Presence and Influence of Human Papillomaviruses (HPV) in Tonsillar Cancer

Presence and Influence of Human Papillomaviruses (HPV) in Tonsillar Cancer Hanna Mellin Dahlstrand and Tina Dalianis Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, 171 76, Stockholm, Sweden

I. II. III. IV.

Introduction Tonsillar Cancer Human Papillomavirus (HPV) Human Papillomavirus (HPV) in Tonsillar Cancer A. Frequency and Type of HPV in Tonsillar Cancer B. HPV and Tonsillar Cancer Patient Features C. HPV and Prognosis in Tonsillar Cancer D. HPV and Radiosensitivity in Tonsillar Cancer E. HPV and Correlation to Cell-Cycle Proteins in Tonsillar Cancer F. HPV and Genetic Instability in Tonsillar Cancer V. HPV and Other Tumors of the Head and Neck VI. HPV Vaccines VII. Conclusions References

Tonsillar cancer is the most common of the oropharyngeal carcinomas and human papillomavirus (HPV) has been found to be present in approximately half of all cases. Patients with HPV-positive tonsillar cancer have been observed to have a better clinical outcome than patients with HPV-negative tonsillar cancer. Moreover, patients with tonsillar cancer and a high viral load have been shown to have a better clinical outcome, including increased survival, compared to patients with a lower HPV load in their tumors. Recent findings show that HPV-positive tumors are not more radiosensitive and do not have fewer chromosomal aberrations than HPV-negative tumors, although some chromosomal differences may exist between HPV-positive and -negative tonsillar tumors. Current experimental and clinical data indicate that an active antiviral cellular immune response may contribute to this better clinical outcome. These data are also in line with the findings that the frequency of tonsillar cancer is increased in patients with an impaired cellular immune system. Thus, therapeutic and preventive HPV-16 antiviral immune vaccination trials may be worthwhile, not only in cervical cancer, but also in tonsillar cancer. ß 2005 Elsevier Inc.

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Copyright 2005, Elsevier Inc. All rights reserved

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I. INTRODUCTION When zur Hausen (zur Hausen, 1976) proposed that cervical cancer might be caused by human papillomavirus (HPV), the scientific community accepted that HPV could potentially be involved in the development of some but definitely not all cervical cancers. Today, it is fully accepted that different types of HPVs are present and instrumental in the induction of almost all cervical cancers as well as some other types of human cancer (zur Hausen, 1996, 1999). In addition, several mechanisms by which HPVs exhibit their oncogenic potential have also been revealed (zur Hausen, 1996, 1999). The possibility that preventive vaccines against HPV may soon reach clinical practice (Koutsky et al., 2002; Lehtinen and Dillner, 2002) has drawn even more attention to the association of HPV with other types of cancer. However, HPV is only present in a proportion of other types of cancer, e.g., head and neck cancer, anogenital cancer, and nonmelanoma skin cancer, and much less is known about the role of HPV in these tumors (Alani and Munger, 1998; de Villiers, 1991, 1997; Gillison et al., 1999; Licitra et al., 2002; Mork et al., 2001; Snijders et al., 1992). Nonetheless, tonsillar carcinoma is of particular interest, since it is the head and neck cancer where HPV is most commonly found (Gillison et al., 2000; Mork et al., 2001; Paz et al., 1997; Snijders et al., 1996). Approximately half of all tonsillar cancers are HPV positive (Andl et al., 1998; Klussmann et al., 2001; Mellin et al., 2000; Paz et al., 1997). In addition, recent reports suggest that patients with HPV-positive tonsillar tumors have a lower risk of relapse and longer survival compared to patients with HPV-negative tonsillar tumors (Gillison et al., 2000; Mellin et al., 2000). These data motivate further comparisons between HPV-positive and HPV-negative tonsillar tumors with regard to clinical outcome, sensitivity to radiotherapy, biology of the tumor, and genetic stability in order to better understand possible options for treatment and vaccination studies. The purpose of this article is to review current knowledge on the status and significance of HPV in tonsillar cancer.

II. TONSILLAR CANCER Cancer of the palatine tonsil, in the lymphoid region called the Waldeyer’s ring, is usually referred to as tonsillar cancer. Tonsillar cancer is the most common of the oropharyngeal malignancies, and 75% of all tonsillar carcinomas are squamous cell carcinomas (Genden et al., 2003). As with all head and neck squamous cell carcinomas (HNSCC), smoking and alcohol abuse are regarded as the main etiological factors for tonsillar

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cancer and are known to account for 80 to 90% of all HNSCC (Decker and Golstein, 1982; Licitra et al., 2002). However, HNSCC and tonsillar cancer also occur in some 15 to 20% of patients without these risk factors (Gillison et al., 2001; Licitra et al., 2002). In many of these instances, viruses are most likely to be involved in the development of HNSCC, and data now indicate that high-risk types of HPV, similar to those observed in cervical cancer, are associated with a subset of HNSCC (Alani and Munger, 1998; de Villiers, 1991; Gillison et al., 2000, 2001; Gissmann et al., 1982; Mork et al., 2001; Naghashfar et al., 1985; Snijders et al., 1992; Syrjanen et al., 1983). Patients with tonsillar cancer do not normally seek health care until the tumor is fairly large and presents symptoms like swallowing-related pain or difficulties in swallowing (Mashberg and Samit, 1995). This is because small tumors generally do not cause any discomfort. Other common first symptoms are pain in the ear or a lump in the neck due to the tumor spreading to the lymph nodes (Mashberg and Samit, 1995). In advanced cases, vital functions such as breathing, eating, and speaking may be significantly affected. Further suffering may be caused by cancer growth in the face and the neck. Later on, the curative treatments of surgery and radiotherapy (Genden et al., 2003; Mellin et al., 2000) can be disabling and disfiguring. Tonsillar cancer is generally treated with (pre-operative or post-operative) full-dose radiotherapy (64 Gy) against the primary tumor and the neck. The extent of the surgical intervention depends on the size of the primary tumor, the presence of metastases in the neck lymph nodes, and the response to the radiotherapy given. Overall survival for patients with oropharyngeal cancer is 67% with stage I, 46% with stage II, 31% with stage III, and 32% with stage IV (Pugliano et al., 1997). However, the overall survival for patients with oropharyngeal cancer is only about 38% (Pugliano et al., 1997). Despite similar histology and stage, as well as standardized treatment, it is not easy to predict the outcome of each individual case. Hence, both predictive and prognostic markers would be of significant clinical value in order to tailor treatment for individual tonsillar cancer patients. This would allow for optimization of therapy to give the most efficient treatment with minimal impact on function and form. Moreover, given the high proportion of HPV-16 associated with tonsillar cancer (see following text), patients could obtain substantial benefit from the use of the same prophylactic and adjuvant therapeutic strategies that are being developed to prevent and/or treat HPV-associated anogenital cancers (for reviews, see Devaraj et al., 2003; Ling et al., 2000). However, before using such treatment, it is important to investigate in which cases this could be an option.

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III. HUMAN PAPILLOMAVIRUS (HPV) There are more than 100 HPV types, and for general reviews on HPV and cancer, as well as, more specifically, head, neck, and oral cancer, see, for example, Zur Hausen (1996), de Villiers (1997), Syrjanen (2003), and Scully (2002). Some HPV types are associated with common warts, while others are associated with chondylomas and papillomas. Finally, there are HPV types such as HPV 16, 18, 31, 33, and others that are associated with malignant tumors. Nevertheless, the genomes of all HPVs are similar and consist of double-stranded circular DNA with a size of 7 to 8 Kb. The genome is enclosed in a 52- to 55-nm viral capsid, and is arbitrarily divided into a noncoding region and two coding regions, the early and late regions. The early region encodes for the early proteins E1-E2 and E4-E7, which are important for pathogenesis and transformation, while the late region encodes for L1 and L2, the two capsid proteins. Of particular interest in this context is that among the HPV types associated with malignant tumors, E6 and E7 are classified as oncogenes. E6 binds to the cellular protein p53 and degrades it, while E7 binds to pRB and abrogates its function (Dyson et al., 1989, 1992; Scheffner et al., 1990). Under these conditions, the intracellular levels of normal p53 and pRB are reduced and this combination results in the inhibition of cell cycle control and facilitation of tumor development (for reviews, see Hanahan and Weinberg, 2000; zur Hausen, 1996). Also of interest is that the L1, the major capsid protein, can self assemble and form viruslike particles (Kirnbauer et al., 1993), which are useful for vaccination against HPV infections.

IV. HUMAN PAPILLOMAVIRUS (HPV) IN TONSILLAR CANCER A. Frequency and Type of HPV in Tonsillar Cancer HPV DNA has been shown to be present in 45 to 100% of all tonsillar tumors (Andl et al., 1998; Dahlgren et al., 2003; Gillison et al., 2000; Koskinen et al., 2003; Mellin et al., 2000; Ringstro¨m et al., 2002; Snijders et al., 1992). The variation depends mainly on the type of material that is available for analysis and the methodology used for detection (as has been discussed) (Mellin, 2002; Mellin et al., 2002). In general, it is easier to  detect HPV in fresh-frozen (70 C) tumors compared to formalin-fixed and paraffin-embedded tumors, where the DNA is degraded and where

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degradation progresses even after storage for decades (Mellin, 2002; Mellin et al., 2000, 2002). The most common and sensitive technique for the detection of HPV is based on polymerase chain reaction (PCR) technology (Mellin, 2002; Mellin et al., 2002). In the past, less sensitive methods such as Southern blots or in situ hybridization techniques have been used (Mellin, 2002; Syrjanen, 1990). Screening for HPV by PCR analysis is usually initially performed using HPV consensus/general primers (e.g., GP5þ/6þ, My9/10, CPI/IIG, FAP59/61) (de Roda Husman et al., 1995; Forslund et al., 1999; Manos et al., 1989; Tieben et al., 1993). These primer sets allow for the amplification of a wide range of HPV types and are useful for screening. Alternatively, they can be type-specific and identify only one specific HPV type (Hagmar et al., 1992). General primers are complementary to sequences (often in L1) in HPV that are highly conserved among many HPV types, while HPV type-specific primers bind to a sequence found in a single HPV type (often in E6 or E7) and do not cross-bind to other HPV types. Instead of an HPV type-specific PCR, HPV typing can also be performed by sequencing the PCR product obtained by a PCR run with general primers (Mellin et al., 2002). For HPV typing, HPV type-specific oligonucleotide probes using either enzyme immunoassays or Southern blot hybridizations are also commonly used (Herrero et al., 2003; van Houten et al., 2001). Without doubt, HPV type 16 is the type predominant in tonsillar cancer (Andl et al., 1998; Gillison et al., 2000; Klussmann et al., 2001; Koskinen et al., 2003; Mork et al., 2001; Paz et al., 1997; Snijders et al., 1992; Strome et al., 2002; Wilczynski et al., 1998). In most reports of HPV-positive tonsillar cancer biopsies, 85 to 100% contain HPV-16 followed by 0 to 7% containing HPV-33. HPV-31, HPV-59, or non-typeable HPVs are found even more rarely. In addition, when using DNA as well as RNA in situ hybridization, the viral genome and its transcription products (performed on HPV-16) have been located in cancer cells and nodal metastases but not to the surrounding stroma of the primary tonsillar tumor or the nodal metastases (Demetrick et al., 1990; Niedobitek et al., 1990; Snijders et al., 1992; Strome et al., 2002; Wilczynski et al., 1998).

B. HPV and Tonsillar Cancer Patient Features Patients with HPV-positive tonsillar tumors are less likely to be heavy smokers and drinkers, although it has been reported that HPV may have a synergistic effect with regard to tumor development in smokers (Gillison et al., 2000; Haraf et al., 1996; Herrero et al., 2003; Koch et al., 1999; Ringstro¨m et al., 2002; Schwartz et al., 1998). A current concern regards the possible sexual transmission of HPV in oral and oropharyngeal

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squamous cell carcinoma (OSCC) (Herrero et al., 2003; Scully, 2002). This is suggested by the significant increase in tonsillar cancer reported among men in the United States from 1973 to 1995 (Frisch et al., 2000b). This may be explained by changes in sexual habits resulting in the increased transmission of HPV (Devaraj et al., 2003). It is also known that individuals with HPV-associated anogenital malignancies have an increased risk for a second primary cancer in the tonsils and oral cavity (Boice et al., 1985; Frisch and Biggar, 1999; Rabkin et al., 1992). In one of these studies, the increased risk was estimated to be 4.3-fold (Frisch and Biggar, 1999). Moreover, a similar study showed an increase in tonsillar cancer in women aged >50 years with a history of in situ cervical cancer, as well as an increased incidence of both tonsillar and tongue cancer in the husbands of cervical cancer patients (Hemminki et al., 2000). In contrast, patients with an HPV-unrelated cancer, e.g., colon cancer or breast cancer, had no increased risk of developing tonsillar cancer (Frisch and Biggar, 1999). Since the histology of the oral mucosa resembles that of the uterine cervix and other lower genital localizations, one can anticipate similar HPV infection patterns in the oral cavity as described for the genital tract (Syrjanen, 2003). HPV infection of the cervix is transmitted by sexual contact and there is a correlation between the prevalence of HPV, the number of sexual partners, and a low age at sexual debut (Oriel, 1971; Schiffman and Brinton, 1995; Syrjanen and Syrjanen, 1990). Orogenital contact has also been suggested to lead to HPV infections (Devaraj et al., 2003; Maden et al., 1992; Schwartz et al., 1998; Scully, 2002; Smith et al., 1998). In cervical cancer, which has been studied in more detail, HPV-involved cancer progression has been shown to be a multistep process. This process includes E6 and E7 transcription (of ‘‘oncogenic’’ HPV types), modification of cellular genes, and possibly also genetic susceptibility, an impaired cellmediated immunity, and co-factors such as smoking (Beskow and Gyllensten, 2002; Hanahan and Weinberg, 2000; Schiffman and Brinton, 1995; zur Hausen, 1996, 1999). Less is known about HPV-induced carcinogenesis at other tumor sites. However, it is reasonable to assume that the pathways are similar but not necessarily identical for tonsillar cancer. In the cervix, the immune system usually clears HPV infections within months or years. Only rarely after 10 to 30 years of latency do HPV infections progress to cervical carcinoma (Beskow and Gyllensten, 2002; Rome et al., 1987; Schiffman and Brinton, 1995; Syrjanen and Syrjanen, 1990). It is possible that this is also true for tonsillar cancer; however, the latency period is still unknown. An impaired cellular-mediated immune system (such as in HIV-infected or transplanted patients) results in an increase in HPV-induced lesions as well as an increase in HPV-associated cancers, including tonsillar cancer

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(Berkhout et al., 1995; Demetrick et al., 1990; de Villiers, 1997; Frisch et al., 2000a; Swoboda and Fabrizii, 1993). Evasion of the cell-mediated immune system is critical for HPV-transformed tumor cells. In cervical cancer, the expression of HLA class I antigens and accessory molecules is often downregulated (Cromme et al., 1994; Stanley, 2001; Stern, 1996). It is possible that similar mechanisms will also be found in tonsillar cancer. The role of humoral immunity in HPV infection is not well understood. In HPV-infected women, an IgG response to HPV-16 and HPV-18 is often observed 4 to 12 months after HPV DNA detection (Carter et al., 1996; Lehtinen and Paavonen, 2001). In the sera of patients with HNSCC, the presence of antibodies against HPV-16 is significantly more frequently observed compared to individuals not having HNSCC (Mork et al., 2001). However, while antibodies against the viral capsid proteins are markers of past or present infection, antibodies against E6 and E7 are markers more clearly associated with malignant disease (Herrero et al., 2003; Lehtinen and Paavonen, 2001).

C. HPV and Prognosis in Tonsillar Cancer In 1998, a survival analysis of 31 patients with tonsillar cancer using tumor pRB expression demonstrated a significantly better survival for patients with pRB-negative tumors (Andl et al., 1998). HPV presence and survival were not analyzed separately. However, there was an indication of a significant correlation between lack of pRB expression and presence of HPV (Andl et al., 1998). It was first reported in 2000 that HPV is a favorable prognostic factor in tonsillar cancer (Gillison et al., 2000; Mellin et al., 2000). In one of these studies (Mellin et al., 2000) on 60 patients with tonsillar cancer, it was found that 52% of the patients with HPV-positive tumors were tumor-free 3 years after diagnosis, as compared to 21% of patients with HPV-negative tumors (Fig. 1). Patients with HPV-positive tumors also exhibited a significantly longer 5-year survival compared to patients with HPV-negative tumors (53.5 compared to 31.5%, p ¼ 0.047, log-rank test). HPV was a favorable prognostic factor independent of tumor stage, age, gender, and grade of differentiation (Mellin et al., 2000). In the study by Gillison et al. (2000) on 253 head and neck cancer patients, 60 had oropharyngeal cancers (mostly tonsillar cancers) and disease-specific survival was significantly improved for the HPV-positive oropharyngeal cancer group compared to the HPV-negative group. However, among patients with non-oropharyngeal cancers, the disease-specific survival was similarly independent of HPV status (Gillison et al., 2000). Accordingly, the prognostic value of HPV did not seem to hold for head and neck cancer in general, but only for tonsillar

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Fig. 1 Number and percent of disease-free patients at 3 years after diagnosis for each stage and HPV status. Reproduced from Mellin et al., 2000, with permission from Wiley.

cancer specifically (Gillison et al., 2000; Paz et al., 1997; Riethdorf et al., 1997; Snijders et al., 1996). Additional reports on HPV as a favorable factor for tonsillar cancer have subsequently been reported (Dahlgren et al., 2003; Friesland et al., 2001; Mellin et al., 2002; Ringstro¨m et al., 2002; Strome et al., 2002). Moreover, in a recent study on the prognostic value of HPV in HNSCC, HPV was not found to have any prognostic value for HNSCC as a whole (Koskinen et al., 2003). However, in this study, all tonsillar tumors were HPV positive and all patients with tonsillar cancer remained alive during the observation period (which for all HNSCC ranged between 1.4 and 89.6 months, mean 24.5 months) (Koskinen et al., 2003). In subsequent studies, the possible importance of the viral load and physical status of HPV on the clinical outcome was evaluated for tonsillar cancer by Mellin et al. (2002) and for HNSCC in general by Koskinen et al. (2003). Mellin et al. (2002) analyzed the presence of HPV in 22 fresh-frozen pretreatment tonsillar samples by general and type-specific PCR using a quantitative PCR. Eleven of the 22 analyzed patients had HPV-16 positive tonsillar cancer; the viral load ranged from 10 to 15,500 HPV-16 copies/cell with a mean of 190 copies/cell. The estimation of viral load in tonsillar cancer was thus in line with the previous study of Klussman et al. (2001). In the six tonsillar cancers and their metastases that were analyzed, the viral copy number per -actin varied between 5.8 and 152.6. Interestingly, Mellin et al. (2002) found that patients with >190 HPV-16 copies in their tumor cells had a significantly longer survival rate than did patients with <60 HPV-16 copies/cell (p ¼ 0.039, log-rank test), as shown in Fig. 2. It is

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possible that a stronger immune response is generated against tumor cells that contain a high viral content, which may explain why patients with a large viral load have a better survival. Koskinen et al. (2003) studied 61 fresh-frozen biopsies obtained at diagnosis from patients with squamous cell carcinoma of the hypopharynx (10), larynx (18), tongue (15), oral cavity (13), and tonsil (5). The frequency of HPV was determined by SPF10 PCR-screening with a general probe hybridization and INNO-LiPA HPV genotyping assay, while HPV quantification was determined by a real-time quantitative PCR (Koskinen et al., 2003). Using these procedures, 61% of the samples were HPV positive; 5/5 (100%) of the tonsillar samples, 11/15 (73%) of the tongue, and around 50% of the samples from the remaining locations were HPV positive, with HPV-16 as the predominant type (85%). There were large individual variations in HPV viral load in general. However, the median copy numbers of E6 DNA in tonsillar specimens were approximately 80,000 times higher than those in nontonsillar HNSCC types (Koskinen et al., 2003). One reason why HPV may be a favorable prognostic factor in tonsillar cancer is because it is present in large quantities and, hence, may induce a better immune response compared to that induced by HPV in nontonsillar locations. However, the number of tonsillar cancer patients in the study of Koskinen et al. (2003) was small (n ¼ 5). Since all five tonsillar cancer patients remained alive and

Fig. 2 Kaplan-Meier graph showing significantly better disease-specific survival in patients with tumors with HPV-16 copies/-actin compared to patients with tumors with HPV-16 copies/-actin in tonsillar cancer (p ¼ 0.039, log-rank test, n ¼ 11). Reproduced from Mellin et al., 2002, with permission from Wiley.

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had HPV-positive tumors, it was not possible to correlate clinical outcome to the presence or absence of HPV or high viral load (Koskinen et al., 2003). Clinical outcome could not be correlated to the physical status of HPV (Koskinen et al., 2003; Mellin et al., 2002). Mellin et al. (2002) studied the physical status of HPV by rliPCR (Kalantari et al., 2001). They found that almost all HPV-16 positive samples were either full length, full length and deleted, or deleted episomal, thus making a comparison between different physical forms impossible. Furthermore, the finding that HPV-16 is mainly episomal in tonsillar cancer was supported by an earlier study where the analysis of physical status was performed by Southern blot and two-dimensional gel electrophoresis. In this study, HPV-16 was found to be episomal, while HPV-33 was both episomal and integrated in tonsillar cancer (Snijders et al., 1992). In the study by Koskinen et al. (2003), the physical status of HPV was based on the assumption that the E2 gene is lost on integration, which is not always the case (Kalantari et al., 2001). A real-time PCR was performed by the amplification of the E2 and E6 genes simultaneously in separate reaction tubes. The presence of equal amounts of E2 and E6 copies was regarded as indicative of the presence of episomal viral genomes, while the presence of E6 amplification without E2 amplification was assumed to be indicative of the virus in an integrated form (Koskinen et al., 2003). Two tonsillar samples were suggested to have episomal forms and three samples were suggested to contain integrated forms of HPV. Since all patients remained alive, it was impossible to correlate the physical status of HPV to clinical outcome. In another study, fluorescence in situ hybridization (FISH) rather than PCR was used to determine the physical state of HPV in HNSCC (including 12 tonsillar carcinomas) (Hafkamp et al., 2003). It was found that 8/12 of the tonsillar tumors exhibited FISH staining, corresponding to integrated HPV-16; 7/8 tumors also harbored episomal HPV (Hafkamp et al., 2003). One tumor contained HPV-16 as well as HPV-18 in integrated and episomal forms (Hafkamp et al., 2003). Although it is not yet clear whether HPV-16 exists mainly in episomal or integrated forms or both, the studies above indicate that episomal HPV-16 can still induce malignant transformation in tonsillar cancer (Hafkamp et al., 2003; Koskinen et al., 2003; Mellin et al., 2002; Snijders et al., 1992). This has been similarly demonstrated in approximately one-third of cervical cancer cases (Cullen et al., 1991; Das et al., 1992; Kalantari et al., 2001; Watts et al., 2002). One mechanism for episomal transformation could be that extra-chromosomal HPV-16 exhibits genetic modifications in the long control region (LCR). This leads to an enhanced activity of the LCR, which may, in turn, influence the promotor activity of E6/E7 transcription (Chen et al., 1997; Dong et al., 1994; May et al., 1994; Watts et al.,

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2001). Whether the LCR is mutated in a similar way in tonsillar cancer is unknown, but it is possible.

D. HPV and Radiosensitivity in Tonsillar Cancer An alternative explanation for HPV as a positive prognostic factor in tonsillar cancer, other than that HPV-positive tumor cells may be more immune sensitive, is that HPV-positive tonsillar tumors are more radiosensitive than HPV-negative tonsillar tumors. Friesland et al. (2001) examined whether the favorable clinical outcome of patients with HPV-positive tonsillar cancer was due to a greater radiosensitivity of HPV-positive tonsillar cancer compared to HPV-negative tonsillar cancer. Forty patients with tonsillar cancer were selected—21 with tumors in complete remission (CR) after radiotherapy and 19 without complete remission (non-CR) (Friesland et al., 2001). The tumors were analyzed for presence of HPV by PCR and for overexpression of p53 by immunohistochemistry (IHC). The evaluation of response to radiotherapy was performed one month after completion of radiotherapy by clinical examination and, when required, using a biopsy from the primary tumor site (Friesland et al., 2001). The response was classified as CR when evidence of the tumor could not be observed and as non-CR when there was viable tumor remaining. Among the 40 patients, 34 had tumors with amplifiable DNA and could be evaluated for HPV status. There were no statistically significant differences in sensitivity to radiotherapy between patients with tumors with a different HPV or p53 status (assayed by IHC) (Friesland et al., 2001). This was unexpected since both CR and HPV are favorable prognostic factors for survival (Friesland et al., 1999, 2001; Mellin et al., 2000). Furthermore, when following the patients for two years, HPV and p53 status could still not be correlated with the radiotherapy response. In absolute numbers, there were more p53-negative, HPV-negative patients in the non-CR group compared to the CR group; however, the number of patients was too small for further statistical analysis (Friesland et al., 2001). A more recent analysis concerned 65 tonsillar cancer patients who had received preoperative radiotherapy (Mellin, 2002). Although a tendency toward a better response was observed for HPV-positive patients (71% in CR) compared to HPV-negative patients (53% in CR), this difference was not statistically significant (p ¼ 0.134, 2 test) (Mellin, 2002). Moreover, when these patients were included in a Kaplan-Meier survival analysis, it was found that a CR induced by radiotherapy may be more crucial than HPV status (p ¼ 0.00002, log-rank test). In the HPV-positive group, the patients in CR had a significantly better survival compared to the

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HPV-positive patients without CR (p ¼ 0.0011, log rank). Similarly, in the HPV-negative group, the patients with CR had a significantly better survival compared to the HPV-negative patients without CR (p ¼ 0.00006, log-rank test). Nevertheless, although not statistically significant, the HPVpositive patients in the CR group had an 82% five-year survival rate compared to 61% in the HPV-negative patients (p ¼ 0.26, log-rank test). In line with these data, a study on HPV and local control after radiotherapy in oropharyngeal cancer reported that HPV-positive tumors seemed to be more sensitive to radiotherapy (Lindel et al., 2001). However, only 14% of the patients were found to be HPV positive in this study and the results were not statistically significant. As has been mentioned, there is still no simple straightforward explanation as to why patients with HPV-positive tonsillar cancer survive better than those with HPV-negative tonsillar cancer. Nonetheless, it is obvious that the explanation cannot be based on differences in radiosensitivity alone. So far there is only a nonsignificant indication in the limited number of cases that have been tested that HPV-positive tonsillar cancer responds slightly better than HPV-negative tonsillar cancer to radiotherapy. It is likely that other tumor biological differences, such as p53 status, between tonsillar cancers harboring oncogenic HPV and tumors lacking HPV may explain differences in radiotherapy sensitivity. This was suggested in a study by Obata et al. (2000) where oropharyngeal cancer with wild-type p53 seemed to respond better to radiotherapy compared to tumors with mutated p53. The role of p53 will be discussed further. In summary, the presence of HPV in tonsillar cancer does not have a major impact on tumor radiosensitivity. The combined data still suggest that the prognostic value of HPV (particularly if correlated to a high viral load) could mainly be due to an immune response to HPV.

E. HPV and Correlation to Cell-Cycle Proteins in Tonsillar Cancer Abrogation of normal p53 function appears to be a vital step for cancer development. This allows the tumor to overcome the normal ‘‘checkpoints’’ of cell regulation and proliferate despite the accumulation of mutations. It is known that E6 (of ‘‘high risk’’ or ‘‘oncogenic’’ HPVs) can bind and degrade p53 (Scheffner et al., 1990; zur Hausen, 1996). Hence, in HNSCC, p53 function may be disabled by the E6 oncogene or by gene aberration in the p53 pathway. Furthermore, E7 of oncogenic HPV types can bind to another vital tumor suppressor pRb, leading to its dysfunction and contributing to dysregulation of the cell cycle (Dyson et al., 1989, 1992; zur Hausen, 1996).

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Generally, in cervical cancer, most cases are HPV positive and p53 is not mutated. However, in the few cases where cervical cancer has been shown to be HPV negative, p53 mutations have been found (Crook et al., 1992). In tonsillar cancer, the p53 status has yet to be proven and the situation is different and more complex since only about 50% of tonsillar cancers are reported to be HPV positive (Gillison et al., 2000; Mellin et al., 2000). Moreover, several reports have been published on the presence of mutated p53 in normal and pre-neoplastic mucosa of the aerodigestive tract of smokers and in the normal mucosa of aged individuals (Dolcetti et al., 1992; Gusterson et al., 1991; Pavelic et al., 1994). Furthermore, it has been reported that p53 mutation is more common in smokers than in former and nonsmokers, and that HPV is more common in the latter group (Koch et al., 1999). It is thus theoretically possible to anticipate the presence of p53 mutations independent of or in combination with HPV in HNSCC in smokers. Lewensohn-Fuchs et al. (1994) reported the occurrence of aberrant p53 expression measured by immunohistochemistry (IHC) in HPV-positive tonsillar cancer (2 cases). In a later sequence analysis of p53 in one of the HPV-positive tonsillar cancers with aberrant p53 expression, an inframe deletion of intron 7 in the p53 gene was demonstrated (Magnusson et al., 1995). Moreover, it was subsequently shown that normal tissue close to HPV-negative, p53 IHC-negative tumors could express aberrant p53 (Munck-Wikland et al., 1997). A number of reports have been published on the combined presence of HPV and mutated p53 in tonsillar cancers (or oropharyngeal cancers with a dominance of tonsillar cancers) (Balz et al., 2003; Friesland et al., 2001; Gillison et al., 2000; Snijders et al., 1994). In general, when p53 is estimated by IHC, p53 overexpression in tonsillar cancer is demonstrated in approximately 50% of all cases, irrespective of HPV status (Friesland et al., 2001; Snijders et al., 1994). However, when p53 is analyzed by sequencing, mutations are not always observed (Balz et al., 2003; Snijders et al., 1994). In one study, 4/8 (50%) of the HPV-positive tonsillar tumors had elevated p53 expression when tested by IHC, but only 1/10 (10%) showed a p53 mutation (in addition to one silent mutation, yielding no change in amino acids and p53 function) (Snijders et al., 1994). Until 1995, around 40% of HNSCC were found to be p53 mutated when using sequencing methods (Sidransky, 1995). However, in a later HNSCC study by Balz et al. (2003), p53 aberrations were found in 80% of the cases. In this instance, sequencing of the entire coding region of p53 was performed and not restricted to exon 5–8, as commonly done previously. In this same study, p53 mutations were shown to be equally frequent in tumors of the hypopharynx (27/30; 90%), the larynx (35/44; 80%), and the oral cavity (11/14; 79%), but less frequent in oropharyngeal tumors (14/33; 45%). Correspondingly, the prevalence of

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HPV-16/18 transcripts was considerably higher in carcinomas of the oropharynx (17/33; 52%) as compared to those of the hypopharynx (8/30; 27%), the oral cavity (4/14; 29%), or the larynx (8/44; 18%) (Balz et al., 2003). Interestingly, as many as 77% of the tumors with wild-type p53 expressed E6, while only 18% of the cases exhibited the combined presence of p53 mutations and E6 transcripts. In another study by Hafkamp et al. (2003), the presence of HPV was observed in 9 out of 16 (53%) oropharyngeal tumors. When these tumors were analyzed by PCR-SSCP for exon 5–8 of the p53 gene, p53 mutations could not be detected (Hafkamp et al., 2003). The fact that tonsillar tumors with HPV demonstrate p53 by IHC and possible p53 overexpression does not exclusively reflect the p53 mutation status (Mineta et al., 1998; Riethdorf et al., 1997). It is known that in normal cells the wild-type p53 tumor suppressor protein has a short half-life and normal p53 is therefore assumed to be undetectable by IHC (Soong et al., 1996). In cells with mutated p53, the level of the protein is stabilized, and the protein appears to be ‘‘overexpressed.’’ This is at least partly due to the inability of mutated p53 to induce transcription of MDM2, a protein that both degrades p53 and inhibits p53 transcription (Haupt et al., 1997; Thut et al., 1995). However, it has also been suggested that wild-type p53 may be stabilized in tumors by disturbances in the degradation of p53 or normally elevated due to the presence of DNA damage (Mineta et al., 1998, and references therein). Furthermore, there may be p53 mutations that do not stabilize p53 sufficiently enough to be detected by IHC. Alternatively, p53 mutations may alter the p53 structure in such a way that the antibody does not react with the epitope of the protein (Saunders et al., 1999). Nevertheless, it has been shown that in 80% of the reported p53 mutations in HNSCC, p53 was also IHC positive, while only 54% of the IHC-positive cases had mutated p53 when examined by sequencing (Riethdorf et al., 1997). With regard to pRB function, in cervical cancer and cervical dysplasia, where pRB is dysfunctional, it has been demonstrated that the cell cycle protein p16INKa is overexpressed (Klaes et al., 2001; Sano et al., 1998). Furthermore, it has been shown that high levels of p16INKa correlate with inactive pRB (Parry et al., 1995). Thus, overexpression of p16INKa can been seen as a surrogate biomarker for the presence of high-risk HPV in these lesions (Sano et al., 1998). Interestingly, in the study by Andl et al. (1998), there was a correlation between the presence of HPV and p16INK4A overexpression with simultaneous pRB downregulation in 6 out of 9 cases. As previously mentioned, Andl and associates also found a correlation between lack of pRB expression and presence of HPV and that the pRB-defective tumors showed an overexpression of p16INK4A (Andl et al., 1998). Similarly, a separate study also demonstrated a correlation between HPV (types 16 and

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33) and an overexpression of p16INK4A (Klussmann et al., 2003). Sixteen out of 18 HPV-positive tonsillar carcinomas showed p16 expression, whereas no HPV-negative tonsillar carcinomas displayed p16 (Klussmann et al., 2003). To conclude, the data show that p53 mutations can exist in HPV-positive HNSCC (including tonsillar cancer), but that they are less common in HPVpositive than HPV-negative HNSCC despite positive p53 by IHC (Andl et al., 1998; Balz et al., 2003; Brachman et al., 1992; Gillison et al., 2000; Hafkampf et al., 2003; Snijders et al., 1994; van Houten et al., 2001). Moreover, it must be mentioned that p53 DNA contact mutations generally have a strong negative impact on clinical outcome in HNSCC (Erber et al., 1998). It cannot therefore be completely excluded that the presence of wild-type p53, despite p53 IHC staining, contributes to the better clinical outcome in patients with HPV-positive tonsillar cancer. Finally, a few current studies in tonsillar cancer have shown a correlation between the presence of HPV, pRB negativity, and overexpression of p16INK4A (Andl et al., 1998; Klussmann et al., 2003), which is in line with recent findings in cervical cancer (Klaes et al., 2001; Sano et al., 1998).

F. HPV and Genetic Instability in Tonsillar Cancer Regardless of the p53 status or sensitivity to radiotherapy, HPV has still been indicated to be a favorable prognostic factor in tonsillar cancer (Dahlgren et al., 2003; Friesland et al., 2001; Gillison et al., 2000; Mellin et al., 2000, 2002, 2003). To find a possible explanation for this, a number of tonsillar cancer biopsies were examined with regard to their genetic instability (Dahlgren et al., 2003; Mellin et al., 2003). The degree of DNA aberration (diploid or aneuploid) was analyzed by Image Cytometry (ICM) (Mellin et al., 2003) and the chromosomal composition was analyzed by comparative genomic hybridization (CGH) (Dahlgren et al., 2003). All tonsillar cancers showed genetic aberrations. However, HPV-positive tonsillar cancers had a tendency to be somewhat less affected than HPV-negative tonsillar cancers when analyzed both by ICM and by CGH (Dahlgren et al., 2003; Mellin et al., 2003). Using ICM, the degree of DNA aberration was examined to study whether HPV positive and negative tumors differed in DNA content and whether the extent of DNA aberration also affected clinical outcome (Mellin et al., 2003). The DNA content was estimated in 58 primary tonsillar tumors. A normal diploid cell nuclear DNA content was referred to as a 2c value (c is the haploid genome equivalent). The fraction (percent) of cancer cells exceeding 2.5c was indicated as the 2.5c exceeding rate (2.5c ER), while the percent of cancer cells exceeding 5c was referred to as the 5c exceeding

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rate (5c ER). Cancer cells with a DNA content value above 2.5c were regarded as either proliferating diploid cells or aneuploid cells, whereas cells with a DNA content value above 5c ER were regarded to be aneuploid (hyperploid). A lesion was classified as diploid if none of the cells exceeded 5c ER and less than 35% of the cells were between 2.5c and 5c ER. Most of the examined tumors showed a high degree of aneuploidy, including a mean of 17.5% of the cells in 5c ER, and only 7 (12%) of the tumors were demonstrated to be diploid. Cancer patients with a 5c ER below the mean value were, to a higher degree, disease-free after 3 years and had a better survival compared to cancer patients with a 5c ER above the mean value. However, these disparities were not statistically significant. HPV-positive tumors tended to have a lower mean 5c ER, 13 as compared to 22% for the HPV-negative tumors (p ¼ 0.066, 2 test). Furthermore, significantly fewer HPV-positive tumors had a 5c ER above the mean value compared to the HPV-negative tumors (p ¼ 0.026, 2 test). Nevertheless, independent of DNA content, patients with HPV-positive cancer were, to a higher degree, disease-free 3 years after diagnosis compared to patients with HPV-negative cancer (Table I) (Mellin et al., 2003). In the study by Dahlgren and associates (Dahlgren et al., 2003), 25 of the original 40 original tonsillar cancer biopsies gave reliable profiles when analyzed by CGH. Of the 25, 15 (60%) were HPV positive. The complete data are summarized in Table II. Among the 15 HPV-positive tonsillar cancers, the gains ranged between 0 and 10 per case (mean 2.3) and the losses between 0 and 6 per case (mean 2.1), which results in an average number of chromosomal aberrations (ANCA) value of 4.5. The most frequently occurring gain in this tumor group was seen on chromosome 3q23-qter, where 11/15 cases (73%) had an increased copy number, whereas the most frequently occurring loss (7 cases, 47%) was seen on chromosome 11q14-q25 (Tables II and III). Amplifications were seen for 3 cases on chromosome 3q with the minimal region 3q24-q27 and chromosomes 8, 9,

Table I HPV Statusa and Meanb Value of 5c ER Correlated to Disease Free 3 Years after Diagnosisc

Disease-free patients

HPVþ and 17.5% in 5c ER

HPVþ and 17.5% in 5c ER

HPV and 17.5% in 5c ER

HPV and 17.5% in 5c ER

Total

2/3 (67%)

9/15 (60%)

2/12 (17%)

2/12 (17%)

15/42 (36%)

aAll HPV-positive tumors were also HPV-16 positive, except for one tumor that contained both HPV-16 and HPV-33. bMean value of 5c ER was 17.5%. cReproduced from Mellin et al. (2003) with permission from Anticancer Research.

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Table II

HPV and CGH Data of Squamous Cell Carcinomas of the Tonsila

Case no.b

HPV

2-2548 3-2549 4-2551

þ þ þ

5-2552 7-2553

þ 

9-2759 10-2557

þ þ

11-2758



13-2559 14-2560 15-2561 17-2562 18-2564 22-2568 23-2569 25-2578 28-2571 30-2572

 þ þ  þ þ   þ þ

32-2579 36-2573 38-2760

  

40-2581 41-2574 44-2576 46-2577

 þ þ þ

DNA losses – – 3pter-p12, 9pter-p12, 11q13-q25, 18pter-p11.2, 18q21-q23 – 3p, 4p, 5, 9p, 10p, 11q13-q25, 13, 15, 18q 13, 16q 2q35-q37, 3p22-p12, 11q, 13, 14q24-q32, 16 11q14-qter, 13, 21 – 11q13-q25 4p, 11q13-q25, 16q – 4q28-q35, 13 7p, 7q11.1-q21, 14q, 16q – 7q22-q36, 10q23-q26 3p, 14 11p, 11q14-q25, 18q21-q23 – 10q, 11q13-q25 4, 11q13-q25, 13, 18q12-q23

2q14.3-q23 10q, 11q – 11q13-q25, 20pter-p11.1

DNA gains 3q13.1-q29 – 3q23-q29, 5pter-p12, 11q12-q13, 12pter-p11.2, 18q11.1-q21

11q12-q13 3q, 7q, 8q24.3, 11q12-q13, 12pter-p11.2, 14, 19q, 20, 22q13-q13 3q21-qter, 8q23-qter 3q, þþ3q24-q27c, 5, 8, þþ9p, þþ9q13-q34, 10, 11p12, 12, 17, 18 1q21-q41, 3q26.1-qter, 8q21.1-qter, 11q13, 18q, 20q – 3q 16p, 17 7q11.2-q31 – 3q, 8, 10, þþ20p, 20q – 3q, 7p21-p11.2, 7q11.1-q22 3q, 20p þþ3q, þþ8, 10p, 20 – 3q, 17, 20q, 22 1pter-p34.3, 1q, 2q11.1-q31, þþ7q, 8q22-q24.3, 9p24-p12, 11q11-q13, 12p, 14q22-q32, 15, 16p, 17q, þþ17q11.2-q12, 18q11.1-12, 19p 8, 12, 17q25 þþ3q 3 3q21-q29, 20q11.1-q13.3

aReproduced from Dahlgren et al. (2003) with permission from Wiley. bCase number and NCBI SKY/CGH database accession number. cAmplified.

and 20p in one tumor each (Table II). Among the 10 HPV-negative samples, the gains ranged between 0 and 16 (mean 4.0) and the losses between 0 and 9 (mean 2.1), resulting in an ANCA value of 6.1. The most frequent gains were seen on chromosomes 3q26.1-qter 7q11.1-22 and 8q24.3, where

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Table III Differences of Gains and Losses of HPVa Positive and Negative Tonsillar Cancerb

All cases HPVþ cases HPV cases

þ3q

þ7q

11q

15 (60%) 11 (73%) 4 (40%)

4 (16%) 0 (0%) 4 (40%)

11 (44%) 7 (47%) 4 (40%)

þ3q/11q 9 (36%) 6 (40%) 3 (30%)

aHPV DNA-positive according to PCR data, with GP5þ/6þ or CPI/IIG primers. bReproduced from Dahlgren et al. 2003 with permission from Wiley.

4 cases (40%) showed copy number increases (Table II and III). Two amplifications were seen in this subgroup, one on chromosome 7q and one on chromosome 17q11.2-q12. The most frequent loss mapped to chromosome 11q14q-q25, where 4 cases (40%) had copy number losses (Tables II and III). In summary, when studying all 25 samples, gains on 3q, 8q, 20q, 11q14-qter, and 13q were common. Furthermore, of the 15 cases with a gain on chromosome 3q, 9 (of which 3 had amplification of 3q24-q27) had loss of chromosome 11q14-q25 as well (Table III). In this study, there was also a better survival for patients with HPV-positive tumors (p ¼ 0.002, log-rank test). In addition, some distinct features of the karyograms of the HPV-positive and -negative groups could be identified (Table III). Presence of 3q24-increase was significantly higher (p ¼ 0.049) in HPV-positive tumors (73%) compared to HPV-negative tumors (40%), and an increase of 7q11.2-q22 was only present in HPV-negative tumors (p ¼ 0.017). The minimal affected region on chromosome 3 was 3q24-qter, which also appeared to be amplified in three HPV-positive samples, while no amplification was seen in the HPV-negative group (Dahlgren et al., 2003). The frequent gain on 3q, particularly in the HPV-positive samples, argues in favor of HPV as a possible etiological agent in tonsillar cancer since gain of 3q is a frequent and early event in cervical cancer where the role of HPV is undisputed (Heselmeyer et al., 1996). Moreover, a very similar pattern with a significant chromosomal gain of 3q (with the smallest region 3q22-25) was also obtained in HPV-positive vulvar cancers (Allen et al., 2002). In vulvar cancer, however, a gain of 8q21 (and not chromosome 7) was observed in HPV-negative cancers, while the chromosome arms of 3p and 11q were lost in both categories of vulvar cancer (Allen et al., 2002). Of functional interest, Redon and associates (2002) concluded, after highresolution amplicon mapping and transcriptional analysis, that cyclin L is mapped to 3q25 and is likely to be involved in head and neck cancers. Second, the RNA component of the human telomerase gene hTERC maps to chromosome band 3q26. The fact that this particular genomic imbalance

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occurs in HPV-positive tumors could point to this gene as a reasonable candidate for functional interactions of HPV proteins with telomerase, as has been demonstrated previously (Veldman et al., 2001). Hence, in line with other studies of HNSCC, the karyotypes were complex and the genomic imbalances were mapped to almost all chromosomes (Struski et al., 2002). The pattern of DNA gains and losses, however, was not random. Common chromosomal imbalances in the entire material were gains of chromosomes 3q24-qter, 8q23-qter, 20q, 11q12-q13, 12p, and 17q, and losses 11q14-q25 and 13. The gains of 3q and 8q in this explicit study of tonsillar cancer were similar to previous observations in HNSCC (Bergamo et al., 2000). Nevertheless, the loss of chromosome 3p, which is common in other reports on HNSCC, was not frequent in these tonsillar cancers (Bockmuhl et al., 1998). This may be due to the fact that the study of Dahlgren et al. (2003) only comprised tonsillar cancer, whereas other reports have included tumors from different locations in the head and neck. Furthermore, gains of 11q12-q13 are common in HNSCC, and several putative oncogenes map to this region. The result that both HPV-positive and HPV-negative tumors contained a high degree of DNA content and that few tumors displayed diploidy was in accordance with what has been observed in cervical cancer (Lorenzato et al., 2001; Rihet et al., 1996; Skyldberg et al., 2001). Nonetheless, independent of genetic instability and chromosomal setup in both the studies of Mellin et al. (2003) and Dahlgren et al. (2003), a better clinical outcome was observed for patients with HPV-positive tumors compared to HPV-negative tumors.

V. HPV AND OTHER TUMORS OF THE HEAD AND NECK The prevalence of HPV in HNSCC varies significantly when comparing different reports (Gillison et al., 2000; Haraf et al., 1996, Klussmann et al., 2001; McKaig et al., 1998; Ringstro¨m et al., 2002; van Houten et al., 2001). However, in general, HPV is more commonly found in HNSCC patients younger than 60 years of age (Ringstro¨m et al., 2002; Snijders et al., 1996). The variation in presence of HPV in HNSCC is most likely explained by differences in the composition of the material, including tumor site, the preparation and storage of the material, as well as the methods applied for analysis (Mellin, 2002; Mellin et al., 2002). However, it is also possible that HPV is detected less frequently in locations other than the tonsil, since it is present in lower copies/cell in the other locations (Koskinen et al., 2003). Nevertheless, the overall prevalence of HPV in HNSCC is 14 to

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35% when detected by PCR technique, 25% by Southern blot hybridization, 18% by in situ hybridization, and 6% by dot blot hybridization (Gillison et al., 2000; Haraf et al., 1996; Klussmann et al., 2001; McKaig et al., 1998; Ringstro¨m et al., 2002; van Houten et al., 2001). Moreover, the two most common locations for HPV after tonsillar cancer are in tongue cancer (19–100%) and laryngeal cancer (10–50%) (Koskinen et al., 2003; Matzow et al., 1998; Mellin, 2002; Syrjanen, 2003). HPV-16 is found in the majority of HNSCC, followed by HPV-33. HPV-31, HPV-18, and others are found less frequently (Gillison et al., 2000; Koskinen et al., 2003). In summary, HPV, more specifically HPV-16, is rather commonly found in HNSCC, in particular, in patients below the age of 60. This information could be of potential interest in future HPV preventive and therapeutic vaccination studies.

VI. HPV VACCINES As has been mentioned, HPV is present in approximately half of all tonsillar tumors and is a favorable prognostic factor for clinical outcome (Dahlgren et al., 2003; Friesland et al., 2001; Gillison et al., 2000; Mellin, 2003; Mellin et al., 2000, 2002). This appears not to be due to differences in radiosensitivity between HPV-positive and HPV-negative tonsillar tumors (Friesland et al., 2001), or to the fact that HPV-positive tumors are somewhat less genetically unstable compared to HPV-negative tonsillar tumors (Dahlgren et al., 2003; Mellin et al., 2003). In fact, patients with a high HPV load in their tonsillar tumors seem to have a longer survival compared to those with a low HPV load (Mellin et al., 2002). In addition, HPV has not been found to be a favorable prognostic factor in other nontonsillar HPV-positive HNSCC tumors (Gillison et al., 2000), where the viral load is much lower, as demonstrated by Koskinen et al. (2003). These findings suggest that an immune response against the virus may contribute toward a better clinical outcome (Mellin et al., 2002). If so, it could be important to enhance this antiviral immune response in HPV-positive tonsillar cancer patients in order to increase disease-free survival and also to possibly limit the extent of the potentially disabling treatment that patients receive. Moreover, it is also possible that the induction of an antiviral immune response could be useful for patients with tonsillar cancer or other HNSCC tumors with a lower HPV load. As also has been discussed, several lines of evidence suggest that cellmediated immune responses are important in controlling both HPV infections and HPV-associated neoplasms (for a review, see Devaraj et al., 2003). First, the prevalence of HPV-related diseases, including both infections and

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neoplasms, is increased in transplant patients and immunodeficiency virus (HIV) infected patients (Schafer et al., 1991). Both these patient types have impaired cellular immunity. Second, animal studies have demonstrated that in immunized animals, the induction of HPV-specific cytotoxic T-cells by HPV peptide vaccination can protect mice from a subsequent challenge with an HPV-16 transformed tumor cell line (Feltkamp et al., 1993). Therefore, HPV vaccines with potential for therapeutic efficacy should generate enhanced HPV-specific cell-mediated immune responses. For prevention, however, it may be sufficient to elicit an antibody response (Breitburd et al., 1995; Heidari et al., 2002; Koutsky et al., 2002; Lehtinen et al., 2003; Suzich et al., 1995; Vlastos et al., 2003; for a review, see Devaraj et al., 2003). Hypothetically, there are at least three different approaches to HPV vaccine development, as reviewed in detail previously (Devaraj et al., 2003). The first approach is to use a prophylactic vaccine and prevent virus from establishing an infection in the epithelium, mainly through the induction of neutralizing antibodies against the viral capsid. In this way, HPV-induced neoplasia will be inhibited. A prophylactic vaccine may, however, be of less benefit to individuals who already have an established infection. The second approach would therefore be to induce a cellular immune response (both CD4þ and CD8þ) to prevent and induce the regression of neoplastic lesions. The strategy used in this kind of therapeutic approach is known as antigen-specific immunotherapy, in which effector cells, particularly T-cells, are primed against HPV epitopes known to be expressed by the neoplastic cells (tumor-specific antigens) such as E6 and E7. Finally, the third approach would be to combine prophylaxis and therapy in one vaccine to cover the needs of people who are newly exposed to high-risk virus and for people with current infection and neoplasia. In the past, HPV vaccine development has been hampered by difficulties in producing HPV in culture and the lack of animal models for HPV infections. However, the discovery that the L1 capsid protein spontaneously assembles to form empty capsids known as virus-like particles (VLPs) (Hagensee et al., 1993; Kirnbauer et al., 1993; Zhou et al., 1991) and the use and development of different animal systems has resulted in the development of potential candidates for future prophylactic and therapeutic vaccines. Knowledge regarding prophylactive vaccines was initially obtained by conducting VLP immunization experiments on cutaneous and mucosal animal papillomaviruses, such as cottontail rabbit papillomavirus (CRPV), canine oral papillomavirus (COPV), and bovine papillomavirus (BPV) (Breithburd et al., 1995; Suzich et al., 1995). VLP vaccination strategies in a murine polyomavirus system were also reported to successfully prevent

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viral infection, not only in mice with a normal immune system but also in T-cell deficient immune mice (Heidari et al., 2002; Vlastos et al., 2003). HPV vaccination development has also benefited from the use of tumorigenic mouse lines, such as C3 and TC-1, that express HPV-16 E6 and E7 proteins. These tumor lines have been used in different in vivo studies, demonstrating the immunogenic potential of E6- and E7-derived products against tumor outgrowth (Feltkamp et al., 1993; Lin et al., 1996). The field of HPV vaccination is now in a much stronger position than one or two decades ago. Successful, preventive strategies using HPV VLPs have been reported (Koutsky et al., 2002; Lehtinen et al., 2003; J. T. Schiller, personal communication). The use of preventive vaccines based on HPV-16 and HPV-18 VLPs, such as those used in clinical trials today, may also be beneficial for the prevention of HPV-positive HNSCC, including tonsillar cancers. However, therapeutic vaccines are much less established, even though experimental systems have been described (Feltkamp et al., 1993; Gerard et al., 2001; Gunn et al., 2001; Lin et al., 1996; Meneguzzi et al., 1991; and for review, see Devaraj et al., 2003). Nevertheless, clinical trials using HPV-16, E6/E7 chimeric VLPs, or HPV-16 specific peptides to boost the immune system against established HPV infection are presently ongoing in patients with cervical cancer (J. T. Schiller and C. Melief, personal communications). It is definitely important to use these strategies in HNSCC as well, particularly in tonsillar cancers, where a high HPV load is a favorable prognostic factor, indicating the potential impact of a strong immune response. If these strategies work, it is possible that these patients may need less traumatic treatment in the future than that required today. In summary, successful preventive vaccination strategies to avoid HPV infection in the cervix and ongoing therapeutic vaccination attempts in cervical cancer should also be applied for the prevention of HPV infection in the head and neck region. Adjuvant treatment in patients with HPV-positive tonsillar tumors should also be attempted.

VII. CONCLUSIONS HPV, with a predominance of HPV-16, is present in approximately half of all tonsillar tumors and is a favorable prognostic factor for clinical outcome (Gillison et al., 2000; Mellin et al., 2000, 2002, 2003). This appears not to be due to differences in radiosensitivity between HPV-positive and HPV-negative tonsillar tumors (Friesland et al., 2001) or to the fact that HPV-positive tumors are somewhat less genetically unstable than HPVnegative tonsillar tumors (Dahlgren et al., 2003; Mellin et al., 2003). The fact that patients with high HPV viral loads in their tumors seem to have a

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longer survival than do patients with lower viral loads suggests that there could be an immune response against the virus that contributes to the better clinical outcome (Mellin et al., 2002). If so, it might be important to therapeutically enhance this antiviral-immune response. This would also emphasize the reason for conducting expanded preventive vaccination trials using preventive anti-HPV-16 vaccines.

ACKNOWLEDGMENTS This work was supported in part by the Swedish Cancer Foundation, the Stockholm Cancer Society, the Stockholm City Council, and Karolinska Institute.

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