Comparison of HPV-positive triage strategies combining extended genotyping with cytology or p16/ki67 dual staining in the Italian NTCC2 study.

BACKGROUND: Each high-risk HPV genotype has different oncogenic potential, and the risk of CIN3+ varies according to genotype. We evaluated the performance of different strategies of HPV-positivity triage combining cytology, p16/ki67 dual staining (DS), and extended genotyping. METHODS: Samples from 3180 consecutive women from the NTCC2 study (NCT01837693) positive for HPV DNA at primary screening, were retrospectively analyzed by the BD Onclarity HPV Assay, which allows extended genotyping. Genotypes were divided into three groups based on the risk of CIN3+. HPV DNA-positive women were followed up for 24 months or to clearance. FINDINGS: Combining the three groups of genotypes with cytology or DS results we identify a group of women who need immediate colposcopy (PPV for CIN3+ from 7.8 to 20.1%), a group that can be referred to 1-year HPV retesting (PPV in those HPV-positive at retesting from 2.2 to 3.8), and a group with a very low 24-month CIN3+ risk, i.e. 0.4%, composed by women cytology or DS negative and positive for HPV 56/59/66 or 35/39/68 or negative with the Onclarity test, who can be referred to 3-year retesting. INTERPRETATION: Among the baseline HPV DNA positive/cytology or DS negative women, the extended genotyping allows to stratify for risk of CIN3+, and to identify a group of women with a risk of CIN3+ so low in the next 24 months that they could be referred to a new screening round after 3 years. FUNDING: Italian Ministry of Health (grant number RF-2009-1536040). Hologic-Genprobe, Roche Diagnostics, and Becton & Dickinson provided financial and non-financial support.

Impact of the Sars-COVID-19 Pandemic on the "Early Diagnosis" of Laryngeal Tumors: Data From Monocentric Tertiary Care Hospital of South Italy.

OBJECTIVE: The aim of this retrospective case-control study is to investigate how the Sars-COVID-19 pandemic has influenced the delay in the early diagnosis of laryngeal cancer in patients who visited the University Hospital "G. Martino" in the last 3 years of the pandemic. It is also proposed to compare the diagnosis, surgical staging, and therapeutic approach offered to patients affected by laryngeal cancer in the years affected by the COVID-19 pandemic compared to cases treated during the 3 years preceding the pandemic METHODOLOGY/PRINCIPAL: A single-center retrospective study was carried out to analyze the effects of the COVID-19 pandemic on the diagnosis and treatment of patients with squamous cell carcinoma of the larynx at our Otorhinolaryngology Division of the "Gaetano Martino" University hospital. The research period, 6 years, was divided into two sub-periods of equal months for which we divided the patients into two groups "Before COVID-19" and "During COVID-19", defined, respectively, from February 2017 to March 2020 and from April 2020 to May 2023. Patients aged 18 years and older with a diagnosis of squamous cell carcinoma, seen and surgically treated at the Otorhinolaryngology division of the "Gaetano Martino" University Hospital and patients referred from other hospitals were included in the study population. Patients affected by malignancies in other sites with secondary laryngeal involvement, patients not amenable to surgical therapy, treated only with radio-chemotherapy therapy, and patients not amenable to surgical therapy were excluded from the study. RESULTS: A total of 141 patients were recruited, divided into 76 patients in the "Before COVID-19" arm and 65 patients in the "During COVID-19" arm, with a male prevalence of 119 patients (84.4%). The mean age of our study population stands at a median of 69 years (percentile 25th 60.00 and percentile 75th 77.00). Smoking and alcohol are two predominant variables in our study population with a prevalence of 89.4% and 79.4%, respectively. A higher number of TNM stage IV cases was detected, 29.2% in the group "During COVID-19" compared to 5.3 detected in the group "Before COVID-19" (P = 0.005). In the "During COVID-19" group, we find an overall percentage of 61.5% for stages II-III-IV versus 36.9% present in the "Before COVID-19" group of patients. An adjuvant radiotherapy treatment was actuated in 30.26% of patients in the "Before COVID-19" versus 41.54% in "During COVID-19". CONCLUSIONS: The most important result of our research was the statistically significant difference in tumor staging at the time of diagnosis between the "Before COVID-19" and "During COVID-19" groups, with a higher mean value in the latter. The current data confirm the previously exposed hypothesis according to which the COVID-19 pandemic has led to a delay in early diagnosis, negatively influencing the staging at the time of the first ENT visit and the subsequent therapeutic approach to be offered to the patient.

Identifying MicroRNAs Suitable for Detection of Breast Cancer: A Systematic Review of Discovery Phases Studies on MicroRNA Expression Profiles.

The analysis of circulating tumor cells and tumor-derived materials, such as circulating tumor DNA, circulating miRNAs (cfmiRNAs), and extracellular vehicles provides crucial information in cancer research. CfmiRNAs, a group of short noncoding regulatory RNAs, have gained attention as diagnostic and prognostic biomarkers. This review focuses on the discovery phases of cfmiRNA studies in breast cancer patients, aiming to identify altered cfmiRNA levels compared to healthy controls. A systematic literature search was conducted, resulting in 16 eligible publications. The studies included a total of 585 breast cancer cases and 496 healthy controls, with diverse sample types and different cfmiRNA assay panels. Several cfmiRNAs, including MIR16, MIR191, MIR484, MIR106a, and MIR193b, showed differential expressions between breast cancer cases and healthy controls. However, the studies had a high risk of bias and lacked standardized protocols. The findings highlight the need for robust study designs, standardized procedures, and larger sample sizes in discovery phase studies. Furthermore, the identified cfmiRNAs can serve as potential candidates for further validation studies in different populations. Improving the design and implementation of cfmiRNA research in liquid biopsies may enhance their clinical diagnostic utility in breast cancer patients.

Human papillomavirus infection in women undergoing in-vitro fertilization: effects on embryo development kinetics and live birth rate.

BACKGROUD: Several studies showed that human papillomavirus (HPV) affects male fertility, but its impact on female fertility and in vitro fertilization (IVF) outcome is not yet clear. METHODS: Objective of this observational, prospective, cohort study was to evaluate the prevalence of HPV infection in women candidate to IVF, and the effects of HPV infection on the kinetic of embryonic development and on IVF outcome. A total number of 457 women candidate to IVF were submitted to HR-HPV test; among them, 326 underwent their first IVF cycle and were included in the analysis on IVF results. RESULTS: 8.9% of women candidate to IVF were HPV-positive, HPV16 being the most prevalent genotype. Among the infertility causes, endometriosis was significantly more frequent in HPV-positive than in negative women (31.6% vs. 10.1%; p < 0.01). Granulosa and endometrial cells resulted HPV-positive in 61% and 48% of the women having HPV-positive cervical swab, respectively. Comparing HPV-positive and negative women at their first IVF cycle, no significant difference was observed in the responsiveness to controlled ovarian stimulation (COS) in terms of number and maturity of retrieved oocytes, and of fertilization rate. The mean morphological embryo score was comparable in the two groups; embryos of HPV-positive women showed a quicker development in the early stages, with a significantly shorter interval between the appearance of pronuclei and their fusion. In the following days, embryo kinetic was comparable in the two groups until the early blastocyst stage, when embryos of HPV-positive women became significantly slower than those of HPV-negative women. Overall, these differences did not affect live birth rate/started cycle, that was comparable in HPV-positive and negative women (22.2 and 28.1%, respectively). CONCLUSIONS: (a) the prevalence of HPV infection in women candidate to IVF is similar to that observed in the general female population of the same age range; (b) HPV infection migrates along the female genital apparatus, involving also the endometrium and the ovary, and perhaps participates in the genesis of pelvic endometriosis; (c) HPV slightly affects the developmental kinetic of in vitro-produced embryos, but does not exert an effect on live birth rate.

The Consensus Project: Participation in cervical cancer screening by the first cohorts of girls offered HPV vaccination at age 15-16 years in Italy.

OBJECTIVE: To evaluate the association between human papillomavirus vaccination status and participation in cervical cancer screening (at age 25) by the first cohorts of girls who were offered vaccination at the age of 15 to 16 years in Italy. METHODS: Women born in 1993, 1994 and 1995 were invited to participate in cervical cancer screening between 2018 and 2020. We report participation in screening by vaccination status in three large areas, Florence province, Piedmont region and Savona province, where the Consensus Project was carried out. The relative risk of participation among vaccinated (≥2 doses) and unvaccinated women was estimated. Odds ratios (OR) of participation by vaccination status were estimated by logistic regression, adjusted by birthplace and birth cohort. RESULTS: Overall, 34,993 women were invited for screening: 13,006 (37.2%) participated and 10,062 of these agreed to participate in the Consensus intervention study. Among the invited women and screening participants, vaccinated women were 51.0% and 60.6%, respectively. Comparing vaccinated and unvaccinated women, the adjusted OR of screening participation was 1.80 (95% confidence interval (CI): 1.72-1.89), 2.17 (95% CI: 1.94-2.42), 1.59 (95% CI: 1.50-1.68) and 1.15 (95% CI: 0.86-1.54) for overall, Florence, Piedmont and Savona, respectively. About 33% of the invited women were unvaccinated and did not participate in screening: 25.8%, 59.5% and 64.2% of women born in Italy, in high migration pressure countries and in advanced development countries, respectively. CONCLUSIONS: Screening participation was higher among vaccinated than unvaccinated women. Active policies are needed to reduce inequalities, targeting the unscreened and unvaccinated population, particularly non-native women, to accelerate cervical cancer elimination in Italy.

An Epidemiological Systematic Review with Meta-Analysis on Biomarker Role of Circulating MicroRNAs in Breast Cancer Incidence.

Breast cancer (BC) is a multifactorial disease caused by an interaction between genetic predisposition and environmental exposures. MicroRNAs are a group of small non-coding RNA molecules, which seem to have a role either as tumor suppressor genes or oncogenes and seem to be related to cancer risk factors. We conducted a systematic review and meta-analysis to identify circulating microRNAs related to BC diagnosis, paying special attention to methodological problems in this research field. A meta-analysis was performed for microRNAs analyzed in at least three independent studies where sufficient data to make analysis were presented. Seventy-five studies were included in the systematic review. A meta-analysis was performed for microRNAs analyzed in at least three independent studies where sufficient data to make analysis were presented. Seven studies were included in the MIR21 and MIR155 meta-analysis, while four studies were included in the MIR10b metanalysis. The pooled sensitivity and specificity of MIR21 for BC diagnosis were 0.86 (95%CI 0.76-0.93) and 0.84 (95%CI 0.71-0.92), 0.83 (95%CI 0.72-0.91) and 0.90 (95%CI 0.69-0.97) for MIR155, and 0.56 (95%CI 0.32-0.71) and 0.95 (95%CI 0.88-0.98) for MIR10b, respectively. Several other microRNAs were found to be dysregulated, distinguishing BC patients from healthy controls. However, there was little consistency between included studies, making it difficult to identify specific microRNAs useful for diagnosis.

Investigating the Role of Circulating miRNAs as Biomarkers in Colorectal Cancer: An Epidemiological Systematic Review.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Primary and secondary preventions are key to reducing the global burden. MicroRNAs (miRNAs) are a group of small non-coding RNA molecules, which seem to have a role either as tumor suppressor genes or oncogenes and to be related to cancer risk factors, such as obesity and inflammation. We conducted a systematic review and meta-analysis to identify circulating miRNAs related to CRC diagnosis that could be selected as biomarkers in a meet-in-the-middle analysis. Forty-four studies were included in the systematic review and nine studies in the meta-analysis. The pooled sensitivity and specificity of miR-21 for CRC diagnosis were 77% (95% CI: 69-84) and 82% (95% CI: 70-90), respectively, with an AUC of 0.86 (95% CI: 0.82-0.88). Several miRNAs were found to be dysregulated, distinguishing patients with CRC from healthy controls. However, little consistency was present across the included studies, making it challenging to identify specific miRNAs, which were consistently validated. Understanding the mechanisms by which miRNAs become biologically embedded in cancer initiation and promotion may help better understand cancer pathways to develop more effective prevention strategies and therapy approaches.

Performance of HPV E6/E7 mRNA assay as primary screening test: Results from the NTCC2 trial.

As the primary screening test, E6/E7 mRNA has shown similar sensitivity for CIN3+ and lower positivity rate than the HPV DNA test. Nevertheless, the overall mRNA positivity is too high for immediate colposcopy, making a triage test necessary. The aim was to estimate the mRNA performance as a primary test with different triage strategies. All HPV DNA-positives were tested for mRNA, cytology and p16/ki67. A sample of HPV DNA-negatives was also tested for mRNA to estimate test specificity. We included all CIN3+ histologically diagnosed within 24 months since recruitment. Of the 41 127 participants, 7.7% were HPV DNA-positive, of which 66.4% were mRNA-positive. Among the HPV DNA-negatives, 10/1108 (0.9%) were mRNA-positive. Overall, 97 CIN3+ were found. If mRNA was used as the primary test, it would miss about 3% of all CIN3+ with a 22% reduction of positivity compared with HPV DNA. The weighted specificity estimate for

Prenatal exposure to PM10 and changes in DNA methylation and telomere length in cord blood.

BACKGROUND: Air pollution exposure in pregnancy can cause molecular level alterations that might influence later disease susceptibility. OBJECTIVES: We investigated DNA methylation (DNAm) and telomere length (TL) in the cord blood in relation to gestational PM10 exposure and explored potential gestational windows of susceptibility. METHODS: Cord blood epigenome-wide DNAm (N = 384) and TL (N = 500) were measured in children of the Italian birth cohort Piccolipiù, using the Infinium Methylation EPIC BeadChip and qPCR, respectively. PM10 daily exposure levels, based on maternal residential address, were estimated for different gestational periods using models based on satellite data. Epigenome-wide analysis to identify differentially methylated probes (DMPs) and regions (DMRs) was conducted, followed by a pathway analysis and replication analysis in an second Piccolipiù dataset. Distributed lag models (DLMs) using weekly exposures were used to study the association of PM10 exposure across pregnancy with telomere length, as well as with the DMPs that showed robust associations. RESULTS: Gestational PM10 exposure was associated with the DNA methylation of more than 250 unique DMPs, most of them identified in early gestation, and 1 DMR. Out of 151 DMPs available in the replication dataset, ten DMPs showed robust associations: eight were associated with exposure during early gestation and 2 with exposure during the whole pregnancy. These exposure windows were supported by the DLM analysis. The PM10 exposure between 15th and 20th gestational week seem to be associated with shorter telomeres at birth, while exposure between 24th and 29th was associated with longer telomeres. DISCUSSION: The early pregnancy period is a potential critical window during which PM10 exposure can influence cord blood DNA methylation. The results from the TL analysis were consistent with previous findings and merit further exploration in future studies. The study underlines the importance of considering gestational windows outside of the predefined trimesters that may not always overlap with biologically relevant windows of exposure.

Red Blood Cell Fatty Acids and Risk of Colorectal Cancer in The European Prospective Investigation into Cancer and Nutrition (EPIC).

BACKGROUND: A growing body of evidence suggests that alterations of dietary fatty acid (FA) profiles are associated with colorectal cancer risk. However, data from large-scale epidemiologic studies using circulating FA measurements to objectively assess individual FA and FA categories are scarce. METHODS: We investigate the association between red blood cell (RBC) membrane FAs and risk of colorectal cancer in a case-control study nested within a large prospective cohort. After a median follow-up of 6.4 years, 1,069 incident colorectal cancer cases were identified and matched to 1,069 controls among participants of the European Prospective Investigation into Cancer and Nutrition (EPIC). The FA composition of RBC phospholipids (in mol%) was analyzed by gas chromatography, and their association with risk of colorectal cancer was estimated by multivariable adjusted conditional logistic regression models. RESULTS: After correction for multiple testing, subjects with higher concentrations of RBC stearic acid were at higher risk for colorectal cancer (OR = 1.23; 95% CI = 1.07-1.42, per 1 mol%). Conversely, colorectal cancer incidence decreased with increasing proportions of RBC n-3 PUFA, particularly eicosapentaenoic acid (0.75; 0.62-0.92, per 1 mol%). The findings for the n-6 PUFA arachidonic acid were inconsistent. CONCLUSIONS: The positive association between prediagnostic RBC stearic acid and colorectal cancer reflects putative differences in FA intake and metabolism between cancer cases and matched controls, which deserve further investigation. The inverse relationship between EPA and colorectal cancer is in line with the repeatedly reported protective effect of fish consumption on colorectal cancer risk. IMPACT: These findings add to the evidence on colorectal cancer prevention.

Determination of saliva epigenetic age in infancy, and its association with parental socio-economic characteristics and pregnancy outcomes.

Epigenetic age acceleration (AA) has been associated with adverse environmental exposures and many chronic conditions. We estimated, in the NINFEA birth cohort, infant saliva epigenetic age, and investigated whether parental socio-economic position (SEP) and pregnancy outcomes are associated with infant epigenetic AA. A total of 139 saliva samples collected at on average 10.8 (range 7-17) months were used to estimate Horvath's DNA methylation age. Epigenetic AA was defined as the residual from a linear regression of epigenetic age on chronological age. Linear regression models were used to test the associations of parental SEP and pregnancy outcomes with saliva epigenetic AA. A moderate positive association was found between DNA methylation age and chronological age, with the median absolute difference of 6.8 months (standard deviation [SD] 3.9). The evidence of the association between the indicators of low SEP and epigenetic AA was weak; infants born to unemployed mothers or with low education had on average 1 month higher epigenetic age than infants of mothers with high education and employment (coefficient 0.78 months, 95% confidence intervals [CIs]: -0.79 to 2.34 for low/medium education; 0.96, 95% CI: -1.81 to 3.73 for unemployment). There was no evidence for association of gestational age, birthweight or caesarean section with infant epigenetic AA. Using the Horvath's method, DNA methylation age can be fairly accurately predicted from saliva samples already in the first months of life. This study did not reveal clear associations between either pregnancy outcomes or parental socio-economic characteristics and infant saliva epigenetic AA.

p16/ki67 and E6/E7 mRNA Accuracy and Prognostic Value in Triaging HPV DNA-Positive Women.

BACKGROUND: The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis. METHODS: Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided. RESULTS: 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both). CONCLUSIONS: p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

Combined use of cytology, p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening.

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.

DNA methylation in repeat negative prostate biopsies as a marker of missed prostate cancer.

BACKGROUND: Men often undergo repeat prostate biopsies because of suspicion of missed cancer. We assessed if (i) methylation of selected genes in prostate tissue vary with aging and (ii) methylation alterations in repeat biopsies predict missed prostate cancer. METHODS: We conducted a case-control study among men who underwent at least two negative prostate biopsies followed by a sampling either positive (cases n = 111) or negative (controls n = 129) for prostate cancer between 1995 and 2014 at the University Hospital (Turin, Italy). Two pathology wards were included for replication purposes. We analyzed methylation of GSTP1, APC, PITX2, C1orf114, GABRE, and LINE-1 in the first two negative biopsies. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the association between genes methylation and prostate cancer. RESULTS: Age at biopsy and time interval between the two negative biopsies were not associated with methylation levels of the selected genes in neither cases nor controls. GSTP1 methylation in the first and in the second negative biopsy was associated with prostate cancer detection [OR per 1% increase: 1.14 (95% CI 1.01-1.29) for the second biopsy and 1.21 (95% CI 1.07-1.37) for the highest methylation level (first or second biopsy)]. A threshold > 10% for GSTP1 methylation corresponded to a specificity of 0.98 (positive likelihood ratio 7.87). No clear association was found for the other genes. Results were consistent between wards. CONCLUSIONS: Our results suggest that GSTP1 methylation in negative prostate biopsies is stable over time and can predict missed cancer with high specificity.

DNA methylation, colon cancer and Mediterranean diet: results from the EPIC-Italy cohort.

The biological mechanisms through which adherence to Mediterranean Diet (MD) protects against colon cancer (CC) are poorly understood. Evidence suggests that chronic inflammation may be implicated in the pathway. Both diet and CC are related to epigenetic regulation. We performed a nested case-control study on 161 pairs from the Italian component of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, in which we looked for the methylation signals in DNA extracted from leucocytes associated with both CC and MD in 995 CpGs located in 48 inflammation genes. The DNA methylation signals detected in this analysis were validated in a subgroup of 47 case-control pairs and further replicated (where validated) in 95 new pairs by means of pyrosequencing. Among the CpG sites selected a-priori in inflammation-related genes, seven CpG sites were found to be associated with CC status and with MD, in line with its protective effect. Only two CpG sites (cg17968347-SERPINE1 and cg20674490-RUNX3) were validated using bisulphite pyrosequencing and, after replication, we found that DNA methylation of cg20674490-RUNX3 may be a potential molecular mediator explaining the protective effect of MD on CC onset. The use of a 'meet-in-the-middle' approach to identify the overlap between exposure and predictive markers of disease is innovative in studies on the relationship between diet and cancer, in which exposure assessment is difficult and the mechanisms through which the nutrients exert their protective effect is largely unknown.

Differentially methylated DNA regions in early childhood wheezing: An epigenome-wide study using saliva.

BACKGROUND: Epigenetics may play a role in wheezing and asthma development. We aimed to examine infant saliva DNA methylation in association with early childhood wheezing. METHODS: A case-control study was nested within the NINFEA birth cohort with 68 cases matched to 68 controls by sex, age (between 6 and 18 months, median: 10.3 months) and season at saliva sampling. Using a bumphunting region-based approach, we examined associations between saliva methylome measured using Illumina Infinium HumanMethylation450k array and wheezing between 6 and 18 months of age. We tested our main findings in independent publicly available data sets of childhood respiratory allergy and atopic asthma, with DNA methylation measured in different tissues and at different ages. RESULTS: We identified one wheezing-associated differentially methylated region (DMR) spanning ten sequential CpG sites in the promoter-regulatory region of PM20D1 gene (family-wise error rate < 0.05). The observed associations were enhanced in children born to atopic mothers. In the publicly available data sets, hypermethylation in the same region of PM20D1 was consistently found at different ages and in all analysed tissues (cord blood, blood, saliva and nasal epithelia) of children with respiratory allergy/atopic asthma compared with controls. CONCLUSION: This study suggests that PM20D1 hypermethylation is associated with early childhood wheezing. Directionally consistent epigenetic alteration observed in cord blood and other tissues at older ages in children with respiratory allergy and atopic asthma provides suggestive evidence that a long-term epigenetic modification, likely operating from birth, may be involved in childhood atopic phenotypes.

Single-nucleotide polymorphisms in DNMT3B gene and DNMT3B mRNA expression in association with prostate cancer mortality.

BACKGROUND: Germline variants in DNA methyltransferase 3B (DNMT3B) may influence DNMT3B enzymatic activity, which, in turn, may affect cancer aggressiveness by altering DNA methylation. METHODS: The study involves two Italian cohorts (NTAT cohort, n = 157, and 1980s biopsy cohort, n = 182) and two U.S. cohorts (Health Professionals Follow-Up Study, n = 214, and Physicians' Health Study, n = 298) of prostate cancer (PCa) patients, and a case-control study of lethal (n = 113) vs indolent (n = 290) PCa with DNMT3B mRNA expression data nested in the U.S. cohorts. We evaluated the association between: three selected DNMT3B variants and global DNA methylation using linear regression in the NTAT cohort, the three DNMT3B variants and PCa mortality using Cox proportional hazards regression in all cohorts, and DNMT3B expression and lethal PCa using logistic regression, with replication in publicly available databases (TCGA, n = 492 and MSKCC, n = 140). RESULTS: The TT genotype of rs1569686 was associated with LINE-1 hypomethylation in tumor tissue (ß = -2.71, 95% CI: -5.41, -0.05). There was no evidence of association between DNMT3B variants and PCa mortality. DNMT3B expression was consistently associated with lethal PCa in the two U.S. cohorts (3rd vs 1st tertile, combined cohorts: OR = 2.04, 95% CI: 1.13, 3.76); the association was replicated in TCGA and MSKCC data (3rd vs 1st tertile, TCGA: HR = 3.00, 95% CI: 1.78, 5.06; MSKCC: HR = 2.22, 95% CI: 1.01, 4.86). CONCLUSIONS: Although there was no consistent evidence of an association between DNMT3B variants and PCa mortality, the TT genotype of rs1569686 was associated with LINE-1 hypomethylation in tumor tissue and DNMT3B mRNA expression was associated with an increased risk of lethal PCa.

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.

Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing.

BACKGROUND: Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for accurate detection of high-grade cervical lesions and cancer. Methylation status has been widely investigated for HPV16, sporadically for other HPV types. METHODS: Objective of this methodological study was to set up molecular methods to test the methylation levels in the twelve oncogenic HPV types by pyrosequencing, minimizing the number of HPV type-specific PCR protocols. Target CpGs were selected on the HPV L1 (two regions, L1 I and L1 II) and L2 genes. Study samples included DNA stored at Turin, Italy, purified by cervical cells collected in Standard Transport Medium or PreservCyt from women who participated in two studies (N = 126 and 140) nested within the regional organized screening programme. PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types. RESULTS: Generation of consensus primers was successful for L1 I and II regions, unsuccessful for L2 region, for which HPV type-specific primers remained necessary. The difference between replicated tests on the same sample was ≤4% in 88%, 77% and 91% of cases when targeting the L1 I, L1 II and L2 regions, respectively. The corresponding intra-class correlation coefficients (ICC) were 0.94, 0.87 and 0.97 respectively. When comparing methylation measures based on consensus and type-specific primers, ICC was 0.97 for the L1 I region and 0.99 the for L1 II region. CONCLUSIONS: The proposed protocols, applying consensus primers suitable to amplify the oncogenic HPV types and minimize the number of PCR reactions, represent a promising tool to quantify viral methylation in women positive for any high risk HPV type. IMPACT: Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.