Distribution of HPV genotypes in cervical intraepithelial lesions and cervical cancer in Tanzanian women

<p>Abstract</p> <p>Background</p> <p>Infection with human papillomavirus (HPV) is associated with uterine cervical intraepithelial neoplasia (CIN) and invasive cancers (ICC). Approximately 80% of ICC cases are diagnosed in under-developed countries. Vaccine development relies on knowledge of HPV genotypes characteristic of LSIL, HSIL and cancer; however, these genotypes remain poorly characterized in many African countries. To contribute to the characterization of HPV genotypes in Northeastern Tanzania, we recruited 215 women from the Reproductive Health Clinic at Kilimanjaro Christian Medical Centre. Cervical scrapes and biopsies were obtained for cytology and HPV DNA detection.</p> <p>Results</p> <p>79 out of 215 (36.7%) enrolled participants tested positive for HPV DNA, with a large proportion being multiple infections (74%). The prevalence of HPV infection increased with lesion grade (14% in controls, 67% in CIN1 cases and 88% in CIN2-3). Among ICC cases, 89% had detectable HPV. Overall, 31 HPV genotypes were detected; the three most common HPV genotypes among ICC were HPV16, 35 and 45. In addition to these genotypes, co-infection with HPV18, 31, 33, 52, 58, 68 and 82 was found in 91% of ICC. Among women with CIN2-3, HPV53, 58 and 84/83 were the most common. HPV35, 45, 53/58/59 were the most common among CIN1 cases.</p> <p>Conclusions</p> <p>In women with no evidence of cytological abnormalities, the most prevalent genotypes were HPV58 with HPV16, 35, 52, 66 and 73 occurring equally. Although numerical constraints limit inference, findings that 91% of ICC harbor only a small number of HPV genotypes suggests that prevention efforts including vaccine development or adjuvant screening should focus on these genotypes.</p

Associations Between Methylation of Paternally Expressed Gene 3 (PEG3), Cervical Intraepithelial Neoplasia and Invasive Cervical Cancer.

Cytology-based screening for invasive cervical cancer (ICC) lacks sensitivity and specificity to discriminate between cervical intraepithelial neoplasia (CIN) likely to persist or progress from cases likely to resolve. Genome-wide approaches have been used to identify DNA methylation marks associated with CIN persistence or progression. However, associations between DNA methylation marks and CIN or ICC remain weak and inconsistent. Between 2008-2009, we conducted a hospital-based, case-control study among 213 Tanzania women with CIN 1/2/3 or ICC. We collected questionnaire data, biopsies, peripheral blood, cervical scrapes, Human papillomavirus (HPV) and HIV-1 infection status. We assessed PEG3 methylation status by bisulfite pyrosequencing. Multinomial logistic regression was used to estimate odds ratios (OR) and confidence intervals (CI 95%) for associations between PEG3 methylation status and CIN or ICC. After adjusting for age, gravidity, hormonal contraceptive use and HPV infection, a 5% increase in PEG3 DNA methylation was associated with increased risk for ICC (OR = 1.6; 95% CI 1.2-2.1). HPV infection was associated with a higher risk of CIN1-3 (OR = 15.7; 95% CI 5.7-48.6) and ICC (OR = 29.5, 95% CI 6.3-38.4). Infection with high risk HPV was correlated with mean PEG3 differentially methylated regions (DMRs) methylation (r = 0.34 p<0.0001), while the correlation with low risk HPV infection was weaker (r = 0.16 p = 0.047). Although small sample size limits inference, these data support that PEG3 methylation status has potential as a molecular target for inclusion in CIN screening to improve prediction of progression. Impact statement: We present the first evidence that aberrant methylation of the PEG3 DMR is an important co-factor in the development of Invasive cervical carcinoma (ICC), especially among women infected with high risk HPV. Our results show that a five percent increase in DNA methylation of PEG3 is associated with a 1.6-fold increase ICC risk. Suggesting PEG3 methylation status may be useful as a molecular marker for CIN screening to improve prediction of cases likely to progress

Neisseria gonorrhoeae Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane

The immunoglobulin A (IgA) protease secreted by pathogenic Neisseria spp. cleaves Lamp1, thereby altering lysosomes in a cell and promoting bacterial intracellular survival. We sought to determine how the IgA protease gains access to cellular Lamp1 in order to better understand the role of this cleavage event in bacterial infection. In a previous report, we demonstrated that the pilus-induced Ca(2+) transient triggers lysosome exocytosis in human epithelial cells. This, in turn, increases the level of Lamp1 at the plasma membrane, where it can be cleaved by IgA protease. Here, we show that porin also induces a Ca(2+) flux in epithelial cells. This transient is similar in nature to that observed in phagocytes exposed to porin. In contrast to the pilus-induced Ca(2+) transient, the porin-induced event does not trigger lysosome exocytosis. Instead, it stimulates exocytosis of early and late endosomes and increases Lamp1 on the cell surface. These results indicate that Neisseria pili and porin perturb Lamp1 trafficking in epithelial cells by triggering separate and distinct Ca(2+)-dependent exocytic events, bringing Lamp1 to the cell surface, where it can be cleaved by IgA protease

Validation of the <i>PEG3</i> pyrosequencing assay.

<p>Defined mixtures (x-axis) of methylated and unmethylated DNAs were prepared and analyzed in quintuplicate by pyrosequencing (y-axis). The results shown represent the mean; error bars indicate standard deviations. The Pearson rho is 0.953 with a p-value of 0.004.</p

Correlation between High and Low Risk HPV status and methylation fraction means at differentially methylated region (DMRs) of <i>PEG3</i>.

<p>High Risk HPV - 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.</p><p>Low/Other Risk HPV – 6, 11, 26,40, 42, 55, 61, 62, 69, 70, 72, 73, 81, 82, 83, 84.</p

Adjusted odds ratios for the associations between mean DMR methylation for <i>PEG3</i>, HPV status and CIN and ICC.

*<p>Controlling for HIV-1 status, HPV positive status, age, and oral contraceptive (OC) use.</p><p>Per 5% methylation increase.</p

Distribution of participant characteristics by case-control status.

*<p>ICC/Cancer subtype: 45 patients had squamous cell carcinoma; 3 had adenosquamous carcinomas.</p><p>High Risk HPV- 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.</p><p>Low/Other Risk HPV – 6,11, 26, 40, 42, 55, 61, 62, 69, 70, 72, 73, 81, 82, 83, 84.</p