Oncogenic HPV types are the major cause of worldwide cervical cancer, but only a small proportion of infected women will develop high-grade cervical intraepithelial neoplasia or cancer (CIN2/3؉). We performed a prospective study including 781 women with normal, atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LGSIL) cytology, and infected or not by high-risk (HR) HPV tested by Hybrid Capture II. Women were followed up every 6 months for a median period of 22 months. Among the HR-HPV-positive women at entry, more than half cleared their virus in 7.5 months; the clearance rate was greater for low viral loads than for high loads and also was higher in women with an initial ASCUS/LGSIL smear than in women with normal cytology. The incidence of cytologic abnormalities strongly depended on baseline viral load and HR-HPV persistence. Maintenance of cytologic abnormalities was associated with the outcome of HR-HPV status (negative100 pg/mL). Conversely, women who were consistently HR-HPV negative or transiently HR-HPV positive, whatever the cytology at baseline was, did not develop CIN2/3؉ during follow-up. Age seemed to affect only the rate of incident HR-HPV infection. In conclusion, our data suggest that women repeatedly tested positive for HR-HPV are at risk of developing CIN2/3؉, even when initial cytology is normal. A high viral load could be used as a short-term marker of progression toward precancerous lesions, although lower load does not inevitably exclude progressive disease.

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Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10 3 cells in normal samples up to 56,354 copies per 10 3 cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10 3 cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.

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Patients with human papillomavirus (HPV)-related oropharyngeal tumors display improved prognosis. The biological basis of this tumor phenotype is poorly understood. We investigated whether increased lymphocyte infiltrate in HPV-positive oropharyngeal squamous cell carcinomas could account for better prognosis. We previously identified, in an Affymetrix GeneChip analysis of 83 HPV-unrelated and 11 HPV-related squamous cell carcinoma of the oropharynx, several candidate genes, including CD8a and CD3f. Their expression was validated in this study by qRT-PCR on an independent clinical series of 144 oropharyngeal tumors. Immunohistochemical staining of tumor specimens was performed to evaluate infiltration of tumor stroma by CD81 and CD41 lymphocytes. The prognostic value of CD8a and CD3f expression levels was measured by Kaplan-Meier and Cox regression model analyses. Immune response-related signaling pathways were found to be deregulated in HPV-positive oropharyngeal tumors. Expression of CD8a, CD3f, granzyme K, CD28 and integrin aL RNAs was upregulated in HPV-positive lesions when compared with HPV-unrelated tumors (p < 0.05). Stroma of HPV-positive tumors was frequently and strongly infiltrated by CD8a-and CD3f-positive T cells. CD8a RNA expression correlated with both improved global (Kaplan-Meier; p 5 0.005; Cox regression: p 5 0.003) and disease-free (Cox regression: p 5 0.04) survival. CD3f RNA expression correlated with improved overall survival (Cox regression: p 5 0.024). These results suggest that an increased cytotoxic T-cell-based antitumor immune response is involved in improved prognosis of patients with HPV-positive tumors.The presence of human papillomaviruses (HPVs; mainly HPV type 16) correlates with the onset and development of about 25% of head and neck squamous cell carcinomas (HNSCC). 1 HPV-positive cancers of the head and neck arise mainly in the oropharynx and define a subgroup of radioand chemosensitive tumors with distinct clinical, pathological and molecular features and improved prognosis with respect to their HPV-negative counterparts. [2][3][4][5] The majority of HPVrelated oropharyngeal squamous cell carcinomas (OSCC) express wild-type TP53, 6,7 which might be involved in the improved response of tumors to treatment. Despite thorough analysis of gene expression profiles of HPV-positive OSCC by several groups, [8][9][10][11] no novel prognostic or predictive biomarker has been identified, and the molecular mechanisms that underlie improved prognosis of HPV-related OSCC are poorly understood.The immune response has an important impact in many HPV-associated tumors and can have either favorable or unfavorable consequences. 12 The importance of the immune response in HPV-related OSCC is less well established. An increased T-cell response against HPV E7 epitopes has been detected in the blood of patients with HPV-positive HNSCC. 13-15 A more recent study has shown that T cells that proliferate and synthesize inflammatory cytokines upon HPV16 E6 and E7 oncoprotein recognition can be isolate...

Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and the development of cervical cancer (CxCa). A rapid and precise diagnosis of the precancerous lesions by conventional cytology or HPV DNA tests remains difficult and often leads to overtreatment. We quantitatively analyzed the HPV16 transcriptome of 80 HPV16 DNA-positive cervical scrapes classified as mild cytologic grade, including no intraepithelial lesion or malignancy (NIL/M; normal, n = 25) and low-grade squamous intraepithelial lesion (LSIL; n = 24), and severe cytologic grade, including high-grade squamous intraepithelial lesion (HSIL; n = 24) and CxCa (n = 7), with novel nucleic acid sequence-based amplification-Luminex assays. In severe lesions, HPV16 E6*II and E1C encoding transcripts were strongly upregulated, whereas spliced E1^E4 and L1 encoding transcripts were markedly downregulated. Using a combination of the four marker transcripts, 100% of CxCa and 67% of HSIL cases were correctly identified as severe, and 74% of LSIL and 92% of NIL/M samples as mild cytologic grade. Compared with a commercially available HPV E6/E7 mRNA assay, the specificity of the marker combination for discriminating severe and mild cytologic lesions increased from 23% to 83%. In conclusion, we identified a novel HPV16 RNA pattern for grading of cervical lesions with a potentially high diagnostic value for the primary screening of CxCa precursors and the triage of cervical lesions.

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