The integration of the high-risk HPV16 and HPV18 types into the cell genome is considered an important step in malignant transformation. The relationship between the physical status of the virus and clinical/pathological parameters was studied by type-specific and multiplex PCR for E6, E2, and E1 sequences in 86 genital tumors from different sites, consisting of 69 invasive carcinomas (including 5 microinvasive carcinomas), 9 carcinomas in situ, 6 severe dysplasias, and 2 moderate dysplasias. Forty tumors contained HPV16 (46.6%), 7 HPV18 (8.1%), and 39 both viruses (45.3%). HPV16 DNA was found either as pure integrant (35.4%), or pure episome (36.7%), or a mixture of both (27.8%). Conversely, all 46 lesions containing HPV18 showed pure integrated forms. The physical status of both types was not related to the tumor site, the tumor/node/metastasis stage, or the histological differentiation grade of the invasive carcinomas. HPV16 integration was significantly associated with invasiveness. Interestingly, in double infections when HPV16 coexisted with HPV18, its genome was found more frequently in episomal form than in single infections where, conversely, it was mostly integrated (P < 0.0001), suggesting a sort of competition for cell integration sites. The complete HPV18 integration, even in pre-neoplastic lesions, indicates a different behavior in genital transformation compared with HPV16 and may reflect a major aggressiveness of this viral type. In conclusion, virus typing in conjunction with the evaluation of the integration status may provide a better prognostic evaluation together with an improved diagnosis.

Abstract. HPV involvement in head and neck (HN) cancer is still under active investigation. Fresh frozen and archival clinical samples from 115 patients affected by HN carcinomas were analysed by PCR-based methods and direct sequencing. HPV types, intra-type variants, physical status, viral load and viral transcript presence were determined. HPV positivity was correlated with the main clinical-pathological features, including smoker and drinker status, and the clinical outcome. Twenty-one tumours were HPV positive (18.3%) with HPV16 being the most frequent type (n=14) followed by HPV6 (n=4), HPV33, HPV35, and HPV58 (n=1, each type). Tonsil carcinomas contained more high-risk HPV types (6/8; 75%) than all other sites (p=0.0004). HPV16 genome was integrated in all analysed tumours, as pure integrated form or mixed with concomitant episomal forms (4 cases). The viral load showed a wide variability (range, 0.7-485 copies per cell) with the highest value detected in a larynx tumour and the lowest one in a case of cancer of the oral cavity. In 9 HPVpositive samples where mRNA was available, transcripts of viral early oncogenes originating by integrated, episomal or mixed forms of the viral genome were found. A statistically significant correlation was evidenced between HPV and tumour differentiation, being the virus more associated with tumour grade G3/G4. Multivariate Cox regression analysis revealed that lymph-node and grade status were significant independent factors for a worse disease-free survival and overall survival, whereas the HPV status was associated with a better overall survival (OR, 0.33; 95% CI, 0.13-0.81; p=0.01). Taken together these results indicate that distinct pathological mechanisms for the malignant transformation in each single HN subsite should be taken in account; HPV molecular analyses should be considered a valid tool to distinguish subsets of oropharyngeal tumours and HPV presence could be useful for the prognostic assessment of HNSCC. IntroductionTumours of the oral cavity and pharynx have been estimated at 643,000 cases for 2002, with 349,000 deaths (1). The incidence of these cancers is traditionally found to be very high in some areas of the world (e.g., France and South India), but in the last decade other areas (e.g., Eastern Europe and Japan) reported an increasing incidence of head and neck (HN) squamous cell carcinomas (SCC) (2). HN cancer constitutes one of the most difficult pathologies to eradicate. Recurrences are frequent and about a half of patients die (1). The heterogeneity of treatment response and the lack of prognostic factors could be the main causes of the adverse clinical outcome. Moreover, the behaviour variability of each individual tumour has also emphasized the difficulties of managing these tumours.Life habits such as the intake of toxic agents present in tobacco smoke, alcohol drink and betel chewing, or poor hygiene remain major risk factors for these tumours. However, the fraction of HN cancers attributable to these risk factors is close to 80-90%, while the...

Screening for human papillomavirus (HPV) types was performed by a PCR‐ based assay on 29 women (mean age 34.0 years, range 21‐48 years). HPV‐DNA was demonstrated in 16 women (55.2%), with a detection rate of 37.9% in the oral cavity and 34.5% in the genital tract. HPV‐16 was the most prevalent genotype (53.8%), followed by HPV‐6, which was present in 34.6% of the positive samples. Other types were more rarely detected. Five subjects showed concurrent genital tract and oral cavity infections but HPV type‐specific concordance was detected in only 3 patients. Multiple HPV infections were found in 9 of the 26 positive samples, where HPV‐6 appeared frequently associated with the other types. These data confirm the occurrence of mixed HPV infections and the wide diffusion of different types of HPV in the genital mucosa and in the oral cavity; they also stress the need to utilize diagnostic methods with a wide typing capacity.

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.

Head and neck cancer represents one of the most challenging diseases as the mortality remains high despite advances in early diagnosis and treatment. Human papillomavirus has been implicated in a third of head and neck squamous cell carcinomas and human papillomavirus type 16 is strongly associated with carcinomas arising from the oropharynx, the tonsil being the preferred infected site. Novel therapeutic approaches including immunotherapy are currently under investigation. Immune vaccines developed against human papillomavirus in the genital area are already available and could simultaneously protect other anatomical localizations; however, prophylactic vaccines are expected to be effective in reducing the incidence of tumors after many years and, therefore, there is an urgent need to improve therapeutic interventions, such as immunotherapy. To date, human papillomavirus therapeutic vaccines are either at the preclinical level or at early phase human trials for genital pathologies. Nevertheless, accumulating evidence from animal and clinical studies suggests that the enhancement of specific and innate immune responses is effective in clearance of the human papillomavirus infection, promoting a cautious optimism regarding the achievement of an efficacious immunotherapy. This article reviews what has been achieved and what remains to be done in the field for the development of future viral vaccines in head and neck tumors.