Purpose: Human papillomavirus 16 (HPV-16) has been implicated as a causative agent in a subset of head and neck squamous cell carcinomas (HNSCC). This study was undertaken to discern the distribution and timing of HPV viral integration during tumorigenesis of the upper respiratory tract.

Experimental Design: A tissue array was assembled from a consecutive group of 176 patients with HNSCCs. The array was evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. Patients with HPV-positive tonsillar cancers who had undergone bilateral tonsillectomies were selected for more complete mapping of viral integration.

Results: HPV-16 was detected in 38 of the 176 (22%) cases by in situ hybridization. When stratified by site of origin, HPV-16 was detected in 37 of 45 cancers arising from the oropharynx but in only 1 of 131 tumors arising from nonoropharyngeal sites (82% versus 0.8%, P < 0.00001). P16 expression was associated with the presence of HPV-16: 31 of 38 HPV-positive tumors exhibited p16 expression, whereas only 9 of the 138 HPV-negative tumors were p16-positive (82% versus 6%, P < 0.00001). In the bilateral tonsil sections, hybridization signals were strictly limited to the invasive cancers and associated dysplasias. P16 staining was widely distributed throughout the nonneoplastic crypt epithelium of individuals with and without tonsillar cancer.

Conclusions: HPV-16 is strongly associated with carcinomas arising from the oropharynx, and integration is tightly coupled to the neoplastic process. Viral integration does not occur as a field alteration throughout normal tonsillar epithelium. P16 expression localizes to HPV-positive cancers, and is intrinsic to the specialized epithelium of the tonsillar crypts. For risk assessment, early cancer detection and disease surveillance, evidence of HPV-16 integration may represent a meaningful finding, whereas high p16 expression, by itself, may not.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of morbidity and mortality with >500,000 new cases worldwide each year (1). Although the incidence of oral cancer has remained stable, the incidence of tonsillar cancer has increased 2% to 3% annually among American men from 1973 to 1995 (2). Human papillomavirus (HPV) DNA has been consistently detected in ∼20% of HNSCCs overall and in 50% of HNSCCs originating in the oropharynx (39). HPV-16 is by far the most frequently identified HPV subtype accounting for >90% of the HPV subtypes isolated from HNSCCs (5, 10). Converging lines of evidence indicate that HPV, like alcohol and tobacco, plays an etiologic role in HNSCC: in HPV-positive HNSCCs HPV-16 genomic DNA is localized to tumor cell nuclei (5, 11), is present in high copy number (6, 12, 13), is frequently integrated (5, 12), and actively transcribes its viral oncoproteins E6 and E7 (9, 14, 15).

Epidemiologic studies have shown that the risk for HPV-induced HNSCC is increased by certain sexual behaviors associated with transmission of high-risk HPV types, and that exposure to HPV-16 is associated with increased risk of developing oropharyngeal cancer (16, 17). In one study, seroreactivity to the HPV-16 viral capsid in sera stored ∼10 years prior to HNSCC diagnosis conferred a 14-fold increased risk of incident oropharyngeal carcinoma, providing strong evidence for a temporal relationship between exposure and risk for HNSCC (8). The tonsils are well-recognized replication sites for important pathogenic viruses (e.g., EBV, adenoviruses, herpes simplex virus, and others) and may also represent a reservoir for HPV following exposure (18). Based on direct brushing of the tonsils from individuals without HNSCC, the prevalence of tonsillar HPV infection is 1% in nonimmunocompromised adults and up to 3% in individuals seropositive for the HIV (19).

The predictive value of tonsillar HPV infection for oropharyngeal cancer is not known, but clearly not every individual with HPV in their oropharynx develops carcinoma. During malignant transformation, integration of HPV DNA into the cellular genome likely represents a critical step in those individuals who harbor HPV in their tonsils (5, 2022). A more complete understanding of the timing and distribution of viral DNA integration has been limited by two obstacles. The first has to do with the unavailability of tonsils at risk for developing tonsillar carcinoma based on a high-risk exposure profile, and of tonsils harboring premalignant lesions (i.e., dysplasia/carcinoma in situ). Premalignant lesions of the tonsils are uncommon, unlikely to be appreciated on routine clinical exam, and are unlikely to be biopsied. The second obstacle has to do with the inherent limitations of traditional PCR-based detection methods in discerning relevant from irrelevant HPV infections, discriminating episomal from integrated virus, and visualizing distribution of viral integration within tissues.

The recent development of a powerful HPV in situ hybridization detection method that uses signal amplification now facilitates a more complete description of the distribution and physical state of high-risk HPV in the tonsils. This amplification method is highly sensitive, permitting visualization of single copies of HPV-16 in infected cells (23); is applicable to formalin-fixed paraffin-embedded clinical samples, facilitating precise spatial localization of viral sequences within tissues; and discriminates between integrated and episomal states of HPV infection (2327). We applied this technique to neoplastic and nonneoplastic tissues of the upper respiratory tract to: (a) confirm the site specificity of HPV-16 infection over a large group of anatomically diverse HNSCCs, (b) discern the distribution of viral integration in tonsils that harbor carcinoma and in the contralateral tonsils sharing the same exposure profile, and (c) establish the reliability of p16 (i.e., p16INK4A gene product) immunohistochemical staining as an alternative method of viral detection.

Patient selection. A tissue microarray was assembled from a consecutive group of 176 patients with HNSCCs from various anatomic sites. Tissue array sections were evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. From this group of 176 patients, 8 patients were selected for a more thorough evaluation of HPV distribution in their tonsils. Selection of this subgroup was based on the presence and availability of: (a) squamous cell carcinomas that were HPV-16-positive by in situ hybridization, (b) concurrent dysplasia of the tonsillar epithelium, and (c) contralateral tonsils removed during bilateral tonsillectomy. For this group of eight patients, HPV-16 in situ hybridization and p16 immunohistochemistry was done on the tonsils harboring carcinoma and on the contralateral tonsils. For tonsils harboring carcinomas, sections were selected to include the invasive carcinoma, associated dysplasia/carcinoma in situ, and histologically nonneoplastic tonsillar epithelium including both the surface and crypt epithelia. Tonsils from eight age-matched patients who had undergone tonsillectomies for nonneoplastic disease were used as controls.

HPV-16 in situ hybridization. HPV-16 detection was done using the in situ hybridization catalyzed signal amplification method for biotinylated probes (DAKO GenPoint, Carpinteria, CA). 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 done by consecutive application of streptavidin-horseradish peroxidase complex, biotinyl tyramide, and streptavidin-horseradish peroxidase complex. Visualization of positive hybridization signals was done by incubation with the chromogenic substrate diaminobenzidine. Positive signals were scored as punctuate and/or diffuse. A punctuate pattern has been correlated with HPV DNA integration into genomic DNA, and a diffuse pattern has been correlated with HPV DNA in its episomal state (2327).

P16 immunohistochemistry. Five-micrometer sections were deparaffinized. Antigen retrieval was done using heat-induced epitope retrieval with 10 mmol/L 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). For HNSCCs, strong nuclear staining together with strong cytoplasmic staining in >25% of the cells was regarded as high p16 expression (28, 29).

Statistical evaluation. Primary tumor location was categorized as a dichotomous variable (oropharynx, nonoropharynx). Factors associated with oropharyngeal tumor location were evaluated by cross-tabulations and analyzed by use of Fisher's exact test. All P values are two-sided.

HPV-16 in situ hybridization in HNSCCs. The tissue array contained tumors from 176 patients with squamous cell carcinomas from various anatomic sites of the head and neck including the oral cavity (n = 61), larynx (n = 54), oropharynx (n = 45), hypopharynx (n = 9), sinonasal cavity (n = 6), and nasopharynx (n = 1). For 22 of the oropharyngeal carcinomas, both the primary carcinoma and its lymph node metastasis were included on the array. HPV-16 was detected in 38 of the 176 (22%) cases by in situ hybridization. When stratified by site of origin, HPV-16 was detected in 37 of 45 tumors from the oropharynx, but in only 1 of the 131 tumors from a nonoropharyngeal site (i.e., sinonasal cavity, 82% versus 0.8%; P < 0.00001; Fisher's exact; Fig. 1). When further stratified by anatomic subsite, all of the HPV-positive oropharyngeal carcinomas arose from either the lingual tonsils (n = 17) or the palatine tonsil (n = 20); whereas four (50%) of the HPV-negative cancers arose from the soft palate, a site unassociated with reticulated crypt epithelium. When primary oropharyngeal tumors were compared with their lymph node metastasis, HPV status was identical for the 22 matched tumor pairs (17 HPV-positive tumors and 5 HPV-negative tumors).

Fig. 1.

Representative examples of a tonsillar carcinoma (left) and nonoropharyngeal carcinoma (right) from the tissue array evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. The tonsillar carcinoma gives a strong hybridization signal for HPV-16 and shows strong p16 staining, whereas the nonoropharyngeal carcinoma does not.

Fig. 1.

Representative examples of a tonsillar carcinoma (left) and nonoropharyngeal carcinoma (right) from the tissue array evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. The tonsillar carcinoma gives a strong hybridization signal for HPV-16 and shows strong p16 staining, whereas the nonoropharyngeal carcinoma does not.

Close modal

P16 immunostaining in HNSCCs. P16 staining has been advocated as a surrogate marker of HPV-16 infection: HPV-16 integration disrupts the retinoblastoma pathway and induces an overexpression of p16 that can be visualized as strong cytoplasmic and nuclear staining by p16 immunohistochemistry (28, 30). We confirmed a strong correlation between HPV-16 status and p16 staining in the 176 HNSCCs. Thirty-one of the 38 HPV-16-positive tumors exhibited strong and diffuse p16 staining, whereas only 9 of the 138 HPV-16-negative tumors were p16-positive (82% versus 7%, P < 0.00001; Fig. 1). P16 expression also correlated with site of origin: 28 of 45 oropharyngeal tumors were p16-positive, whereas only 9 of 131 nonoropharyngeal tumors were p16-positive (62% versus 7%, P < 0.00001).

Distribution of dysplasia in tonsils. Squamous dysplasia was present in the eight tonsils with carcinoma and in one of the contralateral tonsils without invasive carcinoma. Of the nine tonsils that harbored dysplasia, dysplasia involved the tonsillar crypts. In six of these cases, the dysplasia exclusively involved the tonsillar crypts, and in three cases the dysplasia also extended onto the surface epithelium. There were no cases where dysplastic changes were only present on the surface of the tonsils.

Distribution of HPV-16 in tonsils. HPV-16 in situ hybridization and p16 immunohistochemistry were done on full thickness tonsillar sections from eight patients with HPV-16-positive tonsillar carcinomas who had undergone bilateral tonsillectomies, and on the eight control tonsils. A summary of the distribution of HPV-16 in situ hybridization and p16 immunostaining are summarized in Table 1. For all eight of the tonsillar carcinomas, the presence of HPV-16 was confirmed by the presence of an intense hybridization signal visualized as punctuate dots within the nucleus. The signal varied from one or two inconspicuous dots to numerous confluent dots. A diffuse hybridization pattern was not observed. Others have shown that the punctate pattern of hybridization correlates with viral DNA integration, whereas a diffuse signal correlates with episomal HPV (31). The hybridization signals were strictly limited to the carcinoma and the associated dysplastic epithelium. Neither punctate nor diffuse signals were observed in nondysplastic tonsillar epithelium, even in the epithelium directly adjacent to dysplasia (Figs. 2 and 3). HPV-16 was detected in one of the eight contralateral tonsils in an area of dysplasia involving a tonsillar crypt (Fig. 3). HPV was not detected in the other seven contralateral tonsils without dysplasia or in the control tonsils.

Table 1.

Distribution of integrated HPV-16 in tonsils as assessed by in situ hybridization and p16 immunohistochemical staining

HPV-16 in situ hybridizationP16 immunohistochemistry
Ipsilateral tonsil*   
    Dysplasia 8 of 8 8 of 8 
    Invasive carcinoma 8 of 8 8 of 8 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
Contralateral tonsil*   
    Dysplasia 1 of 1 1 of 1 
    Invasive carcinoma 0 of 0 0 of 0 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
Control tonsils   
    Dysplasia 0 of 0 0 of 0 
    Invasive carcinoma 0 of 0 0 of 0 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
HPV-16 in situ hybridizationP16 immunohistochemistry
Ipsilateral tonsil*   
    Dysplasia 8 of 8 8 of 8 
    Invasive carcinoma 8 of 8 8 of 8 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
Contralateral tonsil*   
    Dysplasia 1 of 1 1 of 1 
    Invasive carcinoma 0 of 0 0 of 0 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
Control tonsils   
    Dysplasia 0 of 0 0 of 0 
    Invasive carcinoma 0 of 0 0 of 0 
    Normal crypt epithelium 0 of 8 8 of 8 
    Normal surface epithelium 0 of 8 0 of 8 
*

Patients with tonsillar carcinoma.

Patients without tonsillar carcinoma.

Fig. 2.

HPV-16 viral integration in tonsil harboring a squamous cell carcinoma. The dysplastic changes are limited to the crypt epithelium and sharply demarcated from the surface epithelium (A, H&E stain). HPV-16 in situ hybridization (B) and p16 immunohistochemical staining (C) is strictly confined to the dysplastic tonsillar crypt epithelium and does not extend beyond the altered epithelium onto the adjacent nondysplastic surface epithelium.

Fig. 2.

HPV-16 viral integration in tonsil harboring a squamous cell carcinoma. The dysplastic changes are limited to the crypt epithelium and sharply demarcated from the surface epithelium (A, H&E stain). HPV-16 in situ hybridization (B) and p16 immunohistochemical staining (C) is strictly confined to the dysplastic tonsillar crypt epithelium and does not extend beyond the altered epithelium onto the adjacent nondysplastic surface epithelium.

Close modal
Fig. 3.

HPV-16 viral integration in a contralateral tonsil with squamous dysplasia. By light microscopy, dysplastic changes involving a tonsillar crypt (**) are not apparent in the normal reticulated epithelium of an adjacent crypt (*; A, H&E stain). By in situ hybridization, the HPV-16 hybridization signals localize to the dysplastic crypt epithelium (B).

Fig. 3.

HPV-16 viral integration in a contralateral tonsil with squamous dysplasia. By light microscopy, dysplastic changes involving a tonsillar crypt (**) are not apparent in the normal reticulated epithelium of an adjacent crypt (*; A, H&E stain). By in situ hybridization, the HPV-16 hybridization signals localize to the dysplastic crypt epithelium (B).

Close modal

Distribution of p16 immunostaining in tonsils. Strong p16 staining was noted in all of the invasive tonsillar carcinomas and in all of the dysplasias (Fig. 2). Strong p16 staining, however, was not limited to the neoplastic cells but was consistently observed in the nonneoplastic epithelium, even in the tonsils from the control patients without cancer (Fig. 4). In all tonsillar sections examined, staining was observed in the reticulated crypt epithelium—a specialized epithelium that plays a key role in initiating immune responses and in scattered cells within germinal centers of lymphoid follicles. Staining of the crypt epithelium was focal and generally limited to clusters of nonkeratinizing cells associated with numerous interdigitating lymphocytes (Fig. 4). In the germinal center, the distribution of p16-positive cells mirrored that of the CD21-positive follicular dendritic cells. P16 staining was not observed in the stratified squamous epithelium that covered the surface of the tonsils.

Fig. 4.

P16 immunohistochemical staining in a normal tonsil from a patient without carcinoma. Strong p16 staining is noted in the reticulated crypt epithelium (arrows and inset) and in follicular dendritic cells of germinal centers (*), but it is not seen in the surface epithelium.

Fig. 4.

P16 immunohistochemical staining in a normal tonsil from a patient without carcinoma. Strong p16 staining is noted in the reticulated crypt epithelium (arrows and inset) and in follicular dendritic cells of germinal centers (*), but it is not seen in the surface epithelium.

Close modal

Wildly varied estimates of HPV prevalence in premalignant and malignant lesions of the head and neck have confounded attempts to better understand the role of HPV in HNSCC. In the oral cavity alone, reported estimates in invasive squamous cell carcinomas have ranged from 0% to 100% (3237). Against this background, a growing number of detailed studies point to the tonsils as a preferential site of HPV-induced malignancy: compared with HPV-negative tumors, HPV-positive tumors are more likely to arise from the tonsils than from nonoropharyngeal sites (35, 30). Using a sensitive but specific detection strategy on a large number of HNSCCs across a variety of anatomic sites, we were able to convincingly confirm that HPV-positive tumors are much more likely to arise from the tonsils than HPV-negative tumors by a decisive ratio of 103:1.

In patients with HNSCCs, specific genetic alterations can be widely distributed throughout the mucosa lining the aerodigestive tract even in the absence of overt histopathologic changes of malignancy (3842). This observation has been helpful in clarifying certain puzzling clinical issues relating to tumor spread such as the common occurrence of local tumor recurrences, “skip lesions” and second primary HNSCCs. We found no evidence to support a similar pattern of “field distribution” with regards to HPV-16 DNA integration. As visualized by a highly sensitive in situ signal amplification system, viral DNA integration is sharply restricted to neoplastic cells and does not occur outside of the field of phenotypically altered cells, not even in the epithelium directly adjacent to areas of dysplasia. The absence of widespread viral DNA integration as a background finding in tonsils harboring carcinoma or at risk of developing carcinoma dismisses consideration of HPV-16 as an incidental passenger virus that is inconsequential to head and neck tumorigenesis.

Although HPV-16 is not detected in phenotypically normal tonsillar epithelium, it is detected within the full range of tumor progression from dysplasia to invasion to metastatic dissemination. The detection of HPV-16 hybridization signals in early to advanced stages of tumorigenesis suggests that viral DNA integration represents a critical and necessary step in malignant transformation of the tonsillar epithelium, a step that is tightly coupled to altered cell morphology. Furthermore, the persistence of HPV in metastatic implants dismisses the consideration of a “hit and run” mechanism where the role of HPV is transient and limited to initiation of tumorigenesis.

HPV-positive tumors tend to be poorly differentiated with a distinctly “basaloid” morphology, and they tend to express p16 expression at high levels. To some degree, these features may reflect intrinsic qualities of the tissues targeted by HPV as opposed to specific perturbations of cell morphology and function induced by HPV. HPV DNA integration is consistently centered on the epithelium lining the tonsillar crypts. Like their neoplastic counterpart, this highly specialized reticulated epithelium is characterized by nests and cords of nonkeratinized squamous cells with a prominent basaloid appearance. Moreover, this crypt epithelium consistently shows strong p16 staining. The mechanism of up-regulated p16 expression remains to be elucidated, but the constancy of this finding, even in tonsils from control patients without tonsillar carcinoma, suggests that up-regulated p16 and HPV DNA integration are not equivalent. This observation does not invalidate the concept that functional inactivation of Rb by E7 induces up-regulation of p16 expression (43, 44), but the decoupling p16 staining and HPV DNA integration is worth noting in light of a call for the clinical application of p16 immunostaining as a surrogate marker of HPV-driven neoplasia (28, 30). By itself, the presence of p16 staining, whether it be in cells brushed from the tonsils or aspirated from a cyst in the neck, may not be sufficient evidence of an HPV-associated malignancy. For example, we routinely detect p16 staining in cells aspirated from nonneoplastic branchial cleft cysts of the neck.5

5

D. Cao and W.H. Westra, unpublished observations.

Recognition of HPV-16 as an etiologic factor for an important subset of HNSCCs has prompted considerable interest in HPV-16 detection as a means of assessing cancer risk, detecting early cancers, discerning site of tumor origin for patients with clinically occult primary cancer, monitoring disease recurrence/progression, predicting clinical outcomes, and identifying patients who may benefit from immunology-based therapeutic strategies (5, 8, 30, 4547). Many of these applications require a more precise understanding of the natural history of HPV in the upper respiratory tract. Our findings give support to the growing impression that HPV-16 viral integration not only targets the tonsillar crypt epithelium but is a causal force in initiating and maintaining the malignant phenotype.

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.

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