Morena Trevisan

Prevalence and Follow-Up of Occult HCV Infection in an Italian Population Free of Clinically Detectable Infectious Liver Disease

Background Occult hepatitis C virus infection (OCI) is a recently described phenomenon characterized by undetectable levels of HCV-RNA in serum/plasma by current laboratory assays, with identifiable levels in peripheral blood mononuclear cells (PBMCs) and/or liver tissue by molecular tests with enhanced sensitivity. Previous results from our group showed an OCI prevalence of 3.3% in a population unselected for hepatic disease. The present study aimed to evaluate OCI prevalence in a larger cohort of infectious liver disease-free (ILDF) subjects. Clinical follow-up of OCI subjects was performed to investigate the natural history of the infection. Methods and Findings 439 subjects referred to a Turin Blood Bank for phlebotomy therapy were recruited. They included 314 ILDF subjects, 40 HCV-positive subjects and 85 HBV-positive subjects, of whom 7 were active HBV carriers. Six subjects (4/314 ILDF subjects [1.27%] and 2/7 active HBV carriers [28%]) were positive for HCV-RNA in PBMCs, but negative for serological and virological markers of HCV, indicating OCI. HCV genotypes were determined in the PBMCs of 3/6 OCI subjects two had type 1b; the other had type 2a/2c. OCI subjects were followed up for at least 2 years. After 12 months only one OCI persisted, showing a low HCV viral load (3.73×101 UI/ml). By the end of follow-up all OCI subjects were negative for HCV. No seroconversion, alteration of liver enzyme levels, or reduction of liver synthesis occurred during follow-up. Conclusions This study demonstrated the existence of OCI in ILDF subjects, and suggested a high OCI prevalence among active HBV carriers. Follow-up suggested that OCI could be transient, with a trend toward the decrease of HCV viral load to levels undetectable by conventional methods after 12–18 months. Confirmation studies with a longer follow-up period are needed for identification of the OCI clearance or recurrence rates, and to characterize the viruses involved.

MGMT promoter methylation in plasma of glioma patients receiving temozolomide.

Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays a role in cellular response to alkylating agents. In the present study aimed to: (i) evaluate the concordance between MGMT promoter methylation status in tumor tissue and plasma; (ii) monitor MGMT promoter methylation status in plasma taken before and during temozolomide treatment; (iii) explore the value of MGMT promoter methylation status in plasma as a prognostic/predictive biomarker in glioma patients. We enrolled 58 patients with histologically confirmed glioma at different grades of malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-embedded tumor tissue was available for 48 patients. Blood samples were collected from all patients before temozolomide treatment (baseline) and at each MRI examination for a 12-month period. MGMT promoter methylation status was assessed in both sample types by real time PCR with a specific probe. The frequency of MGMT promoter methylation was 60.4xa0% in tumor tissue and 41.38xa0% in plasma. MGMT promoter methylation status was concordant in the two sample types (Kappaxa0=xa00.75, 95xa0% confidence interval (CI) 0.57–0.93; p valuexa0<0.001). Overall and progression-free survival were longer in patients with methylated MGMT promoter. Mortality was higher in patients with unmethylated MGMT promoter, whether in tumor tissue [hazard ratio (HR) 2.21; 95xa0% CI 0.99–4.95] or plasma (HR 2.19; 95xa0% CI 1.02–4.68). Progression-free survival was shorter in patients with unmethylated MGMT promoter, whether in tissue (HR 2.30; 95xa0% CI 1.19–4.45) or plasma (HR 1.77; 95xa0% CI 0.95–3.30). The cumulative incidence of unmethylated MGMT promoter in plasma at baseline was 58xa0%, and reached virtually 100xa0% at 12xa0months. In conclusion MGMT promoter methylation status in tumor tissue and plasma was highly concordant, and both were associated with longer survival, supporting the role of the detection of methylated MGMT promoter in predicting treatment response. However we suggest caution in using plasma as a surrogate of tumor tissue due to possible false-negative results.

Prenatal Paracetamol Exposure and Wheezing in Childhood: Causation or Confounding?

Background Several studies have reported an increased risk of wheezing in the children of mothers who used paracetamol during pregnancy. We evaluated to what extent this association is explained by confounding. Methods We investigated the association between maternal paracetamol use in the first and third trimester of pregnancy and ever wheezing or recurrent wheezing/asthma in infants in the NINFEA cohort study. Risks ratios (RR) and 95% confidence intervals (CI) were estimated after adjustment for confounders, including maternal infections and antibiotic use during pregnancy. Results The prevalence of maternal paracetamol use was 30.6% during the first and 36.7% during the third trimester of pregnancy. The prevalence of ever wheezing and recurrent wheezing/asthma was 16.9% and 5.6%, respectively. After full adjustment, the RR for ever wheezing decreased from 1.25 [1.07–1.47] to 1.10 [0.94–1.30] in the first, and from 1.26 [1.08–1.47] to 1.10 [0.93–1.29] in the third trimester. A similar pattern was observed for recurrent wheezing/asthma. Duration of maternal paracetamol use was not associated with either outcome. Further analyses on paracetamol use for three non-infectious disorders (sciatica, migraine, and headache) revealed no increased risk of wheezing in children. Conclusion The association between maternal paracetamol use during pregnancy and infant wheezing is mainly, if not completely explained by confounding.

Seasonality modifies methylation profiles in healthy people.

DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (pu200a=u200a0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.

Age and geographic variability of human papillomavirus high-risk genotype distribution in a large unvaccinated population and of vaccination impact on HPV prevalence

BACKGROUNDnThe prevalence of infections with human papillomavirus (HPV) specific genotypes differs by age and areas. Knowledge of these differences will help predicting how prophylactic HPV vaccination and screening program could best be integrated.nnnOBJECTIVESnTo investigate variations in the HPV distribution between areas and ages in Italy and the impact of vaccination on HPV prevalence.nnnSTUDY DESIGNn37,367 women aged 25-60 years who attended cervical screening in eight different areas in Northern and Central Italy were tested for HPV infection with the high-risk hybrid capture (hr-HC2) assay. hr-HC2 positive samples were genotyped by an intensive integrated strategy.nnnRESULTSnhr-HPV types were detected in 79.1% of HC2 positive women. HPV16 was the most frequent type, followed by HPV31, HPV18 and HPV56. A statistically significant variability in HPV type distribution between centres (overall χ84df(2)=195.86p<0.001) was observed. No significant overall difference in the HPV type distribution was observed in the age groups 25-34, 35-44 and 45-60 years. Considering cross-protection, overall 57.6% (95%CI 56.0-59.3) of all infections by hr-HPV types was preventable by vaccination with the bivalent vaccine and 49% (95%CI 46.9-51.1) with the quadrivalent vaccine. The variability between centres was statistically significant with both bivalent (χ7df(2)=43.8, p<0.0001) and quadrivalent vaccine (χ7df(2)=32.9, p<0.0001).nnnCONCLUSIONSnWe observed differences in HPV genotype distribution according to centres but not to age. Results suggest that the higher proportion of HPV16/18 related high grade CIN in younger women could be the result of faster progression and not of earlier infection by these types.

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

BackgroundThe 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.MethodsA 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, Nu2009=u2009150) or grade 3 (CIN3, Nu2009=u2009129). 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.ResultsHPV 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).ConclusionsThis 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.

Temozolomide as salvage treatment for recurrent intracranial ependymomas of the adult: a retrospective study

BACKGROUNDnFew data are available on temozolomide (TMZ) in ependymomas.We investigated the response, survival, and correlation with MGMT promoter methylation in a cohort of patients with adult intracranial ependymoma receiving TMZ as salvage therapy after failure of surgery and radiotherapy.nnnPATIENTS AND METHODSnWe retrieved clinical information from the institutional database and follow-up visits, and response to TMZ on MRI was evaluated according to the MacDonald criteria.nnnRESULTSnEighteen patients (median age, 42 y), with either WHO grade III (10) or grade II (8) ependymoma were evaluable. Tumor location at diagnosis was supratentorial in 11 patients and infratentorial in 7. Progression before TMZ was local in 11 patients, local and spinal in 6 patients, and spinal only in one patient. A median of 8 cycles of TMZ (1-24) was administered. Response to TMZ consisted of complete response (CR) in one (5%) patient, partial response (PR) in 3 (17%) patients, stable disease (SD) in 7 (39%) patients, and progressive disease (PD) in 7 (39%) patients. Maximum response occurred after 3, 10, 14, and 15 cycles, respectively, with neurological improvement in 2 patients. All 4 responding patients were chemotherapy naïve. Both anaplastic (2) and grade II (2) tumors responded. Median progression-free survival and overall survival were 9.69 months (95% CI, 3.22-30.98) and 30.55 months (95% CI, 12.85-52.17), respectively. MGMT methylation was available in 11 patients and was not correlated with response or outcome.nnnCONCLUSIONnTMZ has a role in recurrent chemo-naïve adult patients with intracranial ependymoma, regardless of tumor grade and MGMT methylation. We suggest that, after failure of surgery and radiotherapy, TMZ should be considered as a possible first-line treatment for recurrent ependymoma.

Pre-diagnostic metabolite concentrations and prostate cancer risk in 1077 cases and 1077 matched controls in the European Prospective Investigation into Cancer and Nutrition

BackgroundLittle is known about how pre-diagnostic metabolites in blood relate to risk of prostate cancer. We aimed to investigate the prospective association between plasma metabolite concentrations and risk of prostate cancer overall, and by time to diagnosis and tumour characteristics, and risk of death from prostate cancer.MethodsIn a case-control study nested in the European Prospective Investigation into Cancer and Nutrition, pre-diagnostic plasma concentrations of 122 metabolites (including acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexose and sphingolipids) were measured using targeted mass spectrometry (AbsoluteIDQ p180 Kit) and compared between 1077 prostate cancer cases and 1077 matched controls. Risk of prostate cancer associated with metabolite concentrations was estimated by multi-variable conditional logistic regression, and multiple testing was accounted for by using a false discovery rate controlling procedure.ResultsSeven metabolite concentrations, i.e. acylcarnitine C18:1, amino acids citrulline and trans-4-hydroxyproline, glycerophospholipids PC aa C28:1, PC ae C30:0 and PC ae C30:2, and sphingolipid SM (OH) C14:1, were associated with prostate cancer (pu2009<u20090.05), but none of the associations were statistically significant after controlling for multiple testing. Citrulline was associated with a decreased risk of prostate cancer (odds ratio (OR1SD)u2009=u20090.73; 95% confidence interval (CI) 0.62–0.86; ptrendu2009=u20090.0002) in the first 5xa0years of follow-up after taking multiple testing into account, but not after longer follow-up; results for other metabolites did not vary by time to diagnosis. After controlling for multiple testing, 12 glycerophospholipids were inversely associated with advanced stage disease, with risk reduction up to 46% per standard deviation increase in concentration (OR1SDu2009=u20090.54; 95% CI 0.40–0.72; ptrendu2009=u20090.00004 for PC aa C40:3). Death from prostate cancer was associated with higher concentrations of acylcarnitine C3, amino acids methionine and trans-4-hydroxyproline, biogenic amine ADMA, hexose and sphingolipid SM (OH) C14:1 and lower concentration of glycerophospholipid PC aa C42:4.ConclusionsSeveral metabolites, i.e. C18:1, citrulline, trans-4-hydroxyproline, three glycerophospholipids and SM (OH) C14:1, might be related to prostate cancer. Analyses by time to diagnosis indicated that citrulline may be a marker of subclinical prostate cancer, while other metabolites might be related to aetiology. Several glycerophospholipids were inversely related to advanced stage disease. More prospective data are needed to confirm these associations.

Prenatal exposure to antibiotics and wheezing in infancy: a birth cohort study

The role of prenatal antibiotic exposure in the development of childhood wheezing is debated. We evaluated whether this association could potentially be explained by confounding factors. Antibiotic use in the first and third trimester of pregnancy, wheezing in children aged ≤18u2005months and confounding factors were assessed in singletons participating in the NINFEA (Nascita e Infanzia: gli Effetti dellAmbiente) birth cohort (n=3530 for first-trimester exposure and n=3985 for third-trimester exposure). There was no evidence of an association between antibiotic exposure in the first trimester of pregnancy and ever-wheezing (adjusted risk ratio (RR) 1.02, 95% CI 0.80–1.30) or recurrent wheezing (RR 0.99, 95% CI 0.54–1.82). For the third-trimester exposure, the crude RRs (95% CI) of ever-wheezing and recurrent wheezing were 1.34 (1.10–1.64) and 2.72 (1.80–4.11), respectively, which decreased to 1.12 (0.90–1.39) and 2.09 (1.32–3.29) after adjustment. The RRs of wheezing after genitourinary infections during pregnancy were increased independently of antibiotic treatment. In conclusion, the association between prenatal antibiotic exposure and infant wheezing could be largely explained by confounding factors, in particular respiratory infections during pregnancy. An excess risk of wheezing after antibiotic exposure during the third trimester of pregnancy remains after adjustment. Confounders explain increased risk of infant wheeze after antibiotic exposure in the first but not third trimester http://ow.ly/Tu1U0

Ki-67 proliferation index but not mitotic thresholds integrates the molecular prognostic stratification of lower grade gliomas

Despite several molecular signatures for “lower grade diffuse gliomas” (LGG) have been identified, WHO grade still remains a cornerstone of treatment guidelines. Mitotic count bears a crucial role in its definition, although limited by the poor reproducibility of standard Hematoxylin & Eosin (H&E) evaluation. Phospho-histone-H3 (PHH3) and Ki-67 have been proposed as alternative assays of cellular proliferation. Therefore in the present series of 141 LGG, the molecular characterization (namely IDH status, 1p/19q co-deletion and MGMT promoter methylation) was integrated with the tumor “proliferative trait” (conventional H&E or PHH3-guided mitotic count and Ki-67 index) in term of prognosis definition. Exclusively high PHH3 and Ki-67 values were predictor of poor prognosis (log rank test, P = 0.0281 for PHH3 and P = 0.032 for Ki-67), unlike standard mitotic count. Based on Cox proportional hazard regression analyses, among all clinical (age), pathological (PHH3 and Ki-67) and molecular variables (IDH, 1p/19q codeletion and MGMT methylation) with a prognostic relevance at univariate survival analysis, only IDH expression (P = 0.001) and Ki-67 proliferation index (P = 0.027) proved to be independent prognostic factors. In addition, stratifying by IDH expression status, high Ki-67 retained its prognostic relevance uniquely in the IDH negative patient (P = 0.029) doubling their risk of death (hazard ratio = 2.27). Overall, PHH3 immunostaining is the sole reliable method with a prognostic value to highlight mitotic figures in LGG. Ki-67 proliferation index exceeds PHH3 mitotic count as a predictor of patients prognosis, and should be integrated with molecular markers in a comprehensive grading system for LGG.