TIGER: technical variation elimination for metabolomics data using ensemble learning architecture.

Large metabolomics datasets inevitably contain unwanted technical variations which can obscure meaningful biological signals and affect how this information is applied to personalized healthcare. Many methods have been developed to handle unwanted variations. However, the underlying assumptions of many existing methods only hold for a few specific scenarios. Some tools remove technical variations with models trained on quality control (QC) samples which may not generalize well on subject samples. Additionally, almost none of the existing methods supports datasets with multiple types of QC samples, which greatly limits their performance and flexibility. To address these issues, a non-parametric method TIGER (Technical variation elImination with ensemble learninG architEctuRe) is developed in this study and released as an R package (https://CRAN.R-project.org/package=TIGERr). TIGER integrates the random forest algorithm into an adaptable ensemble learning architecture. Evaluation results show that TIGER outperforms four popular methods with respect to robustness and reliability on three human cohort datasets constructed with targeted or untargeted metabolomics data. Additionally, a case study aiming to identify age-associated metabolites is performed to illustrate how TIGER can be used for cross-kit adjustment in a longitudinal analysis with experimental data of three time-points generated by different analytical kits. A dynamic website is developed to help evaluate the performance of TIGER and examine the patterns revealed in our longitudinal analysis (https://han-siyu.github.io/TIGER_web/). Overall, TIGER is expected to be a powerful tool for metabolomics data analysis.

Blood cell DNA methylation biomarkers in preclinical malignant pleural mesothelioma: The EPIC prospective cohort.

Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer mainly caused by asbestos exposure. Specific and sensitive noninvasive biomarkers may facilitate and enhance screening programs for the early detection of cancer. We investigated DNA methylation (DNAm) profiles in MPM prediagnostic blood samples in a case-control study nested in the European Prospective Investigation into Cancer and nutrition (EPIC) cohort, aiming to characterise DNAm biomarkers associated with MPM. From the EPIC cohort, we included samples from 135 participants who developed MPM during 20 years of follow-up and from 135 matched, cancer-free, controls. For the discovery phase we selected EPIC participants who developed MPM within 5 years from enrolment (n = 36) with matched controls. We identified nine differentially methylated CpGs, selected by 10-fold cross-validation and correlation analyses: cg25755428 (MRI1), cg20389709 (KLF11), cg23870316, cg13862711 (LHX6), cg06417478 (HOOK2), cg00667948, cg01879420 (AMD1), cg25317025 (RPL17) and cg06205333 (RAP1A). Receiver operating characteristic (ROC) analysis showed that the model including baseline characteristics (age, sex and PC1wbc) along with the nine MPM-related CpGs has a better predictive value for MPM occurrence than the baseline model alone, maintaining some performance also at more than 5 years before diagnosis (area under the curve [AUC] < 5 years = 0.89; AUC 5-10 years = 0.80; AUC >10 years = 0.75; baseline AUC range = 0.63-0.67). DNAm changes as noninvasive biomarkers in prediagnostic blood samples of MPM cases were investigated for the first time. Their application can improve the identification of asbestos-exposed individuals at higher MPM risk to possibly adopt more intensive monitoring for early disease identification.

Combined miRNA and SERS urine liquid biopsy for the point-of-care diagnosis and molecular stratification of bladder cancer.

BACKGROUND: Bladder cancer (BC) has the highest per-patient cost of all cancer types. Hence, we aim to develop a non-invasive, point-of-care tool for the diagnostic and molecular stratification of patients with BC based on combined microRNAs (miRNAs) and surface-enhanced Raman spectroscopy (SERS) profiling of urine. METHODS: Next-generation sequencing of the whole miRNome and SERS profiling were performed on urine samples collected from 15 patients with BC and 16 control subjects (CTRLs). A retrospective cohort (BC = 66 and CTRL = 50) and RT-qPCR were used to confirm the selected differently expressed miRNAs. Diagnostic accuracy was assessed using machine learning algorithms (logistic regression, naïve Bayes, and random forest), which were trained to discriminate between BC and CTRL, using as input either miRNAs, SERS, or both. The molecular stratification of BC based on miRNA and SERS profiling was performed to discriminate between high-grade and low-grade tumors and between luminal and basal types. RESULTS: Combining SERS data with three differentially expressed miRNAs (miR-34a-5p, miR-205-3p, miR-210-3p) yielded an Area Under the Curve (AUC) of 0.92 ± 0.06 in discriminating between BC and CTRL, an accuracy which was superior either to miRNAs (AUC = 0.84 ± 0.03) or SERS data (AUC = 0.84 ± 0.05) individually. When evaluating the classification accuracy for luminal and basal BC, the combination of miRNAs and SERS profiling averaged an AUC of 0.95 ± 0.03 across the three machine learning algorithms, again better than miRNA (AUC = 0.89 ± 0.04) or SERS (AUC = 0.92 ± 0.05) individually, although SERS alone performed better in terms of classification accuracy. CONCLUSION: miRNA profiling synergizes with SERS profiling for point-of-care diagnostic and molecular stratification of BC. By combining the two liquid biopsy methods, a clinically relevant tool that can aid BC patients is envisaged.

Functional and clinical implications of genetic structure in 1686 Italian exomes.

To reconstruct the phenotypical and clinical implications of the Italian genetic structure, we thoroughly analyzed a whole-exome sequencing data set comprised of 1686 healthy Italian individuals. We found six previously unreported variants with remarkable frequency differences between Northern and Southern Italy in the HERC2, OR52R1, ADH1B, and THBS4 genes. We reported 36 clinically relevant variants (submitted as pathogenic, risk factors, or drug response in ClinVar) with significant frequency differences between Italy and Europe. We then explored putatively pathogenic variants in the Italian exome. On average, our Italian individuals carried 16.6 protein-truncating variants (PTVs), with 2.5% of the population having a PTV in one of the 59 American College of Medical Genetics (ACMG) actionable genes. Lastly, we looked for PTVs that are likely to cause Mendelian diseases. We found four heterozygous PTVs in haploinsufficient genes (KAT6A, PTCH1, and STXBP1) and three homozygous PTVs in genes causing recessive diseases (DPYD, FLG, and PYGM). Comparing frequencies from our data set to other public databases, like gnomAD, we showed the importance of population-specific databases for a more accurate assessment of variant pathogenicity. For this reason, we made aggregated frequencies from our data set publicly available as a tool for both clinicians and researchers (http://nigdb.cineca.it; NIG-ExIT).

MicroRNA 146a is associated with diabetic complications in type 1 diabetic patients from the EURODIAB PCS.

BACKGROUND: MicroRNA-146a-5p (miR-146a-5p) is a key regulator of inflammatory processes. Expression of miR-146a-5p is altered in target organs of diabetic complications and deficiency of miR-146a-5p has been implicated in their pathogenesis. We investigated if serum miR-146a-5p levels were independently associated with micro/macrovascular complications of type 1 diabetes (DM1). METHODS: A nested case-control study from the EURODIAB PCS of 447 DM1 patients was performed. Cases (n = 294) had one or more complications of diabetes, whereas controls (n = 153) did not have any complication. Total RNA was isolated from all subjects and miR-146a-5p levels measured by qPCR. Both the endogenous controls U6 snRNA and the spike (Cel-miR-39) were used to normalize the results. Logistic regression analysis was carried out to investigate the association of miR-146a-5p with diabetes complications. RESULTS: MiR-146a-5p levels were significantly lower in cases [1.15 (0.32-3.34)] compared to controls [1.74 (0.44-6.74) P = 0.039]. Logistic regression analysis showed that levels of miR-146a-5p in the upper quartile were inversely associated with reduced odds ratio (OR) of all complications (OR 0.34 [95% CI 0.14-0.76]) and particularly with cardiovascular diseases (CVD) (OR 0.31 [95% CI 0.11-0.84]) and diabetic retinopathy (OR 0.40 [95% CI 0.16-0.99]), independently of age, sex, diabetes duration, A1c, hypertension, AER, eGFR, NT-proBNP, and TNF-α. CONCLUSIONS: In this large cohort of DM1 patients, we reported an inverse and independent association of miR-146a-5p with diabetes chronic complications and in particular with CVD and retinopathy, suggesting that miR-146a-5p may be a novel candidate biomarker of DM1 complications.

Genetic and Epigenetic Characterization of a Discordant KMT2A/AFF1-Rearranged Infant Monozygotic Twin Pair.

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment-genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

Consumption of Meat, Fish, Dairy Products, and Eggs and Risk of Ischemic Heart Disease.

BACKGROUND: There is uncertainty about the relevance of animal foods to the pathogenesis of ischemic heart disease (IHD). We examined meat, fish, dairy products, and eggs and risk for IHD in the pan-European EPIC cohort (European Prospective Investigation Into Cancer and Nutrition). METHODS: In this prospective study of 409 885 men and women in 9 European countries, diet was assessed with validated questionnaires and calibrated with 24-hour recalls. Lipids and blood pressure were measured in a subsample. During a mean of 12.6 years of follow-up, 7198 participants had a myocardial infarction or died of IHD. The relationships of animal foods with risk were examined with Cox regression with adjustment for other animal foods and relevant covariates. RESULTS: The hazard ratio (HR) for IHD was 1.19 (95% CI, 1.06-1.33) for a 100-g/d increment in intake of red and processed meat, and this remained significant after exclusion of the first 4 years of follow-up (HR, 1.25 [95% CI, 1.09-1.42]). Risk was inversely associated with intakes of yogurt (HR, 0.93 [95% CI, 0.89-0.98] per 100-g/d increment), cheese (HR, 0.92 [95% CI, 0.86-0.98] per 30-g/d increment), and eggs (HR, 0.93 [95% CI, 0.88-0.99] per 20-g/d increment); the associations with yogurt and eggs were attenuated and nonsignificant after exclusion of the first 4 years of follow-up. Risk was not significantly associated with intakes of poultry, fish, or milk. In analyses modeling dietary substitutions, replacement of 100 kcal/d from red and processed meat with 100 kcal/d from fatty fish, yogurt, cheese, or eggs was associated with ≈20% lower risk of IHD. Consumption of red and processed meat was positively associated with serum non-high-density lipoprotein cholesterol concentration and systolic blood pressure, and consumption of cheese was inversely associated with serum non-high-density lipoprotein cholesterol. CONCLUSIONS: Risk for IHD was positively associated with consumption of red and processed meat and inversely associated with consumption of yogurt, cheese, and eggs, although the associations with yogurt and eggs may be influenced by reverse causation bias. It is not clear whether the associations with red and processed meat and cheese reflect causality, but they were consistent with the associations of these foods with plasma non-high-density lipoprotein cholesterol and for red and processed meat with systolic blood pressure, which could mediate such effects.

Circulating insulin-like growth factor I in relation to melanoma risk in the European prospective investigation into cancer and nutrition.

Insulin-like growth factor-I (IGF-I) regulates cell proliferation and apoptosis, and is thought to play a role in tumour development. Previous prospective studies have shown that higher circulating concentrations of IGF-I are associated with a higher risk of cancers at specific sites, including breast and prostate. No prospective study has examined the association between circulating IGF-I concentrations and melanoma risk. A nested case-control study of 1,221 melanoma cases and 1,221 controls was performed in the European Prospective Investigation into Cancer and Nutrition cohort, a prospective cohort of 520,000 participants recruited from 10 European countries. Conditional logistic regression was used to estimate odds ratios (ORs) for incident melanoma in relation to circulating IGF-I concentrations, measured by immunoassay. Analyses were conditioned on the matching factors and further adjusted for age at blood collection, education, height, BMI, smoking status, alcohol intake, marital status, physical activity and in women only, use of menopausal hormone therapy. There was no significant association between circulating IGF-I concentration and melanoma risk (OR for highest vs lowest fifth = 0.93 [95% confidence interval [CI]: 0.71 to 1.22]). There was no significant heterogeneity in the association between IGF-I concentrations and melanoma risk when subdivided by gender, age at blood collection, BMI, height, age at diagnosis, time between blood collection and diagnosis, or by anatomical site or histological subtype of the tumour (Pheterogeneity≥0.078). We found no evidence for an association between circulating concentrations of IGF-I measured in adulthood and the risk of melanoma.

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9.

BACKGROUND: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. METHODS: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. RESULTS: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer's disease - outcomes for which large-scale trial data were unavailable. CONCLUSIONS: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate.

Methodological issues in a prospective study on plasma concentrations of persistent organic pollutants and pancreatic cancer risk within the EPIC cohort.

BACKGROUND: The use of biomarkers of environmental exposure to explore new risk factors for pancreatic cancer presents clinical, logistic, and methodological challenges that are also relevant in research on other complex diseases. OBJECTIVES: First, to summarize the main design features of a prospective case-control study -nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort- on plasma concentrations of persistent organic pollutants (POPs) and pancreatic cancer risk. And second, to assess the main methodological challenges posed by associations among characteristics and habits of study participants, fasting status, time from blood draw to cancer diagnosis, disease progression bias, basis of cancer diagnosis, and plasma concentrations of lipids and POPs. Results from etiologic analyses on POPs and pancreatic cancer risk, and other analyses, will be reported in future articles. METHODS: Study subjects were 1533 participants (513 cases and 1020 controls matched by study centre, sex, age at blood collection, date and time of blood collection, and fasting status) enrolled between 1992 and 2000. Plasma concentrations of 22 POPs were measured by gas chromatography - triple quadrupole mass spectrometry (GC-MS/MS). To estimate the magnitude of the associations we calculated multivariate-adjusted odds ratios by unconditional logistic regression, and adjusted geometric means by General Linear Regression Models. RESULTS: There were differences among countries in subjects' characteristics (as age, gender, smoking, lipid and POP concentrations), and in study characteristics (as time from blood collection to index date, year of last follow-up, length of follow-up, basis of cancer diagnosis, and fasting status). Adjusting for centre and time of blood collection, no factors were significantly associated with fasting status. Plasma concentrations of lipids were related to age, body mass index, fasting, country, and smoking. We detected and quantified 16 of the 22 POPs in more than 90% of individuals. All 22 POPs were detected in some participants, and the smallest number of POPs detected in one person was 15 (median, 19) with few differences by country. The highest concentrations were found for p,p'-DDE, PCBs 153 and 180 (median concentration: 3371, 1023, and 810 pg/mL, respectively). We assessed the possible occurrence of disease progression bias (DPB) in eight situations defined by lipid and POP measurements, on one hand, and by four factors: interval from blood draw to index date, tumour subsite, tumour stage, and grade of differentiation, on the other. In seven of the eight situations results supported the absence of DPB. CONCLUSIONS: The coexistence of differences across study centres in some design features and participant characteristics is of relevance to other multicentre studies. Relationships among subjects' characteristics and among such characteristics and design features may play important roles in the forthcoming analyses on the association between plasma concentrations of POPs and pancreatic cancer risk.

Separate and combined associations of obesity and metabolic health with coronary heart disease: a pan-European case-cohort analysis.

Aims: The hypothesis of 'metabolically healthy obesity' implies that, in the absence of metabolic dysfunction, individuals with excess adiposity are not at greater cardiovascular risk. We tested this hypothesis in a large pan-European prospective study. Methods and results: We conducted a case-cohort analysis in the 520 000-person European Prospective Investigation into Cancer and Nutrition study ('EPIC-CVD'). During a median follow-up of 12.2 years, we recorded 7637 incident coronary heart disease (CHD) cases. Using cut-offs recommended by guidelines, we defined obesity and overweight using body mass index (BMI), and metabolic dysfunction ('unhealthy') as ≥ 3 of elevated blood pressure, hypertriglyceridaemia, low HDL-cholesterol, hyperglycaemia, and elevated waist circumference. We calculated hazard ratios (HRs) and 95% confidence intervals (95% CI) within each country using Prentice-weighted Cox proportional hazard regressions, accounting for age, sex, centre, education, smoking, diet, and physical activity. Compared with metabolically healthy normal weight people (reference), HRs were 2.15 (95% CI: 1.79; 2.57) for unhealthy normal weight, 2.33 (1.97; 2.76) for unhealthy overweight, and 2.54 (2.21; 2.92) for unhealthy obese people. Compared with the reference group, HRs were 1.26 (1.14; 1.40) and 1.28 (1.03; 1.58) for metabolically healthy overweight and obese people, respectively. These results were robust to various sensitivity analyses. Conclusion: Irrespective of BMI, metabolically unhealthy individuals had higher CHD risk than their healthy counterparts. Conversely, irrespective of metabolic health, overweight and obese people had higher CHD risk than lean people. These findings challenge the concept of 'metabolically healthy obesity', encouraging population-wide strategies to tackle obesity.

Sensitivity to asbestos is increased in patients with mesothelioma and pathogenic germline variants in BAP1 or other DNA repair genes.

Pathogenic germline variants in the BAP1 tumor suppressor gene can cause a cancer syndrome called BAP1 tumor predisposition syndrome (BAP1-TPDS), which is characterized by predisposition to mesothelioma, melanoma, renal cell carcinoma, basal cell carcinoma, and other tumors. Other genes that may predispose to mesothelioma are CDKN2A and DNA repair genes. Asbestos exposure has often been reported in patients with malignant pleural mesothelioma (MPM) and germline variants in BAP1, but this exposure has never been quantified. We aimed to search for germline variants in BAP1 among 25 new Italian probands with suspected BAP1-TPDS, summarize the prevalence of these variants in 39 Italian patients with familial MPM and other tumors recruited over a 5-year period, and compare cumulative asbestos exposure in 14 patients with MPM and pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes with that of 67 patients without germline variants in 94 cancer-predisposing genes. We report here a new pathogenic germline variant in BAP1: c.783 + 2 T > C. The prevalence of pathogenic germline variants in BAP1 was 7.7% among patients with familial MPM (3/39). Patients with pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes showed lower cumulative asbestos exposure than patients without germline variants in 94 cancer-predisposing genes (P = .00002). This suggests an interaction between genetic risk factors and asbestos in the development of mesothelioma.

Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer.

OBJECTIVE: Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC). DESIGN: Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy. RESULTS: Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival. CONCLUSION: This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

MMP23B expression and protein levels in blood and urine are associated with bladder cancer.

Urothelial bladder cancer (UBC) represents a public health problem because of its high incidence/relapse rates. At present, there are no suitable biomarkers for early diagnosis or relapse/progression prognosis. To improve diagnostic accuracy and overcome the disadvantages of current diagnostic strategies, the detection of UBC biomarkers in easily accessible biofluids, such as urine, represents a promising approach compared with painful biopsies. We investigated the levels of MMP23 genes (microarray and qPCR) and protein (western blot and enzyme-linked immunosorbent assay) in a set of samples (blood, plasma and urine) from patients with UBC and controls as biomarkers for this cancer. MMP23B and its pseudogene MMP23A resulted downregulated in blood cells from UBC compared with controls (66 cases, 70 controls; adjusted P-value = 0.02 and 0.03, respectively). In contrast, MMP23B protein levels in plasma (53 UBC, 49 controls) and urine (59 UBC, 57 controls) increased in cases, being statistically significant in urine. MMP23B dosage observed in urine samples was related to both tumor risk classification and grading. As the lack of correlation between mRNA and protein levels could be due to a posttranscriptional regulation mediated by microRNAs (miRNAs), we investigated the expression of urinary miRNAs targeting MMP23B. Five miRNAs resulted differentially expressed between cases and controls. We reported the first evidence of MMP23B secretion in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC as a potential non-invasive biomarker for this cancer. Further analyses are needed to elucidate the mechanism of regulation of MMP23B expression by miRNAs.

Mitochondrial DNA copy number variation, leukocyte telomere length, and breast cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

BACKGROUND: Leukocyte telomere length (LTL) and mitochondrial genome (mtDNA) copy number and deletions have been proposed as risk markers for various cancer types, including breast cancer (BC). METHODS: To gain a more comprehensive picture on how these markers can modulate BC risk, alone or in conjunction, we performed simultaneous measurements of LTL and mtDNA copy number in up to 570 BC cases and 538 controls from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. As a first step, we measured LTL and mtDNA copy number in 96 individuals for which a blood sample had been collected twice with an interval of 15 years. RESULTS: According to the intraclass correlation (ICC), we found very good stability over the time period for both measurements, with ICCs of 0.63 for LTL and 0.60 for mtDNA copy number. In the analysis of the entire study sample, we observed that longer LTL was strongly associated with increased risk of BC (OR 2.71, 95% CI 1.58-4.65, p = 3.07 × 10- 4 for highest vs. lowest quartile; OR 3.20, 95% CI 1.57-6.55, p = 1.41 × 10- 3 as a continuous variable). We did not find any association between mtDNA copy number and BC risk; however, when considering only the functional copies, we observed an increased risk of developing estrogen receptor-positive BC (OR 2.47, 95% CI 1.05-5.80, p = 0.04 for highest vs. lowest quartile). CONCLUSIONS: We observed a very good correlation between the markers over a period of 15 years. We confirm a role of LTL in BC carcinogenesis and suggest an effect of mtDNA copy number on BC risk.

Tumor-associated autoantibodies as early detection markers for ovarian cancer? A prospective evaluation.

Immuno-proteomic screening has identified several tumor-associated autoantibodies (AAb) that may have diagnostic capacity for invasive epithelial ovarian cancer, with AAbs to P53 proteins and cancer-testis antigens (CTAGs) as prominent examples. However, the early detection potential of these AAbs has been insufficiently explored in prospective studies. We performed ELISA measurements of AAbs to CTAG1A, CTAG2, P53 and NUDT11 proteins, for 194 patients with ovarian cancer and 705 matched controls from the European EPIC cohort, using serum samples collected up to 36 months prior to diagnosis under usual care. CA125 was measured using electrochemo-luminiscence. Diagnostic discrimination statistics were calculated by strata of lead-time between blood collection and diagnosis. With lead times ≤6 months, ovarian cancer detection sensitivity at 0.98 specificity (SE98) varied from 0.19 [95% CI 0.08-0.40] for CTAG1A, CTAG2 and NUDT1 to 0.23 [0.10-0.44] for P53 (0.33 [0.11-0.68] for high-grade serous tumors). However, at longer lead-times, the ability of these AAb markers to distinguish future ovarian cancer cases from controls declined rapidly; at lead times >1 year, SE98 estimates were close to zero (all invasive cases, range: 0.01-0.11). Compared to CA125 alone, combined logistic regression scores of AAbs and CA125 did not improve detection sensitivity at equal level of specificity. The added value of these selected AAbs as markers for ovarian cancer beyond CA125 for early detection is therefore limited.

Circulating concentrations of vitamin D in relation to pancreatic cancer risk in European populations.

Evidence from in vivo, in vitro and ecological studies are suggestive of a protective effect of vitamin D against pancreatic cancer (PC). However, this has not been confirmed by analytical epidemiological studies. We aimed to examine the association between pre-diagnostic circulating vitamin D concentrations and PC incidence in European populations. We conducted a pooled nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Nord-Trøndelag Health Study's second survey (HUNT2) cohorts. In total, 738 primary incident PC cases (EPIC n = 626; HUNT2 n = 112; median follow-up = 6.9 years) were matched to 738 controls. Vitamin D [25(OH)D2 and 25(OH)D3 combined] concentrations were determined using isotope-dilution liquid chromatography-tandem mass spectrometry. Conditional logistic regression models with adjustments for body mass index and smoking habits were used to estimate incidence rate ratios (IRRs) and 95% confidence intervals (95%CI). Compared with a reference category of >50 to 75 nmol/L vitamin D, the IRRs (95% CIs) were 0.71 (0.42-1.20); 0.94 (0.72-1.22); 1.12 (0.82-1.53) and 1.26 (0.79-2.01) for clinically pre-defined categories of ≤25; >25 to 50; >75 to 100; and >100 nmol/L vitamin D, respectively (p for trend = 0.09). Corresponding analyses by quintiles of season-standardized vitamin D concentrations also did not reveal associations with PC risk (p for trend = 0.23). Although these findings among participants from the largest combination of European cohort studies to date show increasing effect estimates of PC risk with increasing pre-diagnostic concentrations of vitamin D, they are not statistically significant.

Identification of a novel susceptibility locus at 13q34 and refinement of the 20p12.2 region as a multi-signal locus associated with bladder cancer risk in individuals of European ancestry.

Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1 × 10(-6)), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 × 10(-11)) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 × 10(-10)). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region-the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r(2) = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case-case P ≤ 0.02 for both rs62185668 and rs6108803). Functional analyses are needed to explore the biological mechanisms underlying these novel genetic associations with risk for bladder cancer.

Epigenome-wide association study of adiposity and future risk of obesity-related diseases.

BACKGROUND: Obesity is an established risk factor for several common chronic diseases such as breast and colorectal cancer, metabolic and cardiovascular diseases; however, the biological basis for these relationships is not fully understood. To explore the association of obesity with these conditions, we investigated peripheral blood leucocyte (PBL) DNA methylation markers for adiposity and their contribution to risk of incident breast and colorectal cancer and myocardial infarction. METHODS: DNA methylation profiles (Illumina Infinium® HumanMethylation450 BeadChip) from 1941 individuals from four population-based European cohorts were analysed in relation to body mass index, waist circumference, waist-hip and waist-height ratio within a meta-analytical framework. In a subset of these individuals, data on genome-wide gene expression level, biomarkers of glucose and lipid metabolism were also available. Validation of methylation markers associated with all adiposity measures was performed in 358 individuals. Finally, we investigated the association of obesity-related methylation marks with breast, colorectal cancer and myocardial infarction within relevant subsets of the discovery population. RESULTS: We identified 40 CpG loci with methylation levels associated with at least one adiposity measure. Of these, one CpG locus (cg06500161) in ABCG1 was associated with all four adiposity measures (P = 9.07×10-8 to 3.27×10-18) and lower transcriptional activity of the full-length isoform of ABCG1 (P = 6.00×10-7), higher triglyceride levels (P = 5.37×10-9) and higher triglycerides-to-HDL cholesterol ratio (P = 1.03×10-10). Of the 40 informative and obesity-related CpG loci, two (in IL2RB and FGF18) were significantly associated with colorectal cancer (inversely, P < 1.6×10-3) and one intergenic locus on chromosome 1 was inversely associated with myocardial infarction (P < 1.25×10-3), independently of obesity and established risk factors. CONCLUSION: Our results suggest that epigenetic changes, in particular altered DNA methylation patterns, may be an intermediate biomarker at the intersection of obesity and obesity-related diseases, and could offer clues as to underlying biological mechanisms.

Advances in the Genetics of Hypertension: The Effect of Rare Variants.

Worldwide, hypertension still represents a serious health burden with nine million people dying as a consequence of hypertension-related complications. Essential hypertension is a complex trait supported by multifactorial genetic inheritance together with environmental factors. The heritability of blood pressure (BP) is estimated to be 30-50%. A great effort was made to find genetic variants affecting BP levels through Genome-Wide Association Studies (GWAS). This approach relies on the "common disease-common variant" hypothesis and led to the identification of multiple genetic variants which explain, in aggregate, only 2-3% of the genetic variance of hypertension. Part of the missing genetic information could be caused by variants too rare to be detected by GWAS. The use of exome chips and Next-Generation Sequencing facilitated the discovery of causative variants. Here, we report the advances in the detection of novel rare variants, genes, and/or pathways through the most promising approaches, and the recent statistical tests that have emerged to handle rare variants. We also discuss the need to further support rare novel variants with replication studies within larger consortia and with deeper functional studies to better understand how new genes might improve patient care and the stratification of the response to antihypertensive treatments.