Progressive degradation of serum samples limits proteomic biomarker discovery

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at −80 °C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at −80 and −20 °C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.     

Proteomics-based identification of autoantibody against CDC25B as a novel serum marker in esophageal squamous cell carcinoma

This study was aimed to identify tumor proteins that elicit a humoral response in patients with esophageal squamous cell carcinoma (ESCC). Autologous sera of 15 newly diagnosed patients with ESCC and age- and gender-matched 15 healthy controls were analyzed individually for antibody-based reactivity against proteins from 15 homogenized ESCC tissue mixture resolved by two-dimensional PAGE. One protein spot, which reacted with sera from ESCC patients but not with those from controls, was identified to be CDC25B by mass spectrometry and Western blotting. High expression of CDC25B was detected in ESCC cell lines and primary tumor tissues, but not in normal esophageal tissues. In addition, CDC25B expression was significantly higher in tumor tissue of patients with sera positive CDC25B-Abs than that of patients without CDC25B-Abs. Finally, anti-CDC25B antibodies were readily detectable in sera from 45 of 124 (36.29%) patients with ESCC, 13 of 150 (8.67%) patients with other types of cancer and 0 of 102 (0%) of healthy individuals. Thus, CDC25B autoantibodies may have clinical utility in ESCC screening and diagnosis.     

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.     

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).     

Apolipoprotein A-I as a candidate serum marker for the response to lithium treatment in bipolar disorder

The molecular basis of bipolar disorder (BD) is still unknown as is the mechanism through which lithium, the therapy of choice, exerts its effects in treatment of BD. So far, no biomarkers exist to facilitate diagnosis of BD or treatment evaluation. To investigate whether BD and its treatment with lithium leaves a characteristic signature in the serum proteome, we used SELDI‐TOF MS to analyze individual serum samples from BD patients treated with lithium (BD‐plus‐Li, n=15) or other drugs (BD‐minus‐Li, n=10) and from healthy controls (n=15). Interestingly, features of 28 kDa (one peak) and 14 kDa (three peaks) showed a decreased level in the BD‐minus‐Li group and a level restored to that of the control group in the BD‐plus‐Li group. To reveal the identity of these features, we subjected pooled serum samples from both BD groups to the 2‐D DIGE technology and identified 28 kDa apolipoprotein A‐I (apo A‐I) and three 14 kDa fragments thereof as upregulated in the BD‐plus‐Li group. Immunoturbidimetry, a routine clinical assay, verified the characteristic apo A‐I signature in individual serum samples. In conclusion, we propose apo A‐I as a candidate marker that can visualize response to lithium treatment at the serum protein level.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.     

An integrated approach for comparative proteomic analysis of human bile reveals overexpressed cancer-associated proteins in malignant biliary stenosis

Proteomics is a key tool in the identification of new bile biomarkers for differentiating malignant and nonmalignant biliary stenoses. Unfortunately, the complexity of bile and the presence of molecules interfering with protein analysis represent an obstacle for quantitative proteomic studies in bile samples. The simultaneous need to introduce purification steps and minimize the use of pre-fractionation methods inevitably leads to protein loss and limited quantifications. This dramatically reduces the chance of identifying new potential biomarkers. In the present study, we included differential centrifugation as a preliminary step in a quantitative proteomic workflow involving iTRAQ labeling, peptide fractionation by OFFGEL electrophoresis and LC-MS/MS, to compare protein expression in bile samples collected from patients with malignant or nonmalignant biliary stenoses. A total of 1267 proteins were identified, including a set of 322 newly described bile proteins, mainly belonging to high-density cellular fractions. The subsequent comparative analysis led to a 5-fold increase in the number of quantified proteins over previously published studies and highlighted 104 proteins overexpressed in malignant samples. Finally, immunoblot verifications performed on a cohort of 8 malignant (pancreatic adenocarcinoma, n = 4; cholangiocarcinoma, n = 4) and 5 nonmalignant samples (chronic pancreatitis, n = 3; biliary stones, n = 2) confirmed the results of proteomic analysis for three proteins: olfactomedin-4, syntenin-2 and Ras-related C3 botulinum toxin substrate 1. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Quantitative proteomic analysis by iTRAQ for identification of candidate biomarkers in plasma from acute respiratory distress syndrome patients

Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critical patients. Proteomic analysis of plasma from individuals with ARDS could elucidate new biomarkers for diagnosis and pathophysiology and identify potential ARDS treatment targets. In this study, we recruited 26 patients (15 controls, 11 ARDS). The ARDS group was subdivided into two groups depending on the type of injury: (1) direct lung injury (AD) and (2) indirect lung injury (AI). Using iTRAQ (isobaric tags for relative and absolute quantitation) analysis, we identified 2429 peptides representing 132 plasma proteins. Among these, 16 were differentially expressed in ARDS patients, including 11 overlapping proteins between the AI and AD group and 5 AI-specific proteins. Protein annotation revealed that lipid transport and complement activation were significantly enriched in the biological process category, and lipid transporter, transporter, and serine-type peptidase activities were significantly enriched in the molecular function category. IPA (Ingenuity Pathway Analysis) signaling pathways revealed that the overlapping proteins were involved in a variety of signaling pathways, including those underlying acute phase response; liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X (FXR)/RXR activation; clathrin-mediated endocytosis; atherosclerosis; interleukin (IL)-12; complement system; and cytokine, nitric oxide, and reactive oxygen species production in macrophages. We present the first proteomic analysis of ARDS plasma using the iTRAQ approach. Our data provide new biomarker candidates and shed light on potential pathological mechanisms underlying ARDS.     

Proteomic surveillance of autoantigens in patients with Behcet's disease by a proteomic approach

To promote an understanding of autoimmunity in BD, we surveyed autoAgs in patients with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE‐WB, out of which eight spots were identified. They are enolase‐1, cofilin‐1, vimentin, Rho‐GDI β protein, tubulin‐like protein, and actin‐like proteins. The autoAbs to one of the identified proteins, cofilin‐1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin‐1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%) of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin‐1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs.     

Decline in serum antibodies to methyltetrahydrophthalic anhydride after cessation of exposure.Implications for use as a biomarker

T he association between exposure intensity and serum levels of immunoglobulins E and G against low molecular weight compounds was evaluated. The decay of levels of specific IgE and IgG antibodies was studied after cessation of exposure in workers exposed to the inhalant allergen methyltetrahydrophthalic anhydride in a plant using epoxy resins. Sera have been collected in workers for 18-84 (mean value 54) months after cessation of exposure. Specific IgE and IgG was assessed by RAST and ELISA, respectively. The mean of individual half-times for IgE ( N = 10) and IgG ( N = 8) was 0 9 (range 0 1-1 8) and 0 4 (range 0 2-0 6) years, respectively, after total avoidance of exposure. Corresponding decreases of IgE and IgG were also observed after reduction, but not total elimination, of exposure. No correlation was seen between biologic halftimes of specific IgE and total IgE, atopy, smoking habits or gender. The results indicate that the levels of specific antibodies in sensitized individuals reflect long term exposure, and may persist for years after the end of exposure.     

Undercarboxylated osteocalcin as a biomarker of subclinical atherosclerosis in non-dialysis patients with chronic kidney disease

Background

Studies in recent years have shown that undercarboxylated osteocalcin (uOC) not only maintains bone mineralization, but is also involved in the regulation of atherosclerosis. However, a correlation between uOC and carotid atherosclerosis in non-dialysis patients with chronic kidney disease (CKD) has not been investigated.A total of 240 non-dialysis patients with CKD were included in the study. For these patients, the median estimated glomerular filtration rate (eGFR) was 20.05 (12.43–49.32) ml/min/1.73m². Serum uOC levels were measured using enzyme-linked immunosorbent assay (ELISA). Carotid ultrasonography was performed to assess carotid atherosclerotic plaques and intima–media thickness (IMT) in an attempt to analyze the relationship between uOC level and carotid atherosclerosis.

Results

The uOC levels of non-dialysis patients with CKD were significantly lower than those of healthy controls [28.16 (21.40–45.85) ng/mL vs. 36.42 (28.05–49.28) ng/mL, P < 0.01]. The uOC levels gradually decreased as CKD progressed (P < 0.01). The uOC levels were significantly lower in patients with carotid plaques than in patients without carotid plaques [25.98 (20.14–31.35) ng/mL vs. 31.02 (25.86–36.40) ng/mL, P < 0.01]. uOC level showed significant negative correlation with IMT (r = -0.33, P < 0.01). Logistic regression analysis revealed that after adjustment for various confounding factors, decreased uOC levels were shown to indicate increased possibility of carotid atherosclerotic plaque development in non-dialysis patients with CKD (on every 1 SD decrease in the uOC level, odds ratio 1.70, 95 % confidence interval 1.24–2.98, P < 0.01). Multivariate stepwise regression analysis demonstrated that decreased uOC level (β = -0.163, P < 0.05) was an independent risk factor for increased carotid IMT in non-dialysis patients with CKD.

Conclusion

Serum uOC levels in non-dialysis patients with CKD are significantly lower than those in healthy individuals, and uOC is closely associated with subclinical atherosclerosis in CKD patients.     

A food safety control low mass-range proteomics platform for the detection of illicit treatments in veal calves by MALDI-TOF-MS serum profiling

Performance enhancing agents (PEAs) are illegally used in cattle and other meat producing species to increase food conversion and lean meat production. Due to the very short breeding cycle, veal calves represent the meat producing bovine category mostly subjected to illicit treatments. These chemical agents are difficult to detect by conventional analytical approaches due to the employment of synergistic formulations at very low dosage and given the use of uncharacterized novel compounds. Such a scenario has fostered a strong interest in the discovery of functional molecular biomarkers for the detection of growth promoting agents in meat producing species. A multivariate MALDI-TOF-MS proteomics platform has been developed using bovine serum samples. Analytical performances have been thoroughly evaluated in order to enable reproducible profiles from 10 μL sera samples. We propose univariate and multivariate discrimination models capable to identify calves undergoing illicit treatments. In particular, we found a strong discrimination power associated with a polypeptide fragment from β2-glycoprotein-I. We provide a fundamental proof of concept in the potential application of MALDI-TOF-MS proteomics profiling in the food safety control.     

Serum sulfatides as a novel biomarker for cardiovascular disease in patients with end-stage renal failure

Sulfatides, normal components of serum lipoproteins, may play an important role in cardiovascular disease due to their various modulatory functions in haemostasis. The incidence of cardiovascular disease in patients with end-stage renal failure undergoing maintenance hemodialysis has been reported to be approximately 10 to 30 times higher than that in the general population. To elucidate the possible roles of serum sulfatides in this high incidence, we measured the level of sulfatides in 59 such patients, by converting them to lysosulfatides according to a recently developed quantitative, qualitative, high-throughput technique using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mean level of sulfatides in patients 3.58 ± 1.18 nmol/ml was significantly lower than that in age-matched normal subjects (8.21 ± 1.50 nmol/ml; P < 0.001). Patients receiving maintenance hemodialysis over a longer period had lower levels of sulfatides. When the mean levels of sulfatides were compared between patients with cardiovascular disease (N = 22) and those without the disease (N = 37), the level in the former group 2.85 ± 0.67 nmol/ml was found to be significantly lower than that in the latter group 4.01 ± 1.22 nmol/ml (P < 0.001). These findings reveal a close correlation between low levels of serum sulfatides and a high risk of cardiovascular disease in these patients. Determination of the level of serum sulfatides can contribute to predictions of the incidence of cardiovascular disease in patients with end-stage renal failure undergoing maintenance hemodialysis.     

Quantification of urinary o,o'-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. A comparison with ion-trap tandem mass spectrometry

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o'-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD=0.01 microM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o'-dityrosine concentration was 0.08+/-0.01 microM. Expressed per urinary creatinine concentration, this corresponds to 10.1+/-0.4 micromol/mol creatinine. This is comparable to the previously reported values of 5.8+/-0.3 micromol/mol creatinine in non-smokers night-time urines, and 12.3+/-5 micromol/mol creatinine in day-time urines measured by the ion-trap instrument.     

Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux

Background

The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation – altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure.

Methods

The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure.

Results

The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions.

Conclusions

The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-2) contains supplementary material, which is available to authorized users.     

HnRNPL as a key factor in spermatogenesis: Lesson from functional proteomic studies of azoospermia patients with sertoli cell only syndrome

Sertoli cell only syndrome (SCOS) is one of the main causes leading to the abnormal spermatogenesis. However, the mechanisms for abnormal spermatogenesis in SCOS are still unclear. Here, we analyzed the clinical testis samples of SCOS patients by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to find the key factors contributing to SCOS. Thirteen differential proteins were identified in clinical testis samples between normal spermatogenesis group and SCOS group. Interestingly, in these differential proteins, Heterogeneous nuclear ribonucleoprotein L(HnRNPL) was suggested as a key regulator involved in apoptosis, death and growth of spermatogenic cells by String and Pubgene bioinformatic programs. Down-regulated HnRNPL in testis samples of SCOS patients was further confirmed by immunohistochemical staining and western blotting. Moreover, in vitro and in vivo experiments demonstrated that knockdown of HnRNPL led to inhibited proliferation, increased apoptosis of spermatogenic cell but decreased apoptosis of sertoli cells. Expression of carcinoembryonic antigen-related cell adhesion molecule 1 in GC-1 cells or expression of inducible nitric oxide synthases in TM4 sertoli cells, was found to be regulated by HnRNPL. Our study first shows HnRNPL as a key factor involved in the spermatogenesis by functional proteomic studies of azoospermia patients with sertoli cell only syndrome. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Intestinal fatty acid binding protein (I-FABP) as a possible biomarker of ileitis in patients with ulcerative colitis

BACKGROUND: One of nine types of FABP, intestinal fatty acid binding protein (I-FABP) is primarily limited to the mature enterocytes of the small intestine, with only trace amounts identified in the stomach and large intestine. The aim of this study was to investigate the use of I-FABP as a possible plasma marker of intestinal injury in patients with ulcerative colitis (UC). MATERIAL AND METHODS: The study group consisted of 42 patients (11 females and 31 males) with active ulcerative colitis (UC), aged from 24 to 74 years (mean age: 41.8+/-3.5 years). Plasma I-FABP concentrations and hsCRP were compared using endoscopic pictures scored according to the system developed by Meyers et al., and analysed in the context of inflammatory process extension: pancolitis, or distal or left side colitis. RESULTS: The mean serum I-FABP concentration /mL), whereas individuals with left-side colitis had a mean I-FABP concentration of 61.8+/-8.5 pg/mL. Significant serum I-FABP elevation was observed in UC patients with a severe form of the disease, in contrast to the serum I-FABP concentration in patients with the mild form (260.5+/-60.6 vs. 61.5+/-7.9 pg/mL). CONCLUSION: The elevated serum I-FABP concentration in patients with UC may indicate ileitis. I-FABP may be a useful marker of the extended inflammatory process.     

CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients

Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = ?0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01–1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00–1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.     

Development of an immunoassay for the derived-peptide of chromogranin A,Vasostatin-I (1-76): assessment of severity in patients with sepsis

Context: Proteolytic fragments of chromogranin A (CgA) including the CgA 1-76 fragment (called vasostatin-I [VS-I]) could be a useful biomarker of sepsis, but there is no available immunoassay.

Methods: A sandwich ELISA for VS-I was developed, and plasma VS-I was measured in 30 healthy controls and 60 critically ill patients with sepsis.

Results: The ELISA showed intra- and inter-assay coefficients of variations (CVs) below 4 and 9%. Plasma VS-I was significantly increased compared with controls in patients with sepsis, severe sepsis, and sepsis shock (p < 0.0001). Receiver operating curve (ROC) analyses indicated that plasma VS-I was more sensitive and specific than plasma CgA to diagnose sepsis and to assess its severity.

Conclusions: The measurements of plasma VS-I with this new ELISA may be useful for the clinical investigation of patients with sepsis.