Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high h... more Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional bio...
Background Men often undergo repeat prostate biopsies because of suspicion of missed cancer. We a... more 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...
In this paper, we describe the Prognostic Factors for Mortality in Prostate Cancer (ProMort) stud... more In this paper, we describe the Prognostic Factors for Mortality in Prostate Cancer (ProMort) study and use it to demonstrate how the weighted likelihood method can be used in nested case-control studies to estimate both relative and absolute risks in the competing-risks setting. ProMort is a case-control study nested within the National Prostate Cancer Register (NPCR) of Sweden, comprising 1,710 men diagnosed with low- or intermediate-risk prostate cancer between 1998 and 2011 who died from prostate cancer (cases) and 1,710 matched controls. Cause-specific hazard ratios and cumulative incidence functions (CIFs) for prostate cancer death were estimated in ProMort using weighted flexible parametric models and compared with the corresponding estimates from the NPCR cohort. We further drew 1,500 random nested case-control subsamples of the NPCR cohort and quantified the bias in the hazard ratio and CIF estimates. Finally, we compared the ProMort estimates with those obtained by augmenti...
Background Epigenetics may play a role in wheezing and asthma development. We aimed to examine in... more 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 bump hunting 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 datasets 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 datasets, 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.
Testicular cancer is considered to originate from an impaired differentiation of fetal germ cells... more Testicular cancer is considered to originate from an impaired differentiation of fetal germ cells, but puberty could represent another time window of susceptibility. This study aimed at investigating the association between environmental exposures acting during puberty/adolescence (13 to 19 years of age) and the risk of testicular cancer. We used data of the EPSAM study, a case-control study on germ-cell testicular cancer conducted in the province of Turin, Italy, involving cases diagnosed between 1997 and 2008. Histologically confirmed cases (n=255) and controls (n=459) completed a postal questionnaire focusing in particular on the pubertal period (namely age 13 years) with questions on physical activity (competitive sports, gardening), lifestyle (alcohol consumption, smoking), occupational history, and medical conditions. All analyses were adjusted for the matching variables, cryptorchidism and educational level. Having done at least one competitive sport during puberty (odds rati...
The etiology of testicular cancer is largely unexplained. Research has mainly focused on prenatal... more The etiology of testicular cancer is largely unexplained. Research has mainly focused on prenatal exposures, especially to sex hormones, while less attention has been paid to exposures that may act also postnatally. As baldness has been previously associated with testicular cancer risk we focused on baldness and body hairiness, which are both associated with androgen activity. We used data of the Postnatal Exposures and Male Health (EPSAM) study, a case-control study on testicular cancer conducted in the Province of Turin, Italy, involving cases diagnosed between 1997 and 2008. Information was collected using mailed questionnaires. Analyses included 255 cases and 459 controls. We calculated ORs and 95% CIs to estimate testicular cancer risk among those who developed baldness and among those with body hairiness. We found an inverse association between testicular cancer and baldness (OR: 0.67, 95% CI: 0.46-0.98) and body hairiness (OR: 0.78, 95% CI: 0.53-1.16), although the latter had wider CIs. The inverse association between baldness and testicular cancer is consistent with the results from previous studies. These results suggest that androgens activity may influence testicular cancer risk.
Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for m... more Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may ...
Background: The fetal and infant life are periods of rapid development, characterized by high sus... more Background: The fetal and infant life are periods of rapid development, characterized by high susceptibility to exposures. Birth cohorts provide unique opportunities to study early-life exposures in association with child development and health, as well as, with longer follow-up, the early life origin of adult diseases. Piccolipiù is an Italian birth cohort recently set up to investigate the effects of environmental exposures, parental conditions and social factors acting during pre-natal and early post-natal life on infant and child health and development. We describe here its main characteristics. Methods/design: Piccolipiù is a prospective cohort of expected 3000 newborns, who will be recruiting in six maternity units of five Italian cities (Florence, Rome, Trieste, Turin and Viareggio) since October 2011. Mothers are contacted during pregnancy or at delivery and are offered to participate in the study. Upon acceptance, their newborns are recruited at birth and followed up until at least 18 years of age. At recruitment, the mothers donate a blood sample and complete a baseline questionnaire. Umbilical cord blood, pieces of umbilical cord and heel blood spots are also collected. Postnatal follow-up currently occurs at 6, 12, and 24 months of age using on-line or postal self administered questionnaire; further questionnaires and medical examinations are envisaged. Questionnaires collect information on several factors, including mother's and/or child's environmental exposures, anthropometric measures, reproductive factors, diet, supplements, medical history, cognitive development, mental health and socioeconomic factors. Health promotion materials are also offered to parents. Discussion: Piccolipiù will broaden our understanding of the contribution of early-life factors to infant and child health and development. Several hypotheses on the developmental origins of health can be tested or piloted using the data collected from the Piccolipiù cohort. By pooling these data with those collected by other existing birth cohorts it will be possible to validate previous findings and to study rare exposures and outcomes.
Additional file 2: Table S1. Spearman correlation coefficients of gene-specific methylation level... more Additional file 2: Table S1. Spearman correlation coefficients of gene-specific methylation levels between the two biopsies in cases and controls. Table S2. Predicted values of gene-specific median methylation levels at the first biopsy at selected ages, using quantile regression with age modelled by restricted cubic splines. Table S3. Predicted values of the median differences in gene-specific methylation levels between the two biopsies, at selected time intervals, using quantile regression with time modelled by restricted cubic splines. Table S4. Gene IDs, primer sequences, pyrosequencing assays and PCR annealing temperatures and number of CpG analysed per gene.
• Methylation in CADM1 and MAL is associated with high grade cervical cancer lesions. • Methylati... more • Methylation in CADM1 and MAL is associated with high grade cervical cancer lesions. • Methylation in HPV regions is associated with high grade cervical cancer lesions. • Methylation in HPV regions is associated with age. • Increasing number of methylated genes predicts high grade cervical lesions. a b s t r a c t 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 b 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:
The biological mechanisms through which adherence to Mediterranean Diet (MD) protects A c c e p t... more The biological mechanisms through which adherence to Mediterranean Diet (MD) protects A c c e p t e d M a n u s c r i p t 2 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, 7 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.
Background Germline variants in DNA methyltransferase 3B (DNMT3B) may influence DNMT3B enzymatic ... more 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 (β = −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:
Background: In Sweden, human tissue samples obtained from diagnostic and surgical procedures have... more Background: In Sweden, human tissue samples obtained from diagnostic and surgical procedures have for decades been routinely stored in a formalin-fixed, paraffin-embedded, form. Through linkage with nationwide registers, these samples are available for molecular studies to identify biomarkers predicting mortality even in slowprogressing prostate cancer. However, tissue fixation causes modifications of nucleic acids, making it challenging to extract high-quality nucleic acids from formalin fixated tissues. Methods: In this study, the efficiency of five commercial nucleic acid extraction kits was compared on 30 prostate biopsies with normal histology, and the quantity and quality of the products were compared using spectrophotometry and Agilent's BioAnalyzer. Student's t-test's and Bland-Altman analyses were performed in order to investigate differences in nucleic acid quantity and quality between the five kits. The best performing extraction kits were subsequently tested on an additional 84 prostate tumor tissues. A Spearman's correlation test and linear regression analyses were performed in order to investigate the impact of tissue age and amount of tissue on nucleic acid quantity and quality. Results: Nucleic acids extracted with RNeasy® FFPE and QIAamp® DNA FFPE Tissue kit had the highest quantity and quality, and was used for extraction from 84 tumor tissues. Nucleic acids were successfully extracted from all biopsies, and the amount of tumor (in millimeter) was found to have the strongest association with quantity and quality of nucleic acids. Conclusions: To conclude, this study shows that the choice of nucleic acid extraction kit affects the quantity and quality of extracted products. Furthermore, we show that extraction of nucleic acids from archival formalin-fixed prostate biopsies is possible, allowing molecular studies to be performed on this valuable sample collection.
Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for a... more 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. 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. Generation of consensus primers was successful for L1 I a...
Background/objectives It has been suggested that subfertility and testicular cancer share genetic... more Background/objectives It has been suggested that subfertility and testicular cancer share genetic and environmental risk factors. We studied both subfertility and the strongest known testicular cancer susceptibility gene, the c-KIT ligand (KITLG), whose pathway is involved in spermatogenesis. Methods The EPSAM case-control study is comprised of testicular cancer patients from the Province of Turin, Italy, diagnosed between 1997 and 2008. The present analysis included 245 cases and 436 controls from EPSAM, who were aged 20 years or older at diagnosis/recruitment. The EPSAM questionnaire collected information on factors such as number of children, age at first attempt to conceive, duration of attempt to conceive, use of assisted reproduction techniques, physician-assigned diagnosis of infertility, number of siblings, and self-reported cryptorchidism. Genotyping of the KITLG single nucleotide polymorphism (SNP) rs995030 was performed on the saliva samples of 202 cases and 329 controls.
Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LI... more Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982-1988 and 1993-1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95-2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (<75%) vs. high (>80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03-21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.
DNA methylation is a well-characterized epigenetic modification that plays an important role in t... more 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 (p = 0.04 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.
Background: Markers that can discriminate between indolent and aggressive prostate tumours are ne... more Background: Markers that can discriminate between indolent and aggressive prostate tumours are needed. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumour (NTAT) in association with prostate cancer mortality. Methods: From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostate tumour tissues and used probe real-time PCR to analyse methylation of the glutathione Stransferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters. Results: Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostate tumour tissue for the same two genes as well as with a high Gleason score. The hazard ratio (HR) of prostate cancer mortality was 2.38 (95% confidence interval: 1.23-4.61) for APC methylation, and 2.92 (1.49-5.74) for GSTP1 methylation in NTAT. It changed to 1.91 (1.03-3.56) and 1.60 (0.80-3.19) after adjusting for Gleason score and methylation in prostate tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 4.30 (2.00-9.22), which decreased to 2.40 (1.15-5.01) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.29, 95% CI: 1.27-8.52). Conclusions: Changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker to be evaluated in clinical studies.
Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays 2 a role in ... more Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays 2 a role in cellular response to alkylating agents. In the present study aimed to: i) evaluate the 3 concordance between MGMT promoter methylation status in tumor tissue and plasma; ii) 4 monitor MGMT promoter methylation status in plasma taken before and during 5 temozolomide treatment; iii) explore the value of MGMT promoter methylation status in 6 plasma as a prognostic/predictive biomarker in glioma patients. 7 We enrolled 58 patients with histologically confirmed glioma at different grades of 8 malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-9 embedded tumor tissue was available for 48 patients. Blood samples were collected from all 10 patients before temozolomide treatment (baseline) and at each MRI examination for a 12-11 month period. MGMT promoter methylation status was assessed in both sample types by real 12 time PCR with a specific probe. 13 The frequency of MGMT promoter methylation was 60.4% in tumor tissue and 41.38% in 14 plasma. MGMT promoter methylation status was concordant in the two sample types 15 (Kappa=0.75, 95% confidence interval [CI] 0.57-0.93; p-value <0.001). Overall and 16 progression-free survival were longer in patients with methylated MGMT promoter. 17 Mortality was higher in patients with unmethylated MGMT promoter, whether in tumor 18 tissue (hazard ratio [HR]: 2.21; 95% CI 0.99-4.95) or plasma (HR: 2.19; 95% CI 1.02-4.68). 19 Progression-free survival was shorter in patients with unmethylated MGMT promoter, 20 whether in tissue (HR: 2.30; 95% CI 1.19-4.45) or plasma (HR: 1.77; 95% CI 0.95-3.30). 21 The cumulative incidence of unmethylated MGMT promoter in plasma at baseline was 58%, 22 and reached virtually 100% at 12 months. In conclusion MGMT promoter methylation status 23 in tumor tissue and plasma was highly concordant, and both were associated with longer treatment response. However we suggest caution in using plasma as a surrogate of tumor 1 tissue due to possible false-negative results.
Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high h... more Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional bio...
Background Men often undergo repeat prostate biopsies because of suspicion of missed cancer. We a... more 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...
In this paper, we describe the Prognostic Factors for Mortality in Prostate Cancer (ProMort) stud... more In this paper, we describe the Prognostic Factors for Mortality in Prostate Cancer (ProMort) study and use it to demonstrate how the weighted likelihood method can be used in nested case-control studies to estimate both relative and absolute risks in the competing-risks setting. ProMort is a case-control study nested within the National Prostate Cancer Register (NPCR) of Sweden, comprising 1,710 men diagnosed with low- or intermediate-risk prostate cancer between 1998 and 2011 who died from prostate cancer (cases) and 1,710 matched controls. Cause-specific hazard ratios and cumulative incidence functions (CIFs) for prostate cancer death were estimated in ProMort using weighted flexible parametric models and compared with the corresponding estimates from the NPCR cohort. We further drew 1,500 random nested case-control subsamples of the NPCR cohort and quantified the bias in the hazard ratio and CIF estimates. Finally, we compared the ProMort estimates with those obtained by augmenti...
Background Epigenetics may play a role in wheezing and asthma development. We aimed to examine in... more 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 bump hunting 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 datasets 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 datasets, 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.
Testicular cancer is considered to originate from an impaired differentiation of fetal germ cells... more Testicular cancer is considered to originate from an impaired differentiation of fetal germ cells, but puberty could represent another time window of susceptibility. This study aimed at investigating the association between environmental exposures acting during puberty/adolescence (13 to 19 years of age) and the risk of testicular cancer. We used data of the EPSAM study, a case-control study on germ-cell testicular cancer conducted in the province of Turin, Italy, involving cases diagnosed between 1997 and 2008. Histologically confirmed cases (n=255) and controls (n=459) completed a postal questionnaire focusing in particular on the pubertal period (namely age 13 years) with questions on physical activity (competitive sports, gardening), lifestyle (alcohol consumption, smoking), occupational history, and medical conditions. All analyses were adjusted for the matching variables, cryptorchidism and educational level. Having done at least one competitive sport during puberty (odds rati...
The etiology of testicular cancer is largely unexplained. Research has mainly focused on prenatal... more The etiology of testicular cancer is largely unexplained. Research has mainly focused on prenatal exposures, especially to sex hormones, while less attention has been paid to exposures that may act also postnatally. As baldness has been previously associated with testicular cancer risk we focused on baldness and body hairiness, which are both associated with androgen activity. We used data of the Postnatal Exposures and Male Health (EPSAM) study, a case-control study on testicular cancer conducted in the Province of Turin, Italy, involving cases diagnosed between 1997 and 2008. Information was collected using mailed questionnaires. Analyses included 255 cases and 459 controls. We calculated ORs and 95% CIs to estimate testicular cancer risk among those who developed baldness and among those with body hairiness. We found an inverse association between testicular cancer and baldness (OR: 0.67, 95% CI: 0.46-0.98) and body hairiness (OR: 0.78, 95% CI: 0.53-1.16), although the latter had wider CIs. The inverse association between baldness and testicular cancer is consistent with the results from previous studies. These results suggest that androgens activity may influence testicular cancer risk.
Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for m... more Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may ...
Background: The fetal and infant life are periods of rapid development, characterized by high sus... more Background: The fetal and infant life are periods of rapid development, characterized by high susceptibility to exposures. Birth cohorts provide unique opportunities to study early-life exposures in association with child development and health, as well as, with longer follow-up, the early life origin of adult diseases. Piccolipiù is an Italian birth cohort recently set up to investigate the effects of environmental exposures, parental conditions and social factors acting during pre-natal and early post-natal life on infant and child health and development. We describe here its main characteristics. Methods/design: Piccolipiù is a prospective cohort of expected 3000 newborns, who will be recruiting in six maternity units of five Italian cities (Florence, Rome, Trieste, Turin and Viareggio) since October 2011. Mothers are contacted during pregnancy or at delivery and are offered to participate in the study. Upon acceptance, their newborns are recruited at birth and followed up until at least 18 years of age. At recruitment, the mothers donate a blood sample and complete a baseline questionnaire. Umbilical cord blood, pieces of umbilical cord and heel blood spots are also collected. Postnatal follow-up currently occurs at 6, 12, and 24 months of age using on-line or postal self administered questionnaire; further questionnaires and medical examinations are envisaged. Questionnaires collect information on several factors, including mother's and/or child's environmental exposures, anthropometric measures, reproductive factors, diet, supplements, medical history, cognitive development, mental health and socioeconomic factors. Health promotion materials are also offered to parents. Discussion: Piccolipiù will broaden our understanding of the contribution of early-life factors to infant and child health and development. Several hypotheses on the developmental origins of health can be tested or piloted using the data collected from the Piccolipiù cohort. By pooling these data with those collected by other existing birth cohorts it will be possible to validate previous findings and to study rare exposures and outcomes.
Additional file 2: Table S1. Spearman correlation coefficients of gene-specific methylation level... more Additional file 2: Table S1. Spearman correlation coefficients of gene-specific methylation levels between the two biopsies in cases and controls. Table S2. Predicted values of gene-specific median methylation levels at the first biopsy at selected ages, using quantile regression with age modelled by restricted cubic splines. Table S3. Predicted values of the median differences in gene-specific methylation levels between the two biopsies, at selected time intervals, using quantile regression with time modelled by restricted cubic splines. Table S4. Gene IDs, primer sequences, pyrosequencing assays and PCR annealing temperatures and number of CpG analysed per gene.
• Methylation in CADM1 and MAL is associated with high grade cervical cancer lesions. • Methylati... more • Methylation in CADM1 and MAL is associated with high grade cervical cancer lesions. • Methylation in HPV regions is associated with high grade cervical cancer lesions. • Methylation in HPV regions is associated with age. • Increasing number of methylated genes predicts high grade cervical lesions. a b s t r a c t 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 b 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:
The biological mechanisms through which adherence to Mediterranean Diet (MD) protects A c c e p t... more The biological mechanisms through which adherence to Mediterranean Diet (MD) protects A c c e p t e d M a n u s c r i p t 2 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, 7 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.
Background Germline variants in DNA methyltransferase 3B (DNMT3B) may influence DNMT3B enzymatic ... more 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 (β = −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:
Background: In Sweden, human tissue samples obtained from diagnostic and surgical procedures have... more Background: In Sweden, human tissue samples obtained from diagnostic and surgical procedures have for decades been routinely stored in a formalin-fixed, paraffin-embedded, form. Through linkage with nationwide registers, these samples are available for molecular studies to identify biomarkers predicting mortality even in slowprogressing prostate cancer. However, tissue fixation causes modifications of nucleic acids, making it challenging to extract high-quality nucleic acids from formalin fixated tissues. Methods: In this study, the efficiency of five commercial nucleic acid extraction kits was compared on 30 prostate biopsies with normal histology, and the quantity and quality of the products were compared using spectrophotometry and Agilent's BioAnalyzer. Student's t-test's and Bland-Altman analyses were performed in order to investigate differences in nucleic acid quantity and quality between the five kits. The best performing extraction kits were subsequently tested on an additional 84 prostate tumor tissues. A Spearman's correlation test and linear regression analyses were performed in order to investigate the impact of tissue age and amount of tissue on nucleic acid quantity and quality. Results: Nucleic acids extracted with RNeasy® FFPE and QIAamp® DNA FFPE Tissue kit had the highest quantity and quality, and was used for extraction from 84 tumor tissues. Nucleic acids were successfully extracted from all biopsies, and the amount of tumor (in millimeter) was found to have the strongest association with quantity and quality of nucleic acids. Conclusions: To conclude, this study shows that the choice of nucleic acid extraction kit affects the quantity and quality of extracted products. Furthermore, we show that extraction of nucleic acids from archival formalin-fixed prostate biopsies is possible, allowing molecular studies to be performed on this valuable sample collection.
Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for a... more 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. 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. Generation of consensus primers was successful for L1 I a...
Background/objectives It has been suggested that subfertility and testicular cancer share genetic... more Background/objectives It has been suggested that subfertility and testicular cancer share genetic and environmental risk factors. We studied both subfertility and the strongest known testicular cancer susceptibility gene, the c-KIT ligand (KITLG), whose pathway is involved in spermatogenesis. Methods The EPSAM case-control study is comprised of testicular cancer patients from the Province of Turin, Italy, diagnosed between 1997 and 2008. The present analysis included 245 cases and 436 controls from EPSAM, who were aged 20 years or older at diagnosis/recruitment. The EPSAM questionnaire collected information on factors such as number of children, age at first attempt to conceive, duration of attempt to conceive, use of assisted reproduction techniques, physician-assigned diagnosis of infertility, number of siblings, and self-reported cryptorchidism. Genotyping of the KITLG single nucleotide polymorphism (SNP) rs995030 was performed on the saliva samples of 202 cases and 329 controls.
Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LI... more Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982-1988 and 1993-1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95-2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (<75%) vs. high (>80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03-21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.
DNA methylation is a well-characterized epigenetic modification that plays an important role in t... more 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 (p = 0.04 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.
Background: Markers that can discriminate between indolent and aggressive prostate tumours are ne... more Background: Markers that can discriminate between indolent and aggressive prostate tumours are needed. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumour (NTAT) in association with prostate cancer mortality. Methods: From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostate tumour tissues and used probe real-time PCR to analyse methylation of the glutathione Stransferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters. Results: Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostate tumour tissue for the same two genes as well as with a high Gleason score. The hazard ratio (HR) of prostate cancer mortality was 2.38 (95% confidence interval: 1.23-4.61) for APC methylation, and 2.92 (1.49-5.74) for GSTP1 methylation in NTAT. It changed to 1.91 (1.03-3.56) and 1.60 (0.80-3.19) after adjusting for Gleason score and methylation in prostate tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 4.30 (2.00-9.22), which decreased to 2.40 (1.15-5.01) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.29, 95% CI: 1.27-8.52). Conclusions: Changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker to be evaluated in clinical studies.
Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays 2 a role in ... more Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays 2 a role in cellular response to alkylating agents. In the present study aimed to: i) evaluate the 3 concordance between MGMT promoter methylation status in tumor tissue and plasma; ii) 4 monitor MGMT promoter methylation status in plasma taken before and during 5 temozolomide treatment; iii) explore the value of MGMT promoter methylation status in 6 plasma as a prognostic/predictive biomarker in glioma patients. 7 We enrolled 58 patients with histologically confirmed glioma at different grades of 8 malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-9 embedded tumor tissue was available for 48 patients. Blood samples were collected from all 10 patients before temozolomide treatment (baseline) and at each MRI examination for a 12-11 month period. MGMT promoter methylation status was assessed in both sample types by real 12 time PCR with a specific probe. 13 The frequency of MGMT promoter methylation was 60.4% in tumor tissue and 41.38% in 14 plasma. MGMT promoter methylation status was concordant in the two sample types 15 (Kappa=0.75, 95% confidence interval [CI] 0.57-0.93; p-value <0.001). Overall and 16 progression-free survival were longer in patients with methylated MGMT promoter. 17 Mortality was higher in patients with unmethylated MGMT promoter, whether in tumor 18 tissue (hazard ratio [HR]: 2.21; 95% CI 0.99-4.95) or plasma (HR: 2.19; 95% CI 1.02-4.68). 19 Progression-free survival was shorter in patients with unmethylated MGMT promoter, 20 whether in tissue (HR: 2.30; 95% CI 1.19-4.45) or plasma (HR: 1.77; 95% CI 0.95-3.30). 21 The cumulative incidence of unmethylated MGMT promoter in plasma at baseline was 58%, 22 and reached virtually 100% at 12 months. In conclusion MGMT promoter methylation status 23 in tumor tissue and plasma was highly concordant, and both were associated with longer treatment response. However we suggest caution in using plasma as a surrogate of tumor 1 tissue due to possible false-negative results.
Uploads
Papers by Chiara Grasso