Methylation in host and viral genes as marker of aggressiveness in cervical lesions: Analysis in 543 unscreened women.

OBJECTIVE: The present study aimed to evaluate the association between altered methylation and histologically confirmed high grade cervical intraepithelial neoplasia (hgCIN). METHODS: Methylation levels in selected host (CADM1, MAL, DAPK1) and HPV (L1_I, L1_II, L2) genes were measured by pyrosequencing in DNA samples obtained from 543 women recruited in Curitiba (Brazil), 249 with hgCIN and 294 without cervical lesions. Association of methylation status with hgCIN was estimated by Odds Ratio (OR) with 95% confidence interval (CI). RESULTS: The mean methylation level increased with severity of the lesion in the host and viral genes (p-trend < 0.05), with the exception of L1_II region (p-trend = 0.075). Positive association was found between methylation levels for host genes and CIN2 and CIN3 lesions respectively [CADM1: OR 4.17 (95%CI 2.03-8.56) and OR 9.54 (95%CI 4.80-18.97); MAL: OR 5.98 (95%CI 2.26-15.78) and OR 22.66 (95%CI 9.21-55.76); DAPK1: OR 3.37 (95%CI 0.93-12.13) and OR 6.74 (95%CI 1.92-23.64)]. Stronger risk estimates were found for viral genes [L1_I: OR 10.74 (95%CI 2.66-43.31) and OR 15.00 (95%CI 3.00-74.98); L1_II: OR 73.18 (95%CI 4.07-1315.94) and OR 32.50 (95%CI 3.86-273.65); L2: OR 4.73 (95%CI 1.55-14.44) and OR 10.62 (95%CI 2.60-43.39)]. The cumulative effect of the increasing number of host and viral methylated genes was associated with the risk of CIN2 and CIN3 lesions (p-trend < 0.001). CONCLUSIONS: Our results, empowered by a wide cervical sample series with a large number of hgCIN, supported the role of methylation as marker of aggressiveness.

Case-control study of HLA-G promoter methylation status, HPV infection and cervical neoplasia in Curitiba, Brazil: a pilot analysis.

BACKGROUND: The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has been established, but the mechanisms that favor HPV persistence in cervical cells are still unknown. The diminished capability of the immune system to control and resolve HPV infection is one of several hypotheses. The tolerogenic protein HLA-G has shown aberrant expression in a variety of cancers, which has been suggested as a mechanism for tumor escape from immunosurveillance. In the present study we evaluate the role of epigenetic modification (promoter de-methylation) of the HLA-G gene on susceptibility to HPV infection and development of high-grade cervical lesions. METHODS: A case-control study was carried out in Curitiba, Brazil, between February and June 2010. A total of 789 women aged 15-47 years were recruited: 510 controls with normal cervical cytology, and 279 cases with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN2, N = 150) or grade 3 (CIN3, N = 129). All women were administered a questionnaire by interview, which collected information on demographic and lifestyle factors, and a cervical sample was collected. HPV DNA detection was performed by GP5+/GP6+ primer-mediated PCR. HPV-positive samples were genotyped by multiplex PCR. A pilot analysis of HLA-G promoter methylation was carried out in a subset of the study population (96 cases and 76 controls) by pyrosequencing. HLA-G methylation and HPV infection status of cases and controls were compared, and confounding factors were computed by t Student and non-parametric Wilcoxon tests. Comparison of HLA-G methylation between cases and controls was assessed by the Bonferroni correction. The association of HLA-G methylation with CIN2/3 was evaluated by logistic regression. RESULTS: HPV prevalence was 19.6% in controls and 94.3% in CIN2/3 cases. HPV16, 31, 33, 35 and 18 were the most prevalent types. Methylation analysis of seven CpGs in the HLA-G promoter did not reveal any spontaneous de-methylation events in CIN2/3 cases (mean proportion of methylation: 75.8%) with respect to controls (mean 73.7%; odds ratio 1.01, 95% confidence interval 0.96, 1.07). CONCLUSIONS: This study did not support the hypothesis that spontaneous de-methylation events in the HLA-G promoter play a primary role in promoting escape from immunosurveillance in the development of precancerous cervical lesions.

Antinuclear antibodies in patients with cervical lesions and invasive cervical cancer.

BACKGROUND: Antinuclear antibodies (ANA) have been found in several types of cancer although the meaning of its presence is not completely known. AIM: To study the prevalence of ANA in patients with cervical intraepithelial lesion and invasive cervical cancer. METHODS: A total of 205 women who underwent screening for cervical cancer or treatment at the Erasto Gaertner Cancer Hospital in Curitiba - Brazil, were enrolled in the study. Based on their latest cervical colposcopy-guided biopsy results, they were divided into four groups: CIN-I: 19.4%; CIN-II: 24.0%; CIN-III: 24.0%; and invasive cancer: 32.4%. As control were studied 68 healthy controls. ANA was searched by immunofluorescence in Hep-2 cells evaluating the pattern and titer. RESULTS: Controls had 4/68 (5.8%) of ANA positivity and patients with CIN and invasive cancer had 15.1% (p = 0.001). Patients with CIN-I and CIN-II had the same prevalence of ANA as controls (p = 1.0 and p = 0.11 respectively), but not those with CIN-III (p = 0.03) and invasive cancer (p = 0.05). The most common ANA immunofluorescence pattern was fine speckled pattern (38.7%) and fine dense speckled pattern (38.7%); the mean titer was 1:160. CONCLUSION: ANA is more common in invasive cervical lesions than in controls or non invasive lesions. To understand the meaning of this finding more studies are needed.

MBL serum concentration in women with HPV presenting CIN III lesions.

PURPOSE: This study proposes to assess MBL serum concentrations in HPV positive women that developed high risk preinvasive lesions, CIN III. MATERIALS AND METHODS: A total of 90 consecutive women (mean age 32.2 years, range 18-45 years) with CIN III lesions confirmed by cone biopsy were enrolled as cases at the gynecology cancer department of the Erasto Gaertner Hospital, a center of reference for cancer treatment in Southern Brazil. All the patients were HPV positive. In addition, 81 healthy women, voluntary blood donors, were enrolled as healthy control subjects (mean age 33.3 years, range 14-71 years) with no evidence of HPV infection, tested by hybrid capture, or CIN lesions, from the same geographic area. RESULTS: The median concentration of MBL in the cases was 912 ng/ml (IQR: 100-5820 ng/ml) and in the controls was 1207.3 ng/ml (IQR 100-4320.6 ng/ml). No statistically significant difference was observed between cases and controls. CONCLUSION: This study reports data on MBL serum concentrations in HPV infected women for the first time. The analysis of our findings did not show a statistically significant difference between MBL serum concentrations in HPV women that developed CIN III lesions and healthy controls.

IFNG (+874 T/A) polymorphism and cervical intraepithelial neoplasia in Brazilian women.

Our objective was to investigate the relationship between IFNG (+874 T/A) polymorphism and cervical intraepithelial neoplasia (CIN) in a population of Brazilian women. Ninety-six women, CIN II (48) and CIN III (48) and 50 normal controls, were enrolled in this study. DNA was extracted from blood samples by the salting out method and polymerase chain reaction (PCR) amplified. IFNG genotyping was performed by the PCR-Sequence Specific Primer method, using the Cytokine Genotyping Tray. There were no differences in genotypic frequencies between CIN patients and controls. However, after sample stratification into CIN II and III, a higher frequency of the AA genotype in CIN II versus control group (P = 0.05) was observed. When the comparison was performed between CIN groups, a higher frequency of the AA genotype in CIN II versus CIN III (P = 0.05) was observed. This study suggests that IFNG +874 T/A polymorphism responsible for the genetic differences in interferon (IFN)-gamma production may influence the human papillomavirus (HPV) clearance and cervical malignant progression. Further understanding of the role of this cytokine may contribute to the development of a biomarker of HPV infection and resulting in the improvement of squamous intraepithelial lesions treatment.