Extralesional detection and load of human papillomavirus DNA: a possible marker of preclinical tumor spread in cervical cancer.

OBJECTIVE: Because the interaction between viral DNA products and cellular regulatory mechanisms is the first step leading to cancerous transformation, the detection of its presence in histologically negative lymph nodes may represent a very early biological step in cancer spread. The quantitative estimate may represent and an indirect sign of active cellular replication. MATERIALS AND METHODS: Cervical and lymph nodes tissues of 13 cases of invasive cervical cancer were analyzed for human papillomavirus (HPV)-DNA presence and viral load by HPV typing and quantification by real-time polymerase chain reaction. RESULTS: HPV-DNA was demonstrated in all tissue samples (primary tumor, positive lymph nodes, negative lymph nodes) with the most prevalence of HPV 16 (61.5%) and single-type infection (69.3%), whereas viral load (mean quantity of DNA copies) is statistically different in negative versus positive lymph nodes (p =.005). CONCLUSIONS: Concordance of viral type and lymph nodes viral load may represent as a useful tool in identifying early metastatic risk of tumor spread.

Determination of human papillomavirus (HPV) load and type in high-grade cervical lesions surgically resected from HIV-infected women during follow-up of HPV infection.

BACKGROUND: The role of human papillomavirus (HPV) load and the importance of multiple-strain HPV infections as biomarkers for the development of cervical disease were evaluated in human immunodeficiency virus (HIV)-positive women. METHODS: A total of 108 samples were analyzed, 64 of which were obtained from 16 HIV-positive women who underwent surgical resection of the cervical cone for treatment of a histologically confirmed high-grade cervical intraepithelial neoplasm (cases) and 44 of which were obtained from 22 HIV-positive women who had high-risk HPV but a negative colposcopy result (controls). Each patient underwent periodic examinations at 6-12-month intervals that included colposcopy, Papanicolaou testing, biopsy (if indicated), and cervical brushing for HPV testing. Viral typing was performed by reverse dot-blot hybridization and quantification of viral load by in-house real-time PCR and commercial assays. RESULTS: Analysis of the cervical-brush samples collected when high-grade squamous intraepithelial lesions were diagnosed revealed that all cases had HPV loads that were significantly higher than those of controls (P=.0004 and P=.0003, by PCR and the Hybrid Capture 2 index [Digene], respectively). Decreasing concentrations of HPV load were observed when comparing samples obtained before and after treatment (P<.0001). The number and type of HPV strains that were detected were not statistically different between cases and controls. CONCLUSIONS: The significantly higher HPV load detected in women with high-grade cervical dysplasia, as well as the dramatic decrease in the load after surgical removal of the lesion, suggest that HPV load is a possible prognostic marker of high-grade SIL.

Few modifications of the Cobas Amplicor HIV Monitor 1.5 test allow reliable quantitation of HIV-1 proviral load in peripheral blood mononuclear cells.

Recent studies have suggested that monitoring the amount of HIV provirus in peripheral blood mononuclear cells (PBMCs) may be a useful end point for HAART where, in combination with plasma viral load, it provides additional information as to the possibility of virus eradication. In the present study, a modified version of the Cobas Amplicor HIV-1 Monitor test (CAHIM), currently used to quantify plasma viremia, have been evaluated to also measure the amount of proviral DNA in PBMCs. The analytical and clinical performance of the modified CAHIM test was assessed by quantifying different amounts of a standard HIV-DNA preparation obtained from the 8E5 cell line and by analysing 165 patients and controls samples. In these experiments, the modified test, that showed a linear dynamic range from 1.7 to 4.7 log10 copies/10(6) cells (r = 0.99) with a maximum CV of 20%, proved able to detect and quantify HIV-DNA in all but one clinical samples, with concentrations varying from 1.3 to 3.8 log10 copies/10(6) cells. During anti-retroviral treatment, the assay revealed different proviral DNA time courses associated with viral load changes and inversely correlated with CD4+ cells count. As expected, HIV-DNA was always detectable even when plasma viremia fell below the CAHIM cut-off. The modified CAHIM test specificity was confirmed by testing 20 HIV-negative samples in triplicates. Taken together, the data showed that the modified CAHIM test can be used to monitor HIV proviral DNA changes during HAART and can help in investigating further the clinical use of this marker.