Development and interlaboratory agreement of real-time PCR for HPV16 quantification in liquid-based cervical samples.

High risk HPV infection is the necessary cause for the development of precancerous and cancerous lesions of the cervix. Among HPV, HPV16 represents the most carcinogenic type. Since the determination of HPV16 DNA load could be clinically useful, we assessed quantitative real-time PCR targeting E6HPV16 and albumin genes on two different platforms. Series of SiHa cells diluted in PreservCyt were used to assess repeatability and reproducibility of two in-house real-time PCR techniques run in two different laboratories to determine HPV16 load. Furthermore, 97 HPV16 positive cervical samples were evaluated to estimate inter-center variability using Bland-Alman plots. As a whole, both techniques presented coefficients of variation for HPV16 load measurement similar to those established for other virus quantification with commercial kits. Moreover, the two real-time PCR techniques showed a very good agreement for HPV16 load calculation. Finally, we emphasize that robust HPV16 DNA quantification requires normalization of viral load by the cell number.

Biological and clinical relevance of transcriptionally active human papillomavirus (HPV) infection in oropharynx squamous cell carcinoma.

Human papillomaviruses (HPV) are associated with a subset of head and neck squamous cell carcinoma (HNSCC), particularly HPV16. This study analyzed the presence and genotype of high risk HPVs, viral DNA load and transcription of the E6/E7 mRNAs, in 231 consecutive HNSCC. Twelve out of 30 HPV16 DNA-positive tumors displayed high E6/E7 mRNAs levels and were localized in the oropharyngeal region. While HPV-free and non-transcriptionally active HPV-related patients showed similar 5-years survival rates, E6/E7 expression was associated with a better prognosis. This emphasizes the importance of considering the transcriptional status of HPV-positive tumors for patient stratification. A gene expression profiling analysis of these different types of tumors was carried out. The most significant differentially expressed gene was CDKN2A, a known biomarker for HPV-related cancer. Assessing both the expression level of the E6/E7 mRNAs and of CDKN2A in HNSCC is required to detect active HPV infection. Chromosomic alterations were investigated by Comparative Genomic Hybridation (CGH) analysis of tumors with transcriptionally active HPV and HPV-negative tumors. The loss of the chromosomal region 16q was found to be a major genetic event in HPV-positive lesions. A cluster of genes located in 16q21-24 displayed decreased expression levels, notably APP-BP1 that is involved in the modulation of the transcriptional activity of p53. In conclusion, this study highlights important criteria required to predict clinically active HPV infection, identifies new biological pathways implicated in HPV tumorigenesis and increases the understanding of HPV-HNSCC physiopathology that is required to develop new targets for therapy.

Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma.

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.

HPV prevalence, viral load and physical state of HPV-16 in cervical smears of patients with different grades of CIN.

Human papillomavirus (HPV) infection is the most important event in malignant transformation of human cervical epithelium. We analysed in cervical smears, HPV genotypes with a focus on single/multiple infections, then characteristics of HPV-16 infections (presence of other genotypes, viral load and physical state) according to the grade of histological lesions. The purpose of this study was to know if these parameters could allow to differentiate histological diagnoses. DNA was extracted from 363 cervical samples corresponding to 24 cases without lesion, 96 CIN1, 92 CIN2, 144 CIN3 and 7 cancers. Our results show that HPV-16 was predominant and its prevalence increased with the severity of lesions (CIN1: 27.1%; CIN3: 65.3%). In addition, we showed that the frequency of single infections, as compared with multiple infections, increased with the severity of the lesion (CIN1: 25.0%; CIN3: 54.8%). Among HPV-16 positive samples (n = 170), we found that viral load, determined on cervical samples by real-time PCR, did not vary significantly according to the different CIN grades. Concerning HPV-16 integration, the mixed and integrated HPV-16 forms, already present in women with normal histology, increased to the benefit of pure episomal forms with the severity of lesions (normal cervix: 28.6%; CIN3: 73.8%). Thus, our data raise the question of the viral load as a valuable clinical parameter to discriminate between lesion grades. Moreover, we emphasize integration as an early event in cervical carcinogenesis, increasing with the severity of lesions. Finally, this study underlines the importance of single versus multiple infections linked to the severity of CIN.

[The epidemiology of genital human papillomavirus infections]. / Epidémiologie des infections génitales a papillomavirus.

Human papillomavirus (HPV) genital infections are the most frequent sexually transmitted infections. Their prevalence varies considerably with age and geographic area. The incidence of these infections is particularly high at the onset of the sexual activity. High-risk HPV type 16 is the most prevalent type, and in general, high-risk HPV are more frequent than low-risk HPV. The majority of genital HPV infections is asymptomatic, but can induce lesions in the genital tract: genital warts due to low-risk HPV; high-grade lesions due to high-risk HPV; cancers with a more or less important fraction, depending on the localization, due to high-risk HPV. In particular, high-risk HPV are responsible for almost all cervical carcinomas.