A Simple and Cost-Effective Method for Measuring Hemolysis in Biobank Serum Specimens.

Background: During sampling and processing, blood samples can be affected by hemolysis. Information is lacking regarding hemolysis for biobank samples. There is a need for a method that can easily measure hemoglobin as an indicator of hemolysis in stored samples before they are included in research projects. In this study we present a simple method for estimating hemolysis and investigate the effect of centrifugation speeds and temperatures on sample turbidity that commonly interferes with measurements. Methods: Using a variation of the Beer-Lambert law, we quantified the hemoglobin concentration in 75 long-term stored samples at a wavelength of 414 nm with a NanoDrop™ 8000 spectrophotometer. Owing to interference from turbidity, the samples underwent different treatments post-thawing: centrifugation at 10,000 and 20,000 g at two different temperatures (4°C and 19°C) for 15 minutes. In addition, freshly collected serum samples (n = 20) underwent a single freeze-thaw cycle, with hemoglobin measured prefreeze, post-thaw, and postcentrifugation. Kruskal-Wallis rank sum test groups and pairwise Wilcoxon rank test were used for statistical analysis. Results: A strong effect of centrifugation on the turbidity was shown for the long-term stored samples, however, this effect was independent of the temperature or centrifugation speeds. Centrifugation at 20,000 g for 15 minutes at 19°C reduced the turbidity up to 50%. A single freeze-thaw cycle in the fresh samples increased the optical density at 414 nm slightly, indicating a false increase of hemoglobin concentration. The following centrifugation reduced the concentration to less than the initial sample measurements, suggesting the presence of interference immediately after sampling. Conclusion: We describe here a simple and cost-effective NanoDrop-based method for measuring hemolysis levels intended for use in biobank facilities. We found that centrifugation, but not temperature, is a crucial step to reduce interference from turbidity.

Prediagnostic Serum 25-Hydroxyvitamin D and Mortality Among Bladder Cancer Patients in the Janus Serum Bank Cohort.

PURPOSE: Circulating 25-hydroxyvitamin D (25(OH)D) is inversely associated with overall cancer mortality and selected cancers, while for urothelial bladder cancer (BC) this relationship is unclear. We aimed to examine the association between 25(OH)D and BC mortality. MATERIALS AND METHODS: We used prediagnostic serum from 378 BC cases within the population-based Janus Cohort. Cox regression models estimated hazard ratios (HRs), with 95% confidence intervals (CIs), for the association between 25(OH)D and BC-specific and all-cause mortality. Restricted cubic splines were assessed to examine non-linear risk associations. Analyses were stratified by tumor invasiveness (non-muscle invasive BC (NMIBC) and muscle invasive BC (MIBC)). Additionally, the association between 25(OH)D and all-cause mortality was assessed for 378 cancer-free matched controls. RESULTS: 25(OH)D deficiency (<50 nmol/L) was associated with higher BC-specific mortality (HR 1.87, 95% CI 1.10-3.20), when compared with insufficient levels (50-74 nmol/L). Stratification by tumor invasiveness revealed that this result was evident for NMIBC only, both with respect to BC-specific mortality (HR 2.84, 95% CI 1.14-7.12) and all-cause mortality (HR 1.97, 95% CI 1.06-3.65). No association between 25(OH)D levels and all-cause mortality was found in cancer-free controls. CONCLUSION: 25(OH)D deficiency (<50 nmol/L) prior to a BC diagnosis was associated with increased risk of BC-specific mortality, when compared to insufficient levels (50-74 nmol/L). The results were evident among NMIBC patients only, suggesting a more critical role of vitamin D deficiency in an early stage of the disease.

Vitamin D and Vitamin D-binding protein and risk of bladder cancer: A nested case-control study in the Norwegian Janus Serum Bank Cohort.

BACKGROUND: High circulating levels of vitamin D (25(OH)D) are suggested to reduce the risk of urinary bladder cancer (BC), but the evidence is weak, and several studies lack sufficient adjustment for potential confounders (e.g., smoking, body mass index (BMI), and physical activity). Moreover, few studies have investigated the role of vitamin D-binding protein (DBP) in this context. We conducted a matched nested case-control study including 378 cases and 378 controls within the Norwegian population-based Janus cohort, using serum collected 5-41 years prior to diagnosis, to study 25(OH)D and BC risk, by taking circulating DBP into account. METHODS: Cox regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs), for 25(OH)D, DBP, and the molar ratio of 25(OH)D:DBP, an estimate of unbound (free) 25(OH)D levels. We adjusted for smoking (status and pack-years), BMI, physical activity, education and (mutually) for 25(OH)D and DBP. Restricted cubic splines were employed to examine nonlinear associations. RESULTS: High optimal levels of circulating 25(OH)D (≥100 nmol/L) (HR 0.35, 95% CI 0.19-0.64) were associated with decreased BC risk, when compared with insufficient concentrations (50-74 nmol/L). This association was less pronounced for optimal levels (75-99 nmol/L) (HR = 0.69, 95% CI 0.47-1.01). Moreover, estimated free 25(OH)D, was associated with decreased BC risk for molar ratio 17-21 (HR 0.66, 95% CI 0.44-0.97) and ≥22 (HR 0.50, 95% CI 0.29-0.82), compared to molar ratio 11-16. The HR function for BC risk was not linear, rather reversed u-shaped, with the highest HR at 62.5 nmol/L and 13.5 molar ratio, respectively. CONCLUSION: High levels of total and estimated free 25(OH)D were associated with reduced risk of BC, compared with insufficient concentrations. DBP was not associated with BC risk. We did not observe any impact of DBP or any of the studied lifestyle factors on the association between 25(OH)D and BC.

Prediagnostic circulating levels of sex hormones and survival in esophageal adenocarcinoma.

Sex hormonal differences may contribute to the strong male predominance in esophageal adenocarcinoma (EAC), but whether sex hormone levels influence survival in EAC is unstudied. Our study aimed to assess associations between prediagnostic sex hormone levels and survival in EAC. In a population-based cohort study, 244 male EAC patients from the Janus Serum Bank Cohort in Norway were followed up through 2018. Associations between prediagnostic serum levels of 12 sex hormone measures and disease-specific mortality were assessed using multivariable Cox regression, providing hazard ratios (HR) with 95% confidence intervals (CI) adjusted for age, calendar year, body mass index, tobacco smoking, physical activity and surgical resection. Higher levels of sex hormone-binding globulin (SHBG) indicated decreased disease-specific mortality (HR 0.68, 95% CI 0.44-1.07, highest vs lowest tertile). In stratified analyses by surgery, such associations remained in nonoperated patients (HR 0.58, 95% CI 0.35-0.96, highest vs lowest tertile), but not in operated patients. Higher levels of follicle-stimulating hormone (FSH) were associated with increased disease-specific mortality in an exposure-response pattern; HRs for the middle and highest tertiles vs the lowest tertile were 1.35 (95% CI 0.89-2.05) and 1.61 (95% CI 1.06-2.43), respectively. No clear associations were observed with serum levels of dehydroepiandrosterone sulfate, luteinizing hormone, prolactin, testosterone, 17-OH-progesterone, progesterone, estradiol, androstenedione, testosterone:estradiol ratio or free testosterone index. These findings suggest that higher endogenous levels of SHBG and lower levels of FSH may increase the survival in EAC. The other 10 examined sex hormone measures may not influence the survival.

Competitive learning suggests circulating miRNA profiles for cancers decades prior to diagnosis.

MicroRNAs are regulators of gene expressionand may be key markers in liquid biopsy.Early diagnosis is an effective means to increase patients' overall survival. We generated genome-wide miRNA profiles from serum of patients and controls from the population-based Janus Serum Bank (JSB) and analysed them by bioinformatics and artificial intelligence approaches. JSB contains sera from 318,628 originally healthy persons, more than 96,000 of whom developed cancer. We selected 210 serum samples from patients with lung, colon or breast cancer at three time points prior to diagnosis (up to 32 years prior to diagnosis with median 5 years interval between TPs), one time-point after diagnosis and from individually matched controls. The controls were matched on age and year of all pre-diagnostic sampling time-points for the corresponding case. Using ANOVA we report 70 significantly deregulated markers (adjusted p-value<0.05). The driver for the significance was the diagnostic time point (miR-575, miR-6821-5p, miR-630 with adjusted p-values<10-10). Further, 91miRNAs were differently expressed in pre-diagnostic samples as compared to controls (nominal p < 0.05). Self-organized maps (SOMs)indicated larges effects in lung cancer samples while breast cancer samples showed the least pronounced changes. SOMsalsohighlighted cancer and time point specific miRNA dys-regulation. Intriguingly, a detailed breakdown of the results highlighted that 51% of all miRNAs were highly specific, either for a time-point or a cancer entity. Pathway analysis highlighted 12 pathways including Hipo signalling and ABC transporters.Our results indicate that tumours may be indicated by serum miRNAs decades prior the clinical manifestation.

Lifestyle associated factors and risk of urinary bladder cancer: A prospective cohort study from Norway.

A number of lifestyle associated factors, such as high body mass index (BMI), low physical activity, and related metabolic disorders, are associated with increased risk of cancer at several sites. For urinary bladder cancer (BC), such studies show inconsistent results, which could result from inadequate adjustment for smoking and occupational exposure. In the population-based Janus Cohort (n = 292 851), we investigated the independent and combined impact of BMI, physical activity, blood pressure, and blood lipids on the risk of BC, by thorough adjustment for smoking and potential occupational exposure. We used cox proportional hazard regression to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) for the associations between the lifestyle associated factors and BC risk. The associations observed were dependent on smoking status and gender. Among men, diastolic blood pressure (DBP) (HR 1.07, 95% CI 1.02-1.12) and systolic blood pressure (SBP) (HR 1.04, 95% CI 1.01-1.07) were positively associated with BC risk. Stratification by smoking status revealed a positive association between DBP and BC risk in never smokers (HR 1.14, 95% CI 1.00-1.30), while no association was seen for current and former smokers. A risk score, integrating information across the lifestyle factors was positively associated with BC risk in men (ptrend  = 0.043). In women, physical activity was associated with a decreased BC risk, but only among never smokers (HR 0.65, 95% CI 0.45-0.94). In conclusion, relations between lifestyle associated factors and BC risk were most evident in never smokers, suggesting that smoking dominates the relation in current smokers.

Identification and Validation of Leucine-rich α-2-glycoprotein 1 as a Noninvasive Biomarker for Improved Precision in Prostate Cancer Risk Stratification.

BACKGROUND: More accurate risk assessments are needed to improve prostate cancer management. OBJECTIVE: To identify blood-based protein biomarkers that provided prognostic information for risk stratification. DESIGN SETTING AND PARTICIPANTS: Mass spectrometry was used to identify biomarker candidates from blood, and validation studies were performed in four independent cohorts retrospectively collected between 1988 and 2015. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome objectives were progression-free survival, prostate cancer-specific survival (PCSS), and overall survival. Statistical analyses to assess survival and model performance were performed. RESULTS AND LIMITATION: Serum leucine-rich α-2-glycoprotein 1 (LRG1) was found to be elevated in fatal prostate cancer. LRG1 provided prognostic information independent of metastasis and increased the accuracy in predicting PCSS, particularly in the first 3 yr. A high LRG1 level is associated with an average of two-fold higher risk of disease-progression and mortality in both high-risk and metastatic patients. However, our study design, with a retrospective analysis of samples spanning several decades back, limits the assessment of the clinical utility of LRG1 in today's clinical practice. Thus, independent prospective studies are needed to establish LRG1 as a clinically useful biomarker for patient management. CONCLUSIONS: High blood levels of LRG1 are unfavourable in newly diagnosed high-risk and metastatic prostate cancer, and LRG1 increased the accuracy of risk stratification of prostate cancer patients. PATIENT SUMMARY: High blood levels of leucine-rich α-2-glycoprotein 1 are unfavourable in newly diagnosed high-risk and metastatic prostate cancer.

Ultralow amounts of DNA from long-term archived serum samples produce quality genotypes.

While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3-6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.

Circulating Sex Hormone Levels and Risk of Esophageal Adenocarcinoma in a Prospective Study in Men.

OBJECTIVES: Sex hormones have been hypothesized to explain the strong male predominance in esophageal adenocarcinoma, but evidence is needed. This study examined how circulating sex hormone levels influence future risk of esophageal adenocarcinoma. METHODS: This case-control study was nested in a prospective Norwegian cohort (Janus Serum Bank Cohort), including 244 male patients with esophageal adenocarcinoma and 244 male age-matched control participants. Associations between prediagnostic circulating levels of 12 sex hormones and risk of esophageal adenocarcinoma were assessed using conditional logistic regression. In addition, a random-effect meta-analysis combined these data with a similar prospective study for 5 sex hormones. RESULTS: Decreased odds ratios (ORs) of esophageal adenocarcinoma were found comparing the highest with lowest quartiles of testosterone (OR = 0.44, 95% confidence interval [CI] 0.22-0.88), testosterone:estradiol ratio (OR = 0.37, 95% CI 0.19-0.72), and luteinizing hormone (OR = 0.50, 95% CI 0.30-0.98), after adjustment for tobacco smoking and physical activity. These associations were attenuated after further adjustment for body mass index (OR = 0.56, 95% CI 0.27-1.13 for testosterone; OR = 0.46, 95% CI 0.23-0.91 for testosterone:estradiol ratio; OR = 0.55, 95% CI 0.29-1.08 for luteinizing hormone). No associations were observed for sex hormone-binding globulin, dehydroepiandrosterone sulfate, follicle-stimulating hormone, prolactin, 17-OH progesterone, progesterone, androstenedione, or free testosterone index. The meta-analysis showed an inverse association between testosterone levels and risk of esophageal adenocarcinoma (pooled OR for the highest vs lowest quartile = 0.60, 95% CI 0.38-0.97), whereas no associations were identified for androstenedione, sex hormone-binding globulin, estradiol, or testosterone:estradiol ratio. DISCUSSION: Higher circulating testosterone levels may decrease the risk of esophageal adenocarcinoma in men.

A Collaborative Analysis of Individual Participant Data from 19 Prospective Studies Assesses Circulating Vitamin D and Prostate Cancer Risk.

Previous prospective studies assessing the relationship between circulating concentrations of vitamin D and prostate cancer risk have shown inconclusive results, particularly for risk of aggressive disease. In this study, we examine the association between prediagnostic concentrations of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] and the risk of prostate cancer overall and by tumor characteristics. Principal investigators of 19 prospective studies provided individual participant data on circulating 25(OH)D and 1,25(OH)2D for up to 13,462 men with incident prostate cancer and 20,261 control participants. ORs for prostate cancer by study-specific fifths of season-standardized vitamin D concentration were estimated using multivariable-adjusted conditional logistic regression. 25(OH)D concentration was positively associated with risk for total prostate cancer (multivariable-adjusted OR comparing highest vs. lowest study-specific fifth was 1.22; 95% confidence interval, 1.13-1.31; P trend < 0.001). However, this association varied by disease aggressiveness (P heterogeneity = 0.014); higher circulating 25(OH)D was associated with a higher risk of nonaggressive disease (OR per 80 percentile increase = 1.24, 1.13-1.36) but not with aggressive disease (defined as stage 4, metastases, or prostate cancer death, 0.95, 0.78-1.15). 1,25(OH)2D concentration was not associated with risk for prostate cancer overall or by tumor characteristics. The absence of an association of vitamin D with aggressive disease does not support the hypothesis that vitamin D deficiency increases prostate cancer risk. Rather, the association of high circulating 25(OH)D concentration with a higher risk of nonaggressive prostate cancer may be influenced by detection bias. SIGNIFICANCE: This international collaboration comprises the largest prospective study on blood vitamin D and prostate cancer risk and shows no association with aggressive disease but some evidence of a higher risk of nonaggressive disease.

Low Free Testosterone and Prostate Cancer Risk: A Collaborative Analysis of 20 Prospective Studies.

BACKGROUND: Experimental and clinical evidence implicates testosterone in the aetiology of prostate cancer. Variation across the normal range of circulating free testosterone concentrations may not lead to changes in prostate biology, unless circulating concentrations are low. This may also apply to prostate cancer risk, but this has not been investigated in an epidemiological setting. OBJECTIVE: To examine whether men with low concentrations of circulating free testosterone have a reduced risk of prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Analysis of individual participant data from 20 prospective studies including 6933 prostate cancer cases, diagnosed on average 6.8 yr after blood collection, and 12 088 controls in the Endogenous Hormones, Nutritional Biomarkers and Prostate Cancer Collaborative Group. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Odds ratios (ORs) of incident overall prostate cancer and subtypes by stage and grade, using conditional logistic regression, based on study-specific tenths of calculated free testosterone concentration. RESULTS AND LIMITATIONS: Men in the lowest tenth of free testosterone concentration had a lower risk of overall prostate cancer (OR=0.77, 95% confidence interval [CI] 0.69-0.86; p<0.001) compared with men with higher concentrations (2nd-10th tenths of the distribution). Heterogeneity was present by tumour grade (phet=0.01), with a lower risk of low-grade disease (OR=0.76, 95% CI 0.67-0.88) and a nonsignificantly higher risk of high-grade disease (OR=1.56, 95% CI 0.95-2.57). There was no evidence of heterogeneity by tumour stage. The observational design is a limitation. CONCLUSIONS: Men with low circulating free testosterone may have a lower risk of overall prostate cancer; this may be due to a direct biological effect, or detection bias. Further research is needed to explore the apparent differential association by tumour grade. PATIENT SUMMARY: In this study, we looked at circulating testosterone levels and risk of developing prostate cancer, finding that men with low testosterone had a lower risk of prostate cancer.

Circulating isoflavone and lignan concentrations and prostate cancer risk: a meta-analysis of individual participant data from seven prospective studies including 2,828 cases and 5,593 controls.

Phytoestrogens may influence prostate cancer development. This study aimed to examine the association between prediagnostic circulating concentrations of isoflavones (genistein, daidzein, equol) and lignans (enterolactone and enterodiol) and the risk of prostate cancer. Individual participant data were available from seven prospective studies (two studies from Japan with 241 cases and 503 controls and five studies from Europe with 2,828 cases and 5,593 controls). Because of the large difference in circulating isoflavone concentrations between Japan and Europe, analyses of the associations of isoflavone concentrations and prostate cancer risk were evaluated separately. Prostate cancer risk by study-specific fourths of circulating concentrations of each phytoestrogen was estimated using multivariable-adjusted conditional logistic regression. In men from Japan, those with high compared to low circulating equol concentrations had a lower risk of prostate cancer (multivariable-adjusted OR for upper quartile [Q4] vs. Q1 = 0.61, 95% confidence interval [CI] = 0.39-0.97), although there was no significant trend (OR per 75 percentile increase = 0.69, 95 CI = 0.46-1.05, ptrend = 0.085); Genistein and daidzein concentrations were not significantly associated with risk (ORs for Q4 vs. Q1 = 0.70, 0.45-1.10 and 0.71, 0.45-1.12, respectively). In men from Europe, circulating concentrations of genistein, daidzein and equol were not associated with risk. Circulating lignan concentrations were not associated with the risk of prostate cancer, overall or by disease aggressiveness or time to diagnosis. There was no strong evidence that prediagnostic circulating concentrations of isoflavones or lignans are associated with prostate cancer risk, although further research is warranted in populations where isoflavone intakes are high.

Author Correction: Large-scale intact glycopeptide identification by Mascot database search.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Vitamin D, obesity and leptin in relation to bladder cancer incidence and survival: prospective protocol study.

INTRODUCTION: Bladder cancer (BC) (including renal pelvis, ureter and urethra) is one of the most common urogenital cancers and the fourth most frequent cancer in men in the USA. In Norway, the incidence of BC has increased over the last decades. The age-standardised incidence rates per 100 000 for 2011-2015 were 53.7 in men and 16.5 in women. Compared to the 5-year period 2006-2010, the percentage increase in incidence was 6.1% in men and 12.3% in women. The recurrence rate of BC is over 50%, the highest recurrence rate of any malignancy. Smoking and occupational exposure to aromatic amines are recognised as the major risk factors. Recently, low-serum level of 25-hydroxy vitamin D (25(OH)D) and obesity have been suggested to increase the BC risk, and leptin, which is important in weight regulation, may be involved in bladder carcinogenesis. More knowledge on potential risk factors for BC is necessary for planning and implementing primary prevention measures. METHODS AND ANALYSES: Cohort and nested case-control studies will be carried out using the population-based Janus Serum Bank Cohort consisting of prediagnostic sera, clinical measurement data (body height and weight, body surface area and weight change over time, blood pressure, cholesterol and triglycerides) and self-reported information on lifestyle factors (smoking, physical activity). Participants were followed from cohort inclusion (1972-2003) through 2014. The cohort will be linked to the Cancer Registry of Norway (cancer data), the National Cause of Death Registry (date and cause of death), National Population Registry (vital status) and Statistic Norway (education and occupation). Serum samples will be analysed for 25(OH)D, vitamin D binding protein, leptin, albumin, calcium and parathyroid hormone. Cox regression and conditional logistic regression models and mediation analysis will be used to estimate association between the exposures and BC. ETHICS AND DISSEMINATION: The study has been approved by the Regional Committee for Medical Research Ethics and is funded by the Norwegian Cancer Society. Results will be published in peer-reviewed journals, at scientific conferences and through press releases.

Large-scale intact glycopeptide identification by Mascot database search.

Workflows capable of determining glycopeptides in large-scale are missing in the field of glycoproteomics. We present an approach for automated annotation of intact glycopeptide mass spectra. The steps in adopting the Mascot search engine for intact glycopeptide analysis included: (i) assigning one letter codes for monosaccharides, (ii) linearizing glycan sequences and (iii) preparing custom glycoprotein databases. Automated annotation of both N- and O-linked glycopeptides was proven using standard glycoproteins. In a large-scale study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in 24 serum samples. Thus, a single tool was developed that collectively allows the (i) elucidation of N- and O-linked glycopeptide spectra, (ii) matching glycopeptides to known protein sequences, and (iii) high-throughput, batch-wise analysis of large-scale glycoproteomics data sets.

Sources to variability in circulating human miRNA signatures.

An increasing number of studies propose circulating microRNAs (miRNAs) as biomarkers for a large number of human diseases including cancer, cardiovascular diseases, neurologic pathologies and others. To further validate miRNA as biomarkers it is indispensable to understand the variability of circulating miRNAs in healthy individuals. We determined the longitudinal miRNomes of 90 serum samples from the Janus Serum Bank in Norway, which have been stored between 23 and 40 y at -25 °Celsius. We profiled 3 serum samples with microarrays for 30 individuals, each. For each individual the samples were collected with a time interval of approximately 5 y. This design allowed insights into inter-individual variability, age dependent miRNA variability and the impact of storage length and pre-processing. A significant proportion of the miRNome was affected by the age of the blood donor and a not negligible, albeit small, part of the miRNome by the storage time. A substantial part of miRNAs was differentially abundant between individuals, independent of the time when samples were collected. Stepwise filtering of the 529 miRNAs that were detected in the serum samples showed 168 miRNAs with differential abundance depending on the time point analyzed, 56 miRNAs differentially abundant between individuals, and 169 miRNAs with an abundance depending on the sampling procedure. While these groups of miRNAs contain generally interesting and biologically important miRNAs, the remaining 135 miRNAs constitute very promising biomarker candidates as they show an overall low variability between healthy individuals, a likewise overall low variability across a longer life span, and a high independence of the sampling process and the storage length.

Effect of multiple freeze-thaw cycles on selected biochemical serum components.

BACKGROUND: To maintain the best performance a frozen serum sample should be thawed once to prevent repeated freeze-thaw cycles. Archival biobanks often have one tube of a sample available, causing repeated freeze-thaw cycles when the sample is used in multiple research projects. In this study, we investigated potential effects of freeze-thaw cycles on several biochemical components in serum. METHODS: Serum from 40 fasting donors of both genders, aged 30-60 years, were frozen at -25 °C. Aliquots of the 40 different samples went through 1, 2, 3, 4, 5 and 10 thaws, respectively. They were analyzed after 3 month of storage for 15 serum components including electrolytes and metabolites, proteins and enzymes, lipids, hormones and vitamins. One-way analyses of variance (ANOVA) with repeated measurements and equivalence tests were used to examine differences in component levels. RESULTS: Albumin, aspartate-aminotransferase (ASAT), cholesterol, creatinine, C-reactive protein, glucose, immunoglobulin G, potassium, testosterone, triglycerides, urea and vitamin B12 levels did not show significant difference for pairwise comparisons after 10 repeated thaws. Although albumin, ASAT, bilirubin, potassium, sodium, testosterone and thyroid stimulating hormone (TSH) showed overall statistically significant changes in serum levels, only bilirubin, sodium and TSH were significant for the pairwise comparisons investigated. Clinical significance were shown for albumin, ASAT, bilirubin, sodium and testosterone. CONCLUSIONS: Twelve components (albumin, ASAT, cholesterol, creatinine, C-reactive protein, glucose, immunoglobulin G, potassium, testosterone, triglycerides, urea and vitamin B12) were robust to 10 repeated thaws compared to baseline level. Three components (bilirubin, sodium and TSH) showed statistical significant difference for pairwise comparisons, however, TSH was not clinically affected.

Circulating Folate and Vitamin B12 and Risk of Prostate Cancer: A Collaborative Analysis of Individual Participant Data from Six Cohorts Including 6875 Cases and 8104 Controls.

BACKGROUND: Folate and vitamin B12 are essential for maintaining DNA integrity and may influence prostate cancer (PCa) risk, but the association with clinically relevant, advanced stage, and high-grade disease is unclear. OBJECTIVE: To investigate the associations between circulating folate and vitamin B12 concentrations and risk of PCa overall and by disease stage and grade. DESIGN, SETTING, AND PARTICIPANTS: A study was performed with a nested case-control design based on individual participant data from six cohort studies including 6875 cases and 8104 controls; blood collection from 1981 to 2008, and an average follow-up of 8.9 yr (standard deviation 7.3). Odds ratios (ORs) of incident PCa by study-specific fifths of circulating folate and vitamin B12 were calculated using multivariable adjusted conditional logistic regression. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Incident PCa and subtype by stage and grade. RESULTS AND LIMITATIONS: Higher folate and vitamin B12 concentrations were associated with a small increase in risk of PCa (ORs for the top vs bottom fifths were 1.13 [95% confidence interval (CI), 1.02-1.26], ptrend=0.018, for folate and 1.12 [95% CI, 1.01-1.25], ptrend=0.017, for vitamin B12), with no evidence of heterogeneity between studies. The association with folate varied by tumour grade (pheterogeneity<0.001); higher folate concentration was associated with an elevated risk of high-grade disease (OR for the top vs bottom fifth: 2.30 [95% CI, 1.28-4.12]; ptrend=0.001), with no association for low-grade disease. There was no evidence of heterogeneity in the association of folate with risk by stage or of vitamin B12 with risk by stage or grade of disease (pheterogeneity>0.05). Use of single blood-sample measurements of folate and B12 concentrations is a limitation. CONCLUSIONS: The association between higher folate concentration and risk of high-grade disease, not evident for low-grade disease, suggests a possible role for folate in the progression of clinically relevant PCa and warrants further investigation. PATIENT SUMMARY: Folate, a vitamin obtained from foods and supplements, is important for maintaining cell health. In this study, however, men with higher blood folate levels were at greater risk of high-grade (more aggressive) prostate cancer compared with men with lower folate levels. Further research is needed to investigate the possible role of folate in the progression of this disease.