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Clinical Cancer Research Vol. 9, 6469-6475, December 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Detection of Human Papillomavirus in Cervical Lymph Nodes

A Highly Effective Strategy for Localizing Site of Tumor Origin

Shahnaz Begum1, Maura L. Gillison2, M. Ali Ansari-Lari1, Keerti Shah3 and William H. Westra14

1 Departments of Pathology,
2 Oncology,
3 Molecular Microbiology and Immunology, and
4 Otolaryngology-Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) often come to clinical attention with a neck mass due to metastatic spread to lymph nodes. Treatment is dictated by the subsequent determination of primary tumor site and stage. However, the primary site remains elusive in some patients even after an exhaustive examination. Human papillomavirus type 16 (HPV-16) is an important etiologic agent for HNSCCs that arise within the oropharynx but less so for tumors from nonoropharyngeal sites. Detection of HPV-16 or a surrogate marker may be useful in localizing tumor origin in patients who present with metastatic HNSCC.

Experimental Design: We performed in situ hybridization (ISH) for HPV-16 on lymph node metastases from 68 patients with HNSCC. P16 immunohistochemistry was also performed because HPV-16 integration disrupts the retinoblastoma pathway and induces an overexpression of p16.

Results: HPV-16 was detected in 22 of the 68 (32%) cases by ISH. When stratified by site of origin, HPV-16 was detected in 22 of 31 (71%) metastases from the oropharynx, but in none of the 37 (0%) metastases from other sites (P < 0.001; Fisher’s exact). P16 expression was associated with the presence of HPV-16 by ISH: 21 of 22 HPV-16 positive tumors exhibited p16 expression, whereas only 4 of the 46 HPV-16-negative tumors were p16 positive (95% versus 9%; P < 0.001; Fisher’s exact). P16 expression in the node metastases also correlated with site of tumor origin: 24 of 31 oropharyngeal tumors were p16 positive, whereas only 1 of 37 nonoropharyngeal tumors was p16 positive (77% versus 3%; P < 0.001; Fisher’s exact).

Conclusions: For patients with metastatic HNSCC, detection of HPV-16 is a reliable way to establish origin from the oropharynx, either directly by ISH or indirectly by immunohistochemistry for p16 overexpression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nearly 40,000 patients in the United States (1) and over 500,000 patients worldwide (2) are diagnosed with head and neck squamous cell carcinoma (HNSCC) each year. Despite vast improvements in the diagnostic and therapeutic armament over the past several decades, mortality rates have not changed significantly. Patients tend to present with advanced disease when opportunities for cure are not optimal. Indeed, most patients already have metastatic spread to regional lymph nodes at the time of presentation (3) ; 13% of patients present with a neck mass as the first and only clinical manifestation (4) , and 3% to 9% of the primary tumors continue to elude detection even after clinical, radiographic, endoscopic, and histopathologic evaluation (5) . The treatment of patients with occult primary HNSCCs is challenging and controversial. One option is to blanket the mucosa judged most at risk with wide-field radiation, but such prophylactic treatment is associated with the risk of xerstomia, mucositis, and other complications of radiation.

Human papillomavirus (HPV), particularly the tumorigenic 16 subtype, has been confirmed recently as a causative agent in the development of a subset of HNSCCs (6) . In particular, HPV-16 is present in the majority of tumors arising in the oropharynx, but in only a small percentage of HNSCCs arising in nonoropharyngeal sites (6, 7, 8) . In HPV-positive oropharyngeal carcinomas, transcription of the viral oncoprotein E7 is known to functionally inactivate the Retinoblastoma (Rb) gene product, causing a perturbation of other key components of the Rb pathway (7 , 9, 10, 11) . As one example, functional inactivation of Rb by E7 is known to induce an up-regulation of p16 expression (12 , 13) , reaching levels that can be readily detected by routine immunohistochemistry (IHC). Although p16 IHC has shown promise as an ancillary diagnostic tool in the recognition of HPV-induced neoplasia of the female genital tract (14, 15, 16) , potential applications to tumors of the upper aerodigestive tract have yet to be defined. The purpose of the present study was to determine whether detection of HPV, by direct in situ hybridization (ISH) or by immunohistochemical detection of p16 as a surrogate marker, is helpful in pinpointing the site of tumor origin in those patients with cervical lymph node metastases.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
The surgical pathology files were searched for patients with HNSCC who underwent neck dissections for carcinoma metastatic to regional lymph nodes between 1995 and 2002. Medical records were reviewed to document the primary site of tumor origin. The primary site was defined by histopathologic confirmation of a mass detected on physical examination by a head and neck surgeon. To facilitate comparison between those tumors arising in the oropharynx and those tumors arising from neighboring anatomical sites, cases were selected for additional analysis solely on the basis of primary tumor origin. The group of study cases included 31 metastatic HNSCCs from the oropharynx and 37 metastatic HNSCCs from nonoropharyngeal sites. HPV status of the primary tumors and their metastatic implants was not known at the time of case selection.

In-situ hybridization (ISH).
HPV-16 detection in formalin-fixed and paraffin-embedded tissues was performed using the ISH catalyzed signal amplification method for biotinylated probes (DAKO GenPoint, Carpinteria, CA; Ref. 17 ). This catalyzed signal amplification system permits visualization of single copies of HPV-16 in infected cells (18) . Briefly, 5-µm tissue sections underwent deparaffinization, heat-induced target retrieval in citrate buffer, and digestion using Proteinase K (Roche Diagnostics, Indianapolis, IN). Slides were subsequently hybridized with a biotinylated HPV-16 type-specific probe (DAKO). Signal amplification was performed by consecutive application of streptavidin-horseradish peroxidase complex, biotinyl tyramide, and streptavidin-horseradish peroxidase complex. Visualization of positive hybridization signals was performed by incubation with the chromogenic substrate diaminobenzidine. Interpretation of staining was performed without knowledge of p16 immunohistochemical staining or tumor origin.

Immunohistochemistry (IHC).
Five-µm sections were deparaffinized. Antigen retrieval was performed using heat-induced epitope retrieval with 10 mM citrate buffer. 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, Carpinteria, CA).

Staining was regarded as positive if it was strong and diffuse (>80% of tumor cells), and it was regarded as negative if absent or focal. Only strong, diffuse staining of the cytoplasm and nuclei, and not focal or weak staining, is associated with the presence of high-risk HPV in neoplasia of the female genital tract (14, 15, 16) . Immunohistochemical interpretation was performed without knowledge of HPV status or tumor origin.

HPV Detection and Type Specification.
Five-µm thick sections of formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene, washed with 100% ethanol, and digested in 180 µl of ATL buffer with 40 µl of proteinase K at 55°C overnight (DNEasy; Qiagen Corp., Valencia, CA). Proteinase K was heat inactivated for 10 min at 95°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation, and resuspended in 50 µl of water. Five µl of purified DNA were analyzed for HPV DNA by multiplex PCR targeted to the conserved L1 region of the viral genome by use of PGMY09/11 L1 primer pools. Coamplification of the ß-globin gene was performed as a positive control for the presence of amplifiable DNA in the specimen. PCR products were denatured in 0.13 N NaOH, and hybridized to an immobilized HPV probe array using an extended reverse line-blot assay for HPV genotyping (Roche Molecular Systems, Inc., Alameda, CA) of 37 HPV types classified as "high-risk" (HPV-16, -18, -26, -31, -33, -35, -39, -45, -51, -52, -55, -56, -58, -59, -68, -73, -82, and -83) or of "low"- or "unknown’" -risk (HPV-6, -11, -40, -42, -44, -53, -54, -57, -61, -62, -64, -66, -67, -69, -71, -72, -81, -84, and -89) and ß-globin (19 , 20) . Positive controls consisting of 10 and 100 HPV-16 (SiHa)- or HPV-18 (C4–2)-positive cells diluted in a background of HPV-negative cells (K562) and a negative control (K562 cells) were run within each assay. Samples positive for ß-globin were considered of sufficient quality for analysis.

Statistical Evaluation.
Primary tumor location was categorized as a dichotomous variable (oropharynx or nonoropharynx). Factors associated with oropharyngeal tumor location were evaluated by cross-tabulations and analyzed by use of the Fisher’s exact test. The sensitivity value of a test was defined as the percentage of oropharyngeal tumors that tested positive for HPV-16 by ISH or positive for p16 by IHC. The specificity value was defined as the percentage of nonoropharyngeal tumors that tested negative for HPV-16 by ISH or negative for p16 by IHC. Exact binomial confidence intervals were calculated. The {kappa}-statistic was used to measure agreement between HPV-16 ISH and HPV PCR. Ps are two-sided unless otherwise specified. Statistical analysis was conducted using STATA software, Version 7 (STATA, College Station, TX).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 68 cases of metastatic squamous cell carcinoma to cervical lymph nodes, 31 primary tumors were from the oropharynx, 21 were from the oral cavity, 11 were from the larynx, and 5 were from the hypopharynx. The results of HPV detection using various detection strategies are summarized in Table 1Citation .


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  		Table 1 Human papillomavirus (HPV) detection in lymph node metastases by in situ hybridization, surrogate p16 immunohistochemistry, and E-7 type-specific PCR

 
By ISH, HPV-16 was detected in 22 of 31 (71%) metastases from the oropharynx. In all of the HPV-16 positive cases, hybridization was visualized as a punctate signals within the nuclei. The signal varied from one or two inconspicuous dots to many confluent dots (Fig. 1)Citation . Others have shown that the punctate pattern of hybridization correlates with viral DNA integration and that the number of nuclear dots correlates with the copy number of HPV DNA (21) . In striking contrast, HPV-16 was not detected in any of the 37 metastases from nonoropharyngeal sites (71 versus 0%; P < 0.001; Fisher’s exact). For metastatic squamous cell carcinomas to cervical lymph nodes, the sensitivity of a positive HPV-16 ISH for a diagnosis of an oropharyngeal primary was 70.9% (95% confidence interval, 51.9–85.8%). Conversely, the specificity of a negative HPV-16 ISH for a diagnosis of a primary tumor from a nonoropharyngeal site was 100% (one-sided 97.5% confidence interval, 91–100%).



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  		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.

 
By IHC, p16 overexpression was noted in 24 of the 31 (77%) metastases from the oropharynx. In all of the positive cases, staining intensity was strong, present throughout the cytoplasm and the nucleus, and widely distributed throughout the tumor (Fig. 2)Citation . In 1 case (case 13), staining was weak and focally distributed. In the female genital tract, only strong and diffuse p16 staining is associated with high-risk HPV subtypes (14, 15, 16) ; therefore, we regarded focal and weak staining as negative. P16 overexpression was noted in only 1 (case 38) of the 37 (3%) metastases from nonoropharyngeal sites (77 versus 3%; P < 0.001; Fisher’s exact). The sensitivity of a positive p16 stain for a diagnosis of an oropharyngeal tumor was 77.4% (95% confidence interval, 58.9–90.4%). Conversely, the specificity of a negative p16 stain for a diagnosis of a nonoropharyngeal tumor was 97.4% (95% confidence interval, 86.2–99.9%).



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  		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).

 
The presence of p16 expression by IHC was strongly associated with the presence of HPV-16 by ISH: 21 of 22 HPV-16-positive tumors exhibited p16 overexpression, whereas only 1 of the 46 HPV-16-negative tumors were p16 positive (95.5 versus 2.2%; P < 0.001; Fisher’s exact). There was no correlation between signal intensity by ISH and staining intensity by IHC. In other words, p16 staining was uniformly strong and diffuse even when the ISH pattern was consistent with low copy number (Fig. 3)Citation .



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  		Fig. 3. P16 immunohistochemical staining and human papilloma 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.

 
To better understand the mechanisms of discordance in those p16-positive cases where HPV-16 was not detected by ISH, lymph nodes were analyzed for the presence of HPV DNA by PCR targeted to the conserved L1 region of the HPV genome. Sixty-seven of 68 samples were ß-globin positive by PCR. HPV DNA was detected in 22 (33%) of 67 lymph nodes. All of the HPV types detected were high-risk. HPV16 was found in 21 lymph nodes, 2 of which were also positive for HPV-35 or 73. One lymph node was positive for HPV-82, and 1 for HPV-73. One of 6 p16-positive, HPV-16 ISH-negative cases was positive for high-risk HPV-82. The agreement between HPV-16 ISH and PCR for HPV-16 was high (95.5%; {kappa} = 0.89; P < 0.001). HPV DNA detection by PCR was strongly associated with location of the primary tumor in the oropharynx (P < 0.001; Fisher’s exact) and p16 expression by IHC (P < 0.001; Fisher’s exact).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent progress in uncovering the molecular genetic mechanisms underlying human cancers is translating into more effective strategies for patient care including refinements in tumor diagnosis. For patients presenting with metastatic squamous cell carcinoma of the head and neck, a practical and reliable method for pinpointing the site of tumor origin could obviate the need for sophisticated imaging studies, define anatomical sites for directed biopsy and surgical excision, and better demarcate radiation fields.

To date, the overall impact of novel approaches for tumor localization has been limited. Using a microsatellite-based genetic approach, Califano et al. (22) were able match a distinctive pattern of genetic alterations in the metastatic implant to a specific region of the upper aerodigestive tract and, thus, infer the origin of otherwise occult primary tumors. This matching of genetic fingerprints, however, does not preclude the need for extensive tissue sampling of the upper aerodigestive tract, and it necessitates sophisticated methodologies unavailable to most diagnostic laboratories. Other approaches have taken advantage of the fact that some HNSCCs are caused by certain oncogenic viruses that target specific regions of the upper aerodigestive tract. In effect, detection of a specific virus in the metastasis implicates site of tumor origin. Most notably, detection of EBV in a neck metastasis reliably points to tumor origin from the nasopharynx (23, 24, 25, 26, 27) . Although this approach is readily feasible for diagnostic laboratories with ISH capabilities, application to this point has been limited, as only a small subset of HNSCCs has been linked to a tumorigenic virus.

The potential role of viral probes as a tool to localize tumor origin has been expanded recently with the recognition that the HPV is an important causative factor in the development of a subset of HNSCCs (6) . Reported prevalence rates for HPV in primary oropharyngeal range between 40% and 60% (6 , 8 , 28, 29, 30, 31) . The prevalence rate may even be higher in lymph node metastases, as HPV-related tumors may have an enhanced propensity for metastatic spread (8 , 9) . Indeed, we detected the presence of HPV-16 in 71% of metastatic oropharyngeal carcinomas. Coupled with the absence of HPV-16 in lymph node metastases from other primary sites, HPV-16 ISH appears to be a highly reliable method for discerning the site of origin for metastatic carcinomas from the oropharynx.

Compared to PCR-based techniques, the ISH method is inexpensive and readily feasible for routine use by many diagnostic laboratories. Moreover, direct visualization of viral tissue distribution better substantiates HPV as a causal agent. By contrast, the highly sensitive PCR technique may detect HPV DNA present in the sample that is not specific to tumor cell nuclei and, therefore, not of any pathophysiological significance (11) . One potential limitation of HPV-16 ISH, however, is that ~10% of oropharyngeal carcinomas are associated with high-risk subtypes of HPV other than HPV-16 (6 , 29) . Expansion of the hybridization armament to provide broader coverage of high-risk HPV subtypes could improve sensitivity, but only at the expense of expediency and cost containment. IHC has not been a suitable alternative. The unreliability of viral oncoprotein detection in formalin-fixed tissues has prompted a search for surrogate markers of HPV infection that are more feasible for routine IHC.

Disruption of the Rb pathway is common in HNSCC. In the majority of HNSCCs, pathway disruption occurs via inactivation of the p16 gene, an inhibitor of cyclin-dependent kinase in the Rb pathway. Indeed, loss of p16 function is the most common genetic alteration in HNSCC (32) . Opportunely, p16 is overexpressed in tumors associated with high-risk HPV. The viral oncoprotein E7 binds and functionally inactivates Rb, inducing an increase in p16 (12 , 13) . Importantly, HPV-induced overexpression of p16 reaches levels that are easily discerned by routine IHC. Diffuse p16 staining has been correlated with high-risk HPV in tumors of the female genital tract (14, 15, 16) . In the upper respiratory tract, Klussmann et al. (33) likewise reported a high correlation between p16 IHC staining and the presence of HPV-16 for tonsillar carcinomas.

We found that overexpression of p16 of cervical lymph node metastases serves as a reliable surrogate marker of HPV infection. Twenty-one of 22 (95%) HPV-16-positive tumors exhibited p16 overexpression. Moreover, p16 overexpression was noted in 9% of the tumors where a hybridization signal was not apparent using HPV-16 ISH. Using a PCR-based detection method in these discordant p16-positive/HPV-16-negative tumors, we were able to confirm the presence of HPV-16 or some other high-risk HPV subtype in half of these cases. Thus, the ability to register sizeable increases in p16 expression, regardless of HPV-16 copy number or high-risk HPV subtype, appears to be the near equivalent of actual HPV detection.

As a discriminator of a biologically distinct tumor entity, p16 IHC could have various practical applications in the clinical arena. HPV detection in HNSCC has been correlated with improved survival (6 , 29 , 33) such that p16 IHC may serve as a useful marker of prognoses (33) . Patients with HPV-induced HNSCC may benefit from HPV-targeted treatment strategies (e.g., therapeutic HPV vaccines; Ref. 34 ) such that p16 IHC may help tailor therapy. In the present study, we addressed whether p16 IHC could help pinpoint the site of tumor origin for patients with metastatic HNSCC. In this regard, p16 overexpression in lymph node metastases strongly correlated with tumor origin from the oropharynx. Strong p16 staining was present in 77% of metastases from the oropharynx, but in only 3% of metastases from nonoropharyngeal tumors.

In summary, HPV detection in cervical lymph nodes could help focus the search for and treatment of the primary tumor in patients who present with a lymph node metastases as the initial manifestation of their tumor. To assess the feasibility of this strategy, we focused on patients with well-documented primary tumors who had undergone surgical resections of their metastatic implants. This same strategy could be readily adapted to patients with metastatic HNSCCs during much earlier stages of the diagnostic workup. HPV-16 ISH and p16 IHC can be easily applied to fine needle aspiration specimens (14 , 21 , 35 , 36) . In those patients who present with neck masses, routine HPV analysis coupled with traditional cytopathology could magnify the sum of useful information gleaned from a limited number of aspirated tumor cells.


   FOOTNOTES
 
Grant support: NIH (1R01 DE13121-01A1).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: William H. Westra, The Weinberg Building, Room 2242, The Johns Hopkins Hospital, 401 North Broadway, Baltimore, MD 21231-2410. Phone: (410) 955-2163; Fax: (410) 955-0115; E-mail: wwestra{at}jhmi.edu

Received 4/30/03; revised 9/ 3/03; accepted 9/ 8/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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