MATERIALS AND METHODS Received 4/30/03; revised 9/3/03; accepted 9/8/03. Patients. The surgical pathology files were searched for Grant support: NIH (1R01 DE13121-01A1). The costs of publication of this article were defrayed in part by the patients with HNSCC who underwent neck dissections for car- payment of page charges. This article must therefore be hereby marked cinoma metastatic to regional lymph nodes between 1995 and advertisement in accordance with 18 U.S.C. Section 1734 solely to 2002. Medical records were reviewed to document the primary indicate this fact. site of tumor origin. The primary site was defined by histopatho- Requests for reprints: William H. Westra, The Weinberg Building, Room 2242, The Johns Hopkins Hospital, 401 North Broadway, Balti- logic confirmation of a mass detected on physical examination more, MD 21231-2410. Phone: (410) 955-2163; Fax: (410) 955-0115; by a head and neck surgeon. To facilitate comparison between E-mail: [email protected]. those tumors arising in the oropharynx and those tumors arising

from neighboring anatomical sites, cases were selected for ad- and -89) and ␤-globin (19, 20). Positive controls consisting of ditional analysis solely on the basis of primary tumor origin. The 10 and 100 HPV-16 (SiHa)- or HPV-18 (C4–2)-positive cells group of study cases included 31 metastatic HNSCCs from the diluted in a background of HPV-negative cells (K562) and a oropharynx and 37 metastatic HNSCCs from nonoropharyngeal negative control (K562 cells) were run within each assay. Sam- sites. HPV status of the primary tumors and their metastatic ples positive for ␤-globin were considered of sufficient quality implants was not known at the time of case selection. for analysis. In-situ hybridization (ISH). HPV-16 detection in formalin- Statistical Evaluation. Primary tumor location was cat- fixed and paraffin-embedded tissues was performed using the egorized as a dichotomous variable (oropharynx or nonorophar- ISH catalyzed signal amplification method for biotinylated ynx). Factors associated with oropharyngeal tumor location probes (DAKO GenPoint, Carpinteria, CA; Ref. 17). This cat- were evaluated by cross-tabulations and analyzed by use of the alyzed signal amplification system permits visualization of sin- Fisher’s exact test. The sensitivity value of a test was defined as gle copies of HPV-16 in infected cells (18). Briefly, 5-␮m tissue the percentage of oropharyngeal tumors that tested positive for sections underwent deparaffinization, heat-induced target re- HPV-16 by ISH or positive for p16 by IHC. The specificity trieval in citrate buffer, and digestion using Proteinase K (Roche value was defined as the percentage of nonoropharyngeal tu- Diagnostics, Indianapolis, IN). Slides were subsequently hy- mors that tested negative for HPV-16 by ISH or negative for p16 bridized with a biotinylated HPV-16 type-specific probe by IHC. Exact binomial confidence intervals were calculated. (DAKO). Signal amplification was performed by consecutive The ␬-statistic was used to measure agreement between HPV-16 application of streptavidin-horseradish peroxidase complex, bi- ISH and HPV PCR. Ps are two-sided unless otherwise specified. otinyl tyramide, and streptavidin-horseradish peroxidase com- Statistical analysis was conducted using STATA software, Ver- plex. Visualization of positive hybridization signals was per- sion 7 (STATA, College Station, TX). formed by incubation with the chromogenic substrate diaminobenzidine. Interpretation of staining was performed without knowledge of p16 immunohistochemical staining or RESULTS tumor origin. Of the 68 cases of metastatic squamous cell carcinoma to Immunohistochemistry (IHC). Five-␮m sections were cervical lymph nodes, 31 primary tumors were from the deparaffinized. Antigen retrieval was performed using heat- oropharynx, 21 were from the oral cavity, 11 were from the induced epitope retrieval with 10 mM citrate buffer. Tissue larynx, and 5 were from the hypopharynx. The results of HPV sections were incubated with a mouse monoclonal antibody detection using various detection strategies are summarized against p16 (MTM Laboratories, Heidelberg, Germany) at a in Table 1. 1:500 dilution. The p16 antibody was visualized using the By ISH, HPV-16 was detected in 22 of 31 (71%) me- avidin-biotin-peroxidase technique (DAKO LSAB kit; DAKO tastases from the oropharynx. In all of the HPV-16 positive Cytomation, Carpinteria, CA). cases, hybridization was visualized as a punctate signals Staining was regarded as positive if it was strong and within the nuclei. The signal varied from one or two incon- diffuse (Ͼ80% of tumor cells), and it was regarded as negative spicuous dots to many confluent dots (Fig. 1). Others have if absent or focal. Only strong, diffuse staining of the cytoplasm shown that the punctate pattern of hybridization correlates and nuclei, and not focal or weak staining, is associated with the with viral DNA integration and that the number of nuclear presence of high-risk HPV in neoplasia of the female genital dots correlates with the copy number of HPV DNA (21). In tract (14–16). Immunohistochemical interpretation was per- striking contrast, HPV-16 was not detected in any of the 37 formed without knowledge of HPV status or tumor origin. metastases from nonoropharyngeal sites (71 versus 0%; P Ͻ HPV Detection and Type Specification. Five-␮m thick 0.001; Fisher’s exact). For metastatic squamous cell carcino- sections of formalin-fixed and paraffin-embedded tissues were mas to cervical lymph nodes, the sensitivity of a positive deparaffinized with xylene, washed with 100% ethanol, and HPV-16 ISH for a diagnosis of an oropharyngeal primary was digested in 180 ␮l of ATL buffer with 40 ␮l of proteinase K at 70.9% (95% confidence interval, 51.9– 85.8%). Conversely, 55°C overnight (DNEasy; Qiagen Corp., Valencia, CA). Pro- the specificity of a negative HPV-16 ISH for a diagnosis of teinase K was heat inactivated for 10 min at 95°C. DNA was a primary tumor from a nonoropharyngeal site was 100% purified by phenol-chloroform extraction and ethanol precipita- (one-sided 97.5% confidence interval, 91–100%). tion, and resuspended in 50 ␮l of water. Five ␮l of purified By IHC, p16 overexpression was noted in 24 of the 31 DNA were analyzed for HPV DNA by multiplex PCR targeted (77%) metastases from the oropharynx. In all of the positive to the conserved L1 region of the viral genome by use of cases, staining intensity was strong, present throughout the PGMY09/11 L1 primer pools. Coamplification of the ␤-globin cytoplasm and the nucleus, and widely distributed throughout gene was performed as a positive control for the presence of the tumor (Fig. 2). In 1 case (case 13), staining was weak and amplifiable DNA in the specimen. PCR products were dena- focally distributed. In the female genital tract, only strong and tured in 0.13 N NaOH, and hybridized to an immobilized HPV diffuse p16 staining is associated with high-risk HPV subtypes probe array using an extended reverse line-blot assay for HPV (14–16); therefore, we regarded focal and weak staining as genotyping (Roche Molecular Systems, Inc., Alameda, CA) of negative. P16 overexpression was noted in only 1 (case 38) of 37 HPV types classified as “high-risk” (HPV-16, -18, -26, -31, the 37 (3%) metastases from nonoropharyngeal sites (77 versus -33, -35, -39, -45, -51, -52, -55, -56, -58, -59, -68, -73, -82, and 3%; P Ͻ 0.001; Fisher’s exact). The sensitivity of a positive p16 -83) or of “low”-or“unknown’” -risk (HPV-6, -11, -40, -42, stain for a diagnosis of an oropharyngeal tumor was 77.4% -44, -53, -54, -57, -61, -62, -64, -66, -67, -69, -71, -72, -81, -84, (95% confidence interval, 58.9–90.4%). Conversely, the spec-

Table 1 Human papillomavirus (HPV) detection in lymph node metastases by in situ hybridization, surrogate p16 immunohistochemistry, and E-7 type-specific PCR Case Primary tumor site HPV-16 ISHa P16 IHC HPV L1 PCR 1 Oropharynx Negative Positive 82 2 Oropharynx Negative Positive Negative 3 Oropharynx Positive Positive 16 4 Oropharynx Positive Positive 16 5 Oropharynx Positive Positive 16 6 Oropharynx Positive Positive 16 7 Oropharynx Negative Negative Negative 8 Oropharynx Positive Negative Negative 9 Oropharynx Negative Negative Negative 10 Oropharynx Negative Negative Negative 11 Oropharynx Positive Positive 16 12 Oropharynx Positive Positive 16 13 Oropharynx Negative Negative Negative 14 Oropharynx Negative Negative Negative 15 Oropharynx Positive Positive 16 16 Oropharynx Positive Positive 16/73 17 Oropharynx Positive Positive 16 18 Oropharynx Negative Negative Negative 19 Oropharynx Positive Positive 16 20 Oropharynx Positive Positive 16 21 Oropharynx Positive Positive — 22 Oropharynx Negative Positive 16 23 Oropharynx Positive Positive 16/35 24 Oropharynx Positive Positive 16 25 Oropharynx Positive Positive 16 26 Oropharynx Positive Positive 16 27 Oropharynx Positive Positive 16 28 Oropharynx Positive Positive 16 29 Oropharynx Positive Positive 16 30 Oropharynx Positive Positive Negative 31 Oropharynx Positive Positive 16 32 Oral cavity Negative Negative Negative 33 Oral cavity Negative Negative Negative 34 Oral cavity Negative Negative Negative 35 Oral cavity Negative Negative Negative 36 Oral cavity Negative Negative Negative 37 Oral cavity Negative Negative Negative 38 Oral cavity Negative Positive Negative 39 Oral cavity Negative Negative Negative 40 Oral cavity Negative Negative Negative 41 Oral cavity Negative Negative Negative 42 Oral cavity Negative Negative Negative 43 Oral cavity Negative Negative Negative 44 Oral cavity Negative Negative Negative 45 Oral cavity Negative Negative Negative 46 Oral cavity Negative Negative Negative 47 Oral cavity Negative Negative 16 48 Oral cavity Negative Negative Negative 49 Oral cavity Negative Negative Negative 50 Oral cavity Negative Negative Negative 51 Oral cavity Negative Negative Negative 52 Oral cavity Negative Negative Negative 53 Larynx Negative Negative Negative 54 Larynx Negative Negative 73 55 Larynx Negative Negative Negative 56 Larynx Negative Negative Negative 57 Larynx Negative Negative Negative 58 Larynx Negative Negative Negative 59 Larynx Negative Negative Negative 60 Larynx Negative Negative Negative 61 Larynx Negative Negative Negative 62 Larynx Negative Negative Negative 63 Larynx Negative Negative Negative 64 Hypopharynx Negative Negative Negative 65 Hypopharynx Negative Negative Negative 66 Hypopharynx Negative Negative Negative 67 Hypopharynx Negative Negative Negative 68 Hypopharynx Negative Negative Negative a ISH, in situ hybridization; IHC, immunohistochemistry; —, not done.

Fig. 1 Human papillomavirus (HPV)-16 in situ hybridization in a metastatic squamous cell carcinoma from the oropharynx. The hybridization signal is seen as conspicuous nuclear dots consistent with viral DNA integration. The hybridization signal is present in the tumor cells but not in the surrounding lymphocytes.

ificity of a negative p16 stain for a diagnosis of a nonoropha- HPV-16-positive tumors exhibited p16 overexpression, whereas ryngeal tumor was 97.4% (95% confidence interval, only 1 of the 46 HPV-16-negative tumors were p16 positive 86.2–99.9%). (95.5 versus 2.2%; P Ͻ 0.001; Fisher’s exact). There was no The presence of p16 expression by IHC was strongly correlation between signal intensity by ISH and staining inten- associated with the presence of HPV-16 by ISH: 21 of 22 sity by IHC. In other words, p16 staining was uniformly strong

Fig. 2 P16 immunohistochemical staining in a metastatic squamous cell carcinoma from the oropharynx. The staining is strong and diffuse. This tumor was also positive for human papillomavirus 16 by in situ hybridization (inset).

Fig. 3 P16 immunohistochemical staining and human papil- loma 16 in situ hybridization in a metastatic squamous cell carcinoma from the oropharynx. P16 staining is strong and diffuse although the hybridization signal (arrows) is not conspicuous.

and diffuse even when the ISH pattern was consistent with low pattern of genetic alterations in the metastatic implant to a copy number (Fig. 3). specific region of the upper aerodigestive tract and, thus, infer To better understand the mechanisms of discordance in the origin of otherwise occult primary tumors. This matching of those p16-positive cases where HPV-16 was not detected by genetic fingerprints, however, does not preclude the need for ISH, lymph nodes were analyzed for the presence of HPV DNA extensive tissue sampling of the upper aerodigestive tract, and it by PCR targeted to the conserved L1 region of the HPV ge- necessitates sophisticated methodologies unavailable to most nome. Sixty-seven of 68 samples were ␤-globin positive by diagnostic laboratories. Other approaches have taken advantage PCR. HPV DNA was detected in 22 (33%) of 67 lymph nodes. of the fact that some HNSCCs are caused by certain oncogenic All of the HPV types detected were high-risk. HPV16 was found viruses that target specific regions of the upper aerodigestive in 21 lymph nodes, 2 of which were also positive for HPV-35 or tract. In effect, detection of a specific virus in the metastasis 73. One lymph node was positive for HPV-82, and 1 for HPV- implicates site of tumor origin. Most notably, detection of EBV 73. One of 6 p16-positive, HPV-16 ISH-negative cases was in a neck metastasis reliably points to tumor origin from the positive for high-risk HPV-82. The agreement between HPV-16 nasopharynx (23–27). Although this approach is readily feasible ␬ϭ Ͻ ISH and PCR for HPV-16 was high (95.5%; 0.89; P for diagnostic laboratories with ISH capabilities, application to 0.001). HPV DNA detection by PCR was strongly associated this point has been limited, as only a small subset of HNSCCs Ͻ with location of the primary tumor in the oropharynx (P has been linked to a tumorigenic virus. Ͻ 0.001; Fisher’s exact) and p16 expression by IHC (P 0.001; The potential role of viral probes as a tool to localize tumor Fisher’s exact). origin has been expanded recently with the recognition that the HPV is an important causative factor in the development of a DISCUSSION subset of HNSCCs (6). Reported prevalence rates for HPV in Recent progress in uncovering the molecular genetic mech- primary oropharyngeal range between 40% and 60% (6, 8, anisms underlying human cancers is translating into more ef- 28–31). The prevalence rate may even be higher in lymph node fective strategies for patient care including refinements in tumor metastases, as HPV-related tumors may have an enhanced pro- diagnosis. For patients presenting with metastatic squamous cell pensity for metastatic spread (8, 9). Indeed, we detected the carcinoma of the head and neck, a practical and reliable method presence of HPV-16 in 71% of metastatic oropharyngeal carci- for pinpointing the site of tumor origin could obviate the need nomas. Coupled with the absence of HPV-16 in lymph node for sophisticated imaging studies, define anatomical sites for metastases from other primary sites, HPV-16 ISH appears to be directed biopsy and surgical excision, and better demarcate a highly reliable method for discerning the site of origin for radiation fields. metastatic carcinomas from the oropharynx. To date, the overall impact of novel approaches for tumor Compared to PCR-based techniques, the ISH method is localization has been limited. Using a microsatellite-based ge- inexpensive and readily feasible for routine use by many diag- netic approach, Califano et al. (22) were able match a distinctive nostic laboratories. Moreover, direct visualization of viral tissue

distribution better substantiates HPV as a causal agent. By tumors who had undergone surgical resections of their meta- contrast, the highly sensitive PCR technique may detect HPV static implants. This same strategy could be readily adapted to DNA present in the sample that is not specific to tumor cell patients with metastatic HNSCCs during much earlier stages of nuclei and, therefore, not of any pathophysiological significance the diagnostic workup. HPV-16 ISH and p16 IHC can be easily (11). One potential limitation of HPV-16 ISH, however, is that applied to fine needle aspiration specimens (14, 21, 35, 36). In ϳ10% of oropharyngeal carcinomas are associated with high- those patients who present with neck masses, routine HPV risk subtypes of HPV other than HPV-16 (6, 29). Expansion of analysis coupled with traditional cytopathology could magnify the hybridization armament to provide broader coverage of the sum of useful information gleaned from a limited number of high-risk HPV subtypes could improve sensitivity, but only at aspirated tumor cells. the expense of expediency and cost containment. IHC has not been a suitable alternative. The unreliability of viral oncoprotein REFERENCES detection in formalin-fixed tissues has prompted a search for 1. Greenlee, R. T., Hill-Harmon, M. B., Murray, T., and Thun, M. surrogate markers of HPV infection that are more feasible for Cancer statistics, 2001. Ca-Cancer J. Clin., 51: 15–36, 2001. routine IHC. 2. Kim, E. S., Kies, M., and Herbst, R. S. Novel therapeutics for head Disruption of the Rb pathway is common in HNSCC. In the and neck cancer. Curr. Opin. Oncol., 14: 334–342, 2002. majority of HNSCCs, pathway disruption occurs via inactiva- 3. Sessions, R. B. and Picken C. A. Malignant cervical adenopathy. In: tion of the p16 gene, an inhibitor of cyclin-dependent kinase in C. W. Cummings, J. M. Fredrickson, L. A. Harker, C. J. Krause, M. A. Richardson, and D. E. Schuller (eds.), Otolaryngology-Head and Neck the Rb pathway. Indeed, loss of p16 function is the most Surgery, 3rd ed, pp. 1737–1757. New York: Mosby, 1998. common genetic alteration in HNSCC (32). Opportunely, p16 is 4. Martin, H. R. C. The diagnostic significance of a lump in the neck. overexpressed in tumors associated with high-risk HPV. The Postgrad. Med., 11: 491–500, 1952. viral oncoprotein E7 binds and functionally inactivates Rb, 5. de Braud, F., and al Sarraf, M. Diagnosis and management of inducing an increase in p16 (12, 13). Importantly, HPV-induced squamous cell carcinoma of unknown primary tumor site of the neck. overexpression of p16 reaches levels that are easily discerned by Semin. Oncol., 20: 273–278, 1993. routine IHC. Diffuse p16 staining has been correlated with 6. Gillison, M. L., Koch, W. M., Capone, R. B., Spafford, M., Westra, high-risk HPV in tumors of the female genital tract (14–16). In W. H., Wu, L., Zahurak, M. L., Daniel, R. W., Viglione, M., Symer, D. E., Shah, K. V., and Sidransky, D. Evidence for a causal association the upper respiratory tract, Klussmann et al. (33) likewise re- between human papillomavirus and a subset of head and neck cancers. ported a high correlation between p16 IHC staining and the J. Natl. Cancer Inst., 92: 709–720, 2000. presence of HPV-16 for tonsillar carcinomas. 7. Helbig, M., Andl, T., Kahn, T., Klein-Kuhne, W., Conradt, C., Dietz, We found that overexpression of p16 of cervical lymph A., and Bosch, F. X. The role of oncogenic human papillomaviruses in node metastases serves as a reliable surrogate marker of HPV tonsillar squamous cell carcinomas with functional inactivation of the retinoblastoma protein. HNO, 47: 796–803, 1999. infection. Twenty-one of 22 (95%) HPV-16-positive tumors 8. Paz, I. B., Cook, N., Odom-Maryon, T., Xie, Y., and Wilczynski, exhibited p16 overexpression. Moreover, p16 overexpression S. P. Human papillomavirus (HPV) in head and neck cancer. An was noted in 9% of the tumors where a hybridization signal was association of HPV 16 with squamous cell carcinoma of Waldeyer’s not apparent using HPV-16 ISH. Using a PCR-based detection tonsillar ring. Cancer (Phila.), 79: 595–604, 1997. method in these discordant p16-positive/HPV-16-negative tu- 9. Andl, T., Kahn, T., Pfuhl, A., Nicola, T., Erber, R., Conradt, C., mors, we were able to confirm the presence of HPV-16 or some Klein, W., Helbig, M., Dietz, A., Weidauer, H., and Bosch, F. X. other high-risk HPV subtype in half of these cases. Thus, the Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control. ability to register sizeable increases in p16 expression, regard- Cancer Res., 58: 5–13, 1998. less of HPV-16 copy number or high-risk HPV subtype, appears 10. Wiest, T., Schwarz, E., Enders, C., Flechtenmacher, C., and Bosch, to be the near equivalent of actual HPV detection. F. X. Involvement of intact HPV16 E6/E7 gene expression in head and As a discriminator of a biologically distinct tumor entity, neck cancers with unaltered p53 status and perturbed pRb cell cycle p16 IHC could have various practical applications in the clinical control. Oncogene, 21: 1510–1517, 2002. arena. HPV detection in HNSCC has been correlated with 11. van Houten, V. M. M., Snijders, P. J. F., van den Brekel, M. W. M., Kummer, J. A., Meijer, C. J. L. M., van Leeuwen, B., Denkers, F., improved survival (6, 29, 33) such that p16 IHC may serve as a Smeele, L. E., Snow, G. B., and Brakenhoff, R. H. Biological evidence useful marker of prognoses (33). Patients with HPV-induced that human papillomaviruses are etiologically involved in a subgroup of HNSCC may benefit from HPV-targeted treatment strategies head and neck squamous cell carcinomas. Int. J. Cancer, 93: 232–235, (e.g., therapeutic HPV vaccines; Ref. 34) such that p16 IHC 2001. may help tailor therapy. In the present study, we addressed 12. Khleif, S. N., DeGregori, J., Yee, C. L., Otterson, G. A., Kaye, F. J., whether p16 IHC could help pinpoint the site of tumor origin for Nevins, J. R., and Howley, P. M. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase patients with metastatic HNSCC. In this regard, p16 overex- inhibitor activity. Proc. Natl. Acad. Sci. USA, 93: 4350–4354, 1996. pression in lymph node metastases strongly correlated with 13. Serrano, M., Hannon, G. J., and Beach, D. A new regulatory motif tumor origin from the oropharynx. Strong p16 staining was in cell-cycle control causing specific inhibition of cyclin D/CDK4. present in 77% of metastases from the oropharynx, but in only Nature (Lond.), 366: 704–707, 1993. 3% of metastases from nonoropharyngeal tumors. 14. Negri, G., Egarter-Vigl, E., Kasal, A., Romano, F., Haitel, A., and In summary, HPV detection in cervical lymph nodes could Mian, C. p16INK4a Is a useful marker for the diagnosis of adenocar- cinoma of the cervix uteri and its precursors: an immunohistochemical help focus the search for and treatment of the primary tumor in study with immunocytochemical correlations. Am. J. Surg. Pathol., 27: patients who present with a lymph node metastases as the initial 187–193, 2003. manifestation of their tumor. To assess the feasibility of this 15. Klaes, R., Friedrich, T., Spitkovsky, D., Ridder, R., Rudy, W., strategy, we focused on patients with well-documented primary Petry, U., Dallenbach-Hellweg, G., Schmidt, D., and von Knebel, D. M.

Overexpression of p16(INK4A) as a specific marker for dysplastic and diagnosis of nasopharyngeal carcinoma. Head Neck, 17: 487–493, neoplastic epithelial cells of the cervix uteri. Int. J. Cancer, 92: 276– 1995. 284, 2001. 27. Lee, W. Y., Hsiao, J. R., Jin, Y. T., and Tsai, S. T. Epstein-Barr 16. Sano, T., Oyama, T., Kashiwabara, K., Fukuda, T., and Nakajima, virus detection in neck metastases by in-situ hybridization in fine-needle T. Expression status of p16 protein is associated with human papillo- aspiration cytologic studies: an aid for differentiating the primary site. mavirus oncogenic potential in cervical and genital lesions. Am. J. Head Neck, 22: 336–340, 2000. Pathol., 153: 1741–1748, 1998. 28. Klussmann, J. P., Weissenborn, S. J., Wieland, U., Dries, V., 17. Kerstens, H. M., Poddighe, P. J., and Hanselaar, A. G. A novel in Kolligs, J., Jungehuelsing, M., Eckel, H. E., Dienes, H. P., Pfister, H. J., situ hybridization signal amplification method based on the deposition and Fuchs, P. G. Prevalence, distribution, and viral load of human of biotinylated tyramine. J. Histochem. Cytochem., 43: 347–352, 1995. papillomavirus 16 DNA in tonsillar carcinomas. Cancer (Phila.), 92: 18. Huang, C. C., Qiu, J. T., Kashima, M. L., Kurman, R. J., and Wu, 2875–2884, 2001. T. C. Generation of type-specific probes for the detection of single-copy 29. Mellin, H., Dahlgren, L., Munck-Wikland, E., Lindholm, J., Rab- human papillomavirus by a novel in situ hybridization method. Mod. bani, H., Kalantari, M., and Dalianis, T. Human papillomavirus type 16 Pathol., 11: 971–977, 1998. is episomal and a high viral load may be correlated to better prognosis 19. Gravitt, P. E., Peyton, C. L., Apple, R. J., and Wheeler, C. M. in tonsillar cancer. Int. J. Cancer, 102: 152–158, 2002. Genotyping of 27 human papillomavirus types by using L1 consensus 30. Mork, J., Lie, A. K., Glattre, E., Hallmans, G., Jellum, E., Koskela, PCR products by a single-hybridization, reverse line blot detection P., Moller, B., Pukkala, E., Schiller, J. T., Youngman, L., Lehtinen, M., method. J. Clin. Microbiol., 36: 3020–3027, 1998. and Dillner, J. Human papillomavirus infection as a risk factor for 20. Peyton, C. L., Gravitt, P. E., Hunt, W. C., Hundley, R. S., Zhao, squamous-cell carcinoma of the head and neck. N. Engl. J. Med., 344: M. F., Apple, R. J., and Wheeler, C. M. Determinants of genital human 1125–1131, 2001. papillomavirus detection in a US population. J. Infect. Dis., 183: 1554– 31. Wilczynski, S. P., Lin, B. T., Xie, Y., and Paz, I. B. Detection of 1564, 2001. human papillomavirus DNA and oncoprotein overexpression are asso- 21. Samama, B., Plas-Roser, S., Schaeffer, C., Chateau, D., Fabre, M., ciated with distinct morphological patterns of tonsillar squamous cell and Boehm, N. HPV DNA detection by in situ hybridization with carcinoma. Am. J. Pathol., 152: 145–156, 1998. catalyzed signal amplification on thin-layer cervical smears. J. Histo- 32. Reed, A. L., Califano, J., Cairns, P., Westra, W. H., Jones, R. M., chem. Cytochem., 50: 1417–1420, 2002. Koch, W., Ahrendt, S. A., Eby, Y., Sewell, D., Nawroz, H., Bartek, J., 22. Califano, J., Westra, W. H., Koch, W., Meininger, G., Reed, A., and Sidransky, D. High frequency of p16 (CDKN2/MTS-1/INK4A) Yip, L., Boyle, J. O., Lonardo, F., and Sidransky, D. Unknown primary inactivation in head and neck squamous cell carcinoma. Cancer Res., head and neck squamous cell carcinoma: molecular identification of the 56: 3630–3633, 1994. site of origin. J. Natl. Cancer Inst., 91: 599–604, 1999. 33. Klussmann, J. P., Gultekin, E., Weissenborn, S. J., Wieland, U., 23. Nakao, K., Yuge, T., Mochiki, M., Nibu, K., and Sugasawa, M. Dries, V., Dienes, H. P., Eckel, H. E., Pfister, H. J., and Fuchs, P. G. Detection of epstein-barr virus in metastatic lymph nodes of patients Expression of p16 protein identifies a distinct entity of tonsillar carci- with nasopharyngeal carcinoma and a primary unknown carcinoma. nomas associated with human papillomavirus. Am. J. Pathol., 162: Arch. Otolaryngol. Head Neck Surg., 129: 338–340, 2003. 747–753, 2003. 24. Feinmesser, R., Miyazaki, I., Cheung, R., Freeman, J. L., Noyek, 34. McNeil, C. HPV in oropharyngeal cancers: new data inspire hope A. M., and Dosch, H. M. Diagnosis of nasopharyngeal carcinoma by for vaccines. J. Natl. Cancer Inst., 92: 680–681, 2000. DNA amplification of tissue obtained by fine-needle aspiration. N. Engl. 35. Murphy, N., Ring, M., Killalea, A. G., Uhlmann, V., O’Donovan, J. Med., 326: 17–21, 1992. M., Mulcahy, F., Turner, M., McGuinness, E., Griffin, M., Martin, C., 25. Chan, M. K., Huang, D. W., Ho, Y. H., and Lee, J. C. Detection of Sheils, O., and O’Leary, J. J. p16(INK4A) as a marker for cervical Epstein-Barr virus-associated antigen in fine needle aspiration smears dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep (TM) from cervical lymph nodes in the diagnosis of nasopharyngeal carci- smears. J. Clin. Pathol., 56: 56–63, 2003. noma. Acta Cytol., 33: 351–354, 1989. 36. Vocaturo, A., Marandino, F., Celata, E., Caffo, A., Moauro, M., 26. Macdonald, M. R., Freeman, J. L., Hui, M. F., Cheung, R. K., Vocaturo, G., and Donnorso, R. P. Colourimetric signal amplification of Warde, P., McIvor, N. P., Irish, J., and Dosch, H. M. Role of Epstein- in situ hybridization assay for human papillomavirus DNA detection in Barr virus in fine-needle aspirates of metastatic neck nodes in the cytological samples. J. Exp. Clin. Cancer Res., 21: 239–246, 2002.

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