Reliable Biomarkers For Early Detection Research Articles

Monoclonal Antibody Production Against Vimentin by Whole Cell Immunization in a Mouse Model.

BackgroundPancreatic carcinoma is the fourth-leading cause of cancer death in the United States and due to its late presentation, only few patients would be candidates for the curative treatment of pancreactomy. Monoclonal antibodies have brought hope to targeted therapy.ObjectivesTo identify new biomarkers, a panel of monoclonal antibodies was generated against newly established cell line, Faraz-ICR from a patient with pancreatic acinar cell carcinoma.Material and MethodsBalb/c female mice were immunized with Faraz-ICR cell line and their spleenocytes fused with SP2/0 myeloma cell line. Highly reactive hybridoma producing antibodies against Faraz-ICR was detected using ELISA, immunofluorescence staining and flow cytometry. Western blot and 2D immunoblot were utilized for further characterization of the target antibodies.ResultsAmong highly reactive clones, the reactivity of 7C11 clone was assessed in comparison to other epithelial tumors. The antibody isotype was IgM that reacted with a 55 kDa protein in western blot analysis. To further characterize the target antigen, immunoproteome of the Faraz-ICR cell line was performed. By LC-MS analysis, the target of 7C11 clone was identified to be vimentin.ConclusionsPancreatic cancer is a highly lethal malignancy with no reliable biomarker for early detection and diagnosis. In this study, by establishing a pancreatic acinar carcinoma cell line, a panel of monoclonal antibodies was generated to identify specific or associated cancer targets. Furthermore, 7C11 mAb was introduced that can specifically recognizes vimentin as a tumor marker. This antibody may serve as a new tool for prognostic and therapeutic strategies.

Diagnostična vrednost označevalcev pri sepsi

Background: Sepsis is a life-threatening organ dysfunction that arises when a host responds insufficiently to an infection as bacteria enter the bloodstream. In recent years, SOFA scoring system has been used to identify poor organ functioning. Microbiological blood tests represent a gold standard in sepsis diagnostics. Reliable biomarkers for early detection of sepsis would greatly facilitate rapid and efficient treatment of sepsis.Methods: In a prospective non-interventional study we studied the diagnostic value of C-reactive protein (CRP), procalcitonin (PCT), neutrophil CD64 index, neutrophil granulocyte count and immature neutrophil count in patients with sepsis and patients with severe infection without sepsis. A total of 46 consecutive intensive-care-unit patients admitted for severe infection and 10 healthy controls were included. The patients were treated routinely according to the principles of good clinical practice.Results: Statistically significant differences between the two groups of patients have been established for the CD64 index, the PCT and the immature neutrophil count, whereas the differences in the CRP and the neutrophil granulocyte count are statistically non-significant. The highest diagnostic values were measured for the immature neutrophil count (AUC 0.91) and PCT (AUC 0.84). The combination of biomarkers has been shown to have same predictive values as the immature neutrophil count and the PCT.

SERUM SOLUBLE CD25 IN HEPATOCELLULAR CARCINOMA, SHALL WE BE ABLE TO CHANGE THE NATURAL HISTORY?

Background. Although hepatocellular carcinoma (HCC) is one of the most common malignancy related mortality worldwide, it can be curable if detected in early stages. Emergence of a new marker that can early detect HCC could help in early treatment and therefore ameliorate the outcome.Objective. The aim of the research is to evaluate the performance of serum soluble CD25 (sCD25) in the prediction of early HCC and compare it to α-fetoprotein (AFP).Methods. Serum levels of sCD25 and AFP were measured in three groups of population; HCC group (40 patients), cirrhosis without HCC control group (20 patients) and healthy control group (20 patients). HCC group contained 20 early and 20 late stage patients according to Barcelona Clinic Liver Cancer (BCLC) staging system (stage 0/A and B-D respectively). Levels of both biomarkers were compared in all groups. Predictive yield of both biomarkers for early HCC was evaluated using ROC curve analysis.Results. Level of sCD25 was significantly higher in patients with HCC than in both cirrhotic controls and healthy controls (P<0.0001and 0.013 respectively). For prediction of early HCC in patients with cirrhosis, the optimal sCD25 cut-off level was 7.15 ng/ml with sensitivity and specificity of 90% and 60% respectively (AUC=0.717; P=0.019) while sensitivity and specificity of AFP were 70% and 85% respectively at a cut-off value of 9.85 ng/ml (AUC=0.781; P=0.002) in the same settings.Conclusion. sCD25 seems to be a reliable biomarker for early detection of HCC and therefore could enhance the outcome.

Potential serum biomarkers for early detection of diabetic nephropathy

AimDiabetic nephropathy (DN) is considered as one of the diabetic complications affecting up to 40% of patients with type 1 or type 2 diabetes. In clinical practice, the frequently used markers of renal disease and progression are serum creatinine, estimated glomerular filtration rate (eGFR) and albuminuria. The aim of this study is to determine new biomarkers in human serum which are promising for early detection of DN. MethodsThis study included 50 patients with type 2 diabetes mellitus (T2DM) and 25 clinically healthy individuals. The patients were divided into two groups; group I included 25 T2DM patients with normoalbuminuria, and group II consisted of 25 T2DM patients with microalbuminuria. In all groups, neutrophil gelatinase-associated lipocalin (NGAL), β-trace protein (βTP) and microRNA- 130b (miR-130b) were estimated. ResultsThe serum levels of NGAL and βTP were significantly elevated in T2DM patients with microalbuminuria (group II) compared with T2DM patients with normoalbuminuria (group I) and control subjects but there was no significant difference between group I and control subjects. Serum miR-130b level was significantly decreased in patients with T2DM (groups I and II) compared with healthy control subjects, with a higher decrease in their levels in group II compared with group I. ConclusionOur results suggest that serum NGAL and βTP as tubular and glomerular markers respectively, together with serum miR-130b may be independent and reliable biomarkers for early detection of DN in patients with T2DM.

Identification of key metabolic changes during liver fibrosis progression in rats using a urine and serum metabolomics approach

Reversibility of hepatic fibrosis is an intrinsic response to chronic injury, and with on-going damage, fibrosis can progress to its end-stage consequence, cirrhosis. Non-invasive and reliable biomarkers for early detection of liver fibrosis are needed. Based on the CCl4-induced liver fibrosis rat model, urinary and serum metabolic profiling performed by LC-QTOF-MS associated with histological progression were utilized to identify liver fibrosis-specific potential biomarkers for early prediction and to reveal significant fibrotic pathways and their dynamic changes in different stages of liver fibrosis. Finally, nine differential metabolites in urine and ten in serum were selected and identified involving the most relevant metabolic pathways. Perturbations of tryptophan, valine, leucine, isoleucine, and citrate (TCA) cycle metabolites, along with sphingolipid and glycerophospholipid metabolites, occurred from the onset of liver fibrosis. Furthermore, dysregulation of valine and bile acid biosynthesis metabolites occurred in the intermediate and advanced stages. More importantly, among these metabolites, urinary kynurenic acid, 5-hydroxyindoleacetyl glycine, 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid and serum sphinganine, sphingomyelin, L-leucine, L-tryptophan, and LysoPC(17:0) changed at all time points and may serve as potential early biomarkers for the diagnosis of hepatic fibrosis and as therapeutic targets. Overall, this work evaluates the potential of these metabolites for the early detection of liver fibrosis.

Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma.

Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

Diagnostic biomarkers in murine Cryptosporidiosis: dose- and age-related infection

Increasing prevalence of Cryptosporidium raises the importance to explore different aspects of its infection. In the absence of reproducible in vitro culturing, animal model is the only experimental method to study Cryptosporidium. Our study evaluated Cryptosporidium infection using coproscopy, copro-antigen and copro-DNA for early detection of murine cryptosporidiosis. Hundred and forty albino mice (neonates and adult) were divided into two groups, control group received sterile PBS solution, and infected groups were inoculated with molecularly characterized Cryptosporidium parvum oocysts and further subdivided into three subgroups for infectious dose response detection. Mice fecal samples were collected every 4h on the first day and then daily and examined for fecal oocysts, copro-antigen and copro-DNA. Four mice from each subgroup were killed at 12, 24 and 48h post-infection (P-I), and their intestines were examined for cryptosporidial mucosal DNA. Cryptosporidium copro-antigen and copro-DNA were detected 4 and 8h P-I in infected neonatal and adult mice, respectively, and intestinal mucosal DNA was detected after 12h in both. Microscopy was able to detect oocysts 48h P-I. Inoculated C. parvum oocysts were recovered in feces of infected mice without genotypic changes. Neonate mice showed higher susceptibility for cryptosporidial infection than adults without statistical differences for the given infectious doses. Both copro-immunoassay and copro-nPCR assays can early detect Cryptosporidium infection; however, nPCR was able to identify Cryptosporidium species, making nPCR a reliable biomarker for early detection in murine model.

Restoring walking after total knee arthroplasty: Symmetries of 3D body centre of mass trajectory and mechanical work

INTRODUCTION Osteoarthritis (OA) is a degenerative joint disease constantly increasing in elderly population and the knee is one of the most affected joints. It contributed together with other musculoskeletal disorders to the 2nd greatest cause of disability in the WHO Global Burden of Disease [1] and patients no more responding to conservative treatments undergo total knee-arthroplasty (TKA) to remove pain and permit an asymptomatic walking. Radiological reports and questionnaires are the current diagnostic tools employed both for the diagnosis and monitoring of OA progression. However, substantial discordance exists between current diagnostic outcomes using those tools and OA symptoms [2]. Further investigation need to identify more reliable biomarkers for early detection of the disease, correct timing for TKA and post-surgery monitoring. Therefore in this study we aim to describe quantitatively the gait of OA patients undergoing to the TKA, with a specific focus on the 3D body centre of mass (BcOM) trajectory (Locomotion 3D Lissajous Countours [3]). METHODS 7 patients with unilateral knee OA (68.9 ± 4.8 years, 69.1 ± 9.1 kg, 1.60 ± 0.07 m) were tested for 4 times: before surgery (PRE), after 2 months (POST2), after 6 months (POST6) and after 12 months (POST12) from TKA. Each time patients were asked to i) complete a WOMAC questionnaire, ii) walk on a 5-force plates (Bertec, Columbus, OH, U.S.A) corridor to estimate spontaneous velocity (vs), and iii) walk for 1 min on a treadmill at 3 selected velocities (vs, v1 and v2). Two velocities, lower than vs, were calculated starting from the Froude Number (Fr): v1 = -30% Fr and v2 = -15% Fr, respectively). A 18 marker model was used to calculate (VICON, Oxford UK) the 3D BCoM trajectory, its symmetries, stride frequency (SF), double contact (DC) time, right and left duty factor, mechanical external and internal work (Wext and Wint respectively), total work (Wtot) and energy recovery (RE). RESULTS WOMAC indices improved significantly already after 6 months and vs significantly increased from 0.89 ± 0.14 (PRE) to 1.13 ± 0.07 m/s (POST12). The figure shows the average BCoM profiles during walking (pointed arrows refer to vertical (z) and progression (x) axes), where upper boundary reaches a higher vertical position (3%) in the POST12 condition compared to the PRE condition and a more symmetrical shape, significantly in x and z directions. For the same walking speed SF decreased significantly already after two months from TKA, DC time decreased to 20% of the stride length and duty factor also assumed lower values after surgery. Patients after TKA improved their locomotor energy recovery mechanisms, reducing mechanical external work by 10%. WINT was constant along the months, while RE slightly increased after TKA. DISCUSSION The group of quantitatively analysed variables, suggest as patients with prosthesis tend to re-establish their natural locomotion pattern after TKA. Body centre of mass analysis during walking, including symmetry and mechanical energy evaluations, quantitatively assess osteoarthritis disease level and can be employed to monitor patients before and after pharmacological and surgical interventions. The same methodology could be applicable to neurological pathologies and to patients affected by other orthopaedic pathologies affecting other limb joints, like hip or ankle. The effects of different prosthesis or surgical techniques on a single pathology/joint could be equally investigated.

Controversies in Candida management

Invasive candidiasis (IC) is associated with increased morbidity and mortality. Although advances in diagnosis and management of this infection have been reached, there remain several controversies. The aim of this review is to tackle some of these controversies and shed light on studies that support the different sides of the arguments. Regarding central line-associated candidaemia, the current evidence appears to be more in favour of early central line removal whenever possible. Otherwise, antifungal agents such as echinocandins or polyenes should be the preferred agents. In the setting of infection with Candida parapsilosis and in light of the high minimum inhibitory concentrations (MICs) to echinocandins, azoles have been considered the preferred treatment agents. However, a recent study appears to indicate that empirical echinocandin use was not associated with a worse outcome when the isolated species was C. parapsilosis. Different strategies of antifungal treatment have been considered, namely prophylactic, empirical, pre-emptive or directed therapies. Whilst there is consensus on the need for prophylaxis in high-risk cancer patients, especially haematological malignancy and stem cell transplant populations, it remains debatable whether prophylaxis is of benefit in very low birthweight infants and in intensive care unit (ICU) patients. In the era of antifungal resistance and where antifungal stewardship has been advocated, pre-emptive therapy based on predictive models with various Candida risk scores and sensitive non-culture-based biomarkers such as β-d-glucan appears to be a more cost-effective approach. Future efforts should be directed to optimise clinical predictive models and reliable biomarkers for early detection of IC.

An electrochemical nanobiosensor for plasma miRNA-155, based on graphene oxide and gold nanorod, for early detection of breast cancer.

Circulating miRNAs are emerging as novel reliable biomarkers for early detection of cancer diseases. Through combining the advantages of electrochemical methods and nanomaterials with the selectivity of the oligo-hybridization-based biosensors, a novel electrochemical nanobiosensor for plasma miR-155 detection have demonstrated here, based on thiolated probe-functionalized gold nanorods (GNRs) decorated on the graphene oxide (GO) sheet on the surface of the glassy carbon electrode (GCE). The reduction signals of a novel intercalating label Oracet Blue (OB), were measured by differential pulse voltammetry (DPV) method. The transmission electron microscope (TEM) imaging, UV-vis spectrophotometry, cyclic voltammetry (CV), field emission scanning electron microscope (FE-SEM) imaging and energy dispersive spectroscopy (EDS) were proved the right synthesis of the GNRs and correct assembly of the modified electrode. The electrochemical signal had a linear relationship with the concentration of the target miRNA ranging from 2.0 fM to 8.0 pM, and the detection limit was 0.6 fM. Furthermore, the nanobiosensor showed high Specificity, and was able to discriminate sharply between complementary target miRNA, single-, three-base mismatch, and non-complementary miRNA. Alongside the outstanding sensitivity and selectivity, this nanobiosensor had great storage ability, reproducibility, and showed a decent response in the real sample analysis with plasma. In conclusion, the proposed electrochemical nanobiosensor could clinically be used in the early detection of the breast cancer, by direct detection of the plasma miR-155 in real clinical samples, without a need for sample preparation, RNA extraction and/or amplification.

Clinical significance of serum miR-196a in cervical intraepithelial neoplasia and cervical cancer.

Previous studies have reported that miR-196a is upregulated in cervical cancer tissues and cell lines. However, whether serum miR-196a is increased in patients with cervical cancer or cervical intraepithelial neoplasia (CIN), and its potential clinical value remained unknown. In total, 105 cervical cancer patients, 86 CIN patients, and 50 healthy volunteers were recruited. Quantitative reverse transcription-polymerase chain reaction was performed to compare the serum levels miR-196a in all participants. The associations between serum miR-196a and CIN grade/clinicopathological parameters of cervical cancer were also examined. A survival analysis was performed using the Kaplan-Meier method. Univariate and multivariate analyses were conducted to explore the independent risk factors for cervical cancer. Our results revealed that serum miR-196a levels were higher in patients with cervical cancer (P < 0.01) and CIN (P < 0.05) compared to those in healthy controls. Serum miR-196a was associated with CIN grade and various cervical cancer parameters including tumor size (P = 0.031), lymph node metastasis (P = 0.018), FIGO stage (P = 0.004), and grade (P = 0.011). Cervical cancer patients with higher serum miR-196a levels had a poorer overall survival rate (P = 0.004). Multivariate analysis revealed that high serum miR-196a was an independent predictor for poor survival of cervical cancer (HR = 3.510; 95%CI = 1.961-6.874; P = 0.025). In conclusion, our findings suggest that serum miR-196a overexpression is associated with CIN grade and cervical cancer progression. Therefore, serum miR-196a may be a reliable biomarker for early detection and prognosis of cervical cancer.

Identification of genes in ulcerative colitis associated colorectal cancer based on centrality analysis of co-expression network

PreviousColorectal cancer (CRC) is awell-recognized complication of Ulcerative colitis (UC) and patients with UC have ahigher incidence of CRC than the general population. Early detection and mechanism of colitis-associated colorectal cancer (CAC) is still challenging. The aim of present study is to identify genes associated with CAC by centrality analysis of co-expression networks. Co-expression networks of CRC and UC were constructed by empirical Bayes approach based on top 200 gene signatures which identified by the model of genome-wide relative significance and genome-wide global significance across multiple datasets. Centrality of degree, stress centrality, betweenness centrality and closeness centrality of co-expression networks were selected to explore hub genes presented in CRC and UC. Validation of mRNA expression in CRC patients was conducted by real-time quantitative Polymerase Chain Reaction (qPCR). Pathway analysis was conducted based on Kyoto Encyclopedia of Genes and Genomes database. We found 21 common genes, such as SLC4A4 and AQP8, both existed in CRC and UC top 200 genes. By accessing centralities analyses of co-expression networks, HPGD and AQP8 were common hub genes in CRC and UC, and various centralities analyses of the same gene were not consistent. Patients with alteration of AQP8 have significantly reduced the survival rate according to real-time qPCR results. Our study displayed genes associated with CAC (AQP8 and HPGD), and they might be reliable biomarkers for early detection and therapies of CAC.

Diagnostic and short-term prognostic utility of plasma pro-enkephalin (pro-ENK) for acute kidney injury in patients admitted with sepsis in the emergency department.

Acute kidney injury (AKI) aggravates the prognosis of patients with sepsis. Reliable biomarkers for early detection of AKI in this setting are lacking. Enkephalins influence kidney function, and may have a role in AKI from sepsis. We utilized a novel immunoassay for plasma proenkephalin (pro-ENK), a stable surrogate marker for endogenous enkephalins, in patients hospitalized with sepsis, in order to assess its clinical utility. In an observational retrospective study we enrolled 101 consecutive patients admitted to the emergency department (ED) with suspected sepsis. Plasma levels of pro-ENK and neutrophil gelatinase-associated lipocalin (NGAL) were evaluated at ED arrival for their association with presence and severity of AKI and 7-day mortality. pro-ENK was inversely correlated to creatinine clearance (r=-0.72) and increased with severity of AKI as determined by RIFLE (risk, injury, failure, loss of function, end-stage renal disease) stages (p<0.0001; pro-ENK median [interquartile range, IQR]) pmol/l: no AKI: 71 [41-97]; risk: 72 [51-120]; injury: 200 [104-259]; failure: 230 [104-670]; loss of function: 947 [273-811]. The majority of septic patients without AKI or at risk had pro-ENK concentrations within the normal range. While NGAL was similarly associated with AKI severity, it was strongly elevated already in septic patients without AKI. pro-ENK added predictive information to NGAL for detecting kidney dysfunction (added χ (2) 10.0, p=0.0016). Admission pro-ENK outperformed creatinine clearance in predicting 7-day mortality (pro-ENK: χ (2) 13.4, p<0.001, area under curve, AUC 0.69; creatinine clearance: χ (2) 4, p=0.045, AUC: 0.61), and serial measurement improved prediction. Use of pro-ENK in septic patients can detect the presence and severity of AKI. Moreover, pro-ENK is highly predictive of short-term mortality and could enable early identification of patients at risk of death.

The possible involvement of microRNAs in preeclampsia and gestational diabetes mellitus

Great obstetrical syndromes is a collective name for several complications of pregnancy that affect >15% of pregnancies. They may confer adverse pregnancy outcomes and maternal and fetal morbidity, and require close medical monitoring and treatment. The etiology, pathogenesis, and outcome of these syndromes are obscure in the majority of cases. All appear during mid-to-late pregnancy with no reliable biomarkers for early detection and possibly prevention at present. This article focuses on the quest for early reliable markers for preeclampsia and gestational diabetes mellitus (GDM) development, mainly on the involvement of microRNA in the pathogenesis and its possible role as an early biomarker for disease development.

Dimethylnitrosamine-induced reduction in the level of poly-ADP-ribosylation of histone proteins of blood lymphocytes--a sensitive and reliable biomarker for early detection of cancer.

Poly-ADP-ribosylation (PAR) is a post-translational modification of mainly chromosomal proteins. It is known to be strongly involved in several molecular events, including nucleosome-remodelling and carcinogenesis. In this investigation, it was attempted to evaluate PAR level as a reliable biomarker for early detection of cancer in blood lymphocyte histones. PAR of isolated histone proteins was monitored in normal and dimethylnitrosamine (DMN)-exposed mice tissues using a novel ELISA-based immuno-probe assay developed in our laboratory. An inverse relationship was found between the level of PAR and period of DMN exposure in various histone proteins of blood lymphocytes and spleen cells. With the increase in the DMN exposure period, there was reduction in the PAR level of individual histones in both cases. It was also observed that the decrease in the level of PAR of histones resulted in progressive relaxation of genomic DNA, perhaps triggering activation of genes that are involved in initiation of transformation. The observed effect of carcinogen on the PAR of blood lymphocyte histones provided us with a handy tool for monitoring biochemical or physiological status of individuals exposed to carcinogens without obtaining biopsies of cancerous tissues, which involves several medical and ethical issues. Obtaining blood from any patient and separating blood lymphocytes are routine medical practices involving virtually no medical intervention, post-procedure medical care or trauma to a patient. Moreover, the immuno-probe assay is very simple, sensitive, reliable and cost-effective. Therefore, combined with the ease of preparation of blood lymphocytes and the simplicity of the technique, immuno-probe assay of PAR has the potential to be applied for mass screening of cancer. It appears to be a promising step in the ultimate goal of making cancer detection simple, sensitive and reliable in the near future.

Autoantibodies to Ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models

BackgroundPancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy with only a 5% 5-year survival rate. Reliable biomarkers for early detection are still lacking. The goals of this study were (a) to identify early humoral responses in genetically engineered mice (GEM) spontaneously developing PDAC; and (b) to test their diagnostic/predictive value in newly diagnosed PDAC patients and in prediagnostic sera.Methods and resultsThe serum reactivity of GEM from inception to invasive cancer, and in resectable or advanced human PDAC was tested by two-dimensional electrophoresis Western blot against proteins from murine and human PDAC cell lines, respectively. A common mouse-to-human autoantibody signature, directed against six antigens identified by MALDI-TOF mass spectrometry, was determined. Of the six antigens, Ezrin displayed the highest frequency of autoantibodies in GEM with early disease and in PDAC patients with resectable disease. The diagnostic value of Ezrin-autoantibodies to discriminate PDAC from controls was further shown by ELISA and ROC analyses (P < 0.0001). This observation was confirmed in prediagnostic sera from the EPIC prospective study in patients who eventually developed PDAC (with a mean time lag of 61.2 months between blood drawing and PDAC diagnosis). A combination of Ezrin-autoantibodies with CA19.9 serum levels and phosphorylated α-Enolase autoantibodies showed an overall diagnostic accuracy of 0.96 ± 0.02.ConclusionsAutoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature.

Abstract 2486: Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat.

Abstract The development of sensitive, specific, and reliable biomarkers for early detection of breast cancer and the discovery of novel chemopreventive agents are two strategies with promising clinical applications. It has been postulated that the risk of breast cancer can be influenced by certain dietary compounds such as the type and amount of dietary fats ingested. We have shown that a diet high in an n-3:n-6 ratio (10:1 and 25:1), that provided 30% calories from fat (10% monounsaturated, 10% polyunsaturated, and 10% saturated fatty acids), significantly reduced rat mammary cancer induced by 1-methyl-1-nitrosourea(MNU). In the present study we used a proteomic approach to gain insights into the mechanisms of protection by a high n-3:n-6 ratio with the goal of identifying biomarkers that can be employed to monitor efficacy of chemopreventive agents. The plasma from six rats from each of three groups, a control group(n-3:n-6=1:1) and two groups fed a high n-3 diet(n-3:n-6=10:1 and 25:1) was pooled and submitted to the isobaric Tags for Relative and Absolute Quantitation(iTRAQ) method, which includes matrix-assisted laser desorption/ionization tandem mass spectroscopy(MALDITOF/TOF) to identify and quantify proteins that were regulated as a result of n-3 fatty acid supplementation. There were 148 proteins identified in our study with 95% confidence(unused score 1.3 or greater) by ProteinPilot 4.0. In plasma of rats fed 10:1 n-3:n-6, and 25:1 n-3:n-6 the number of proteins that met our criteria(p&amp;lt;0.05, error factor≤2) were 10 and 14 proteins, respectively. Our results illustrated that many proteins were modified in a manner consistent with chemoprevention. Validation of the iTRAQ by Western Blotting showed up-regulation of vitamin D binding protein and gelsolin postulated to have a tumor suppressive effect through modulation of inflammation and actin depolarization, respectively. We also observed that 14-3-3 sigma, a well known tumor suppressor in breast tissue, was increased in a ratio dependent fashion. In contrast, we observed down-regulation of alpha-1b-glycoprotein which has been reported to be elevated in the serum of breast cancer patients. These proteins may serve in future studies as candidate biomarkers in early detection and clinical chemoprevention trials with well-defined ratios of n-3:n-6 fatty acids. Support: Susan G. Komen Foundation(KG081632) Citation Format: Christine G. Skibinski, Henry J. Thompson, Arunangshu Das, Andrea Manni, Karam El-Bayoumy. Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2486. doi:10.1158/1538-7445.AM2013-2486

Global DNA Hypomethylation in Peripheral Blood Mononuclear Cells as a Biomarker of Cancer Risk

Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25-4.53) vs. 5.13 (95% CI, 5.03-5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24-4.51) vs. 5.08 (95% CI, 5.05-5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52-32.63; P = 0.013). Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status.

Identification of GABRA1 and LAMA2 as new DNA methylation markers in colorectal cancer

Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.

Presence of evolutionary conserved gene expression signatures during progression of Barrett's esophagus to esophageal adenocarcinoma in human and rat

Esophageal adenocarcinoma (EAC) in Barrett's esophagus (BE) patients is thought to arise from a sequence of growth‐promoting genetic alterations due to chronic esophagitis. However the molecular events underlying the pathogenesis of BE and EAC remain largely unclear. To define the biological mechanisms of EAC development, we performed a cross‐species comparison of gene expression data from human and rat tissues, seeking the evolutionarily conserved gene expression patterns during tumor progression across both species. We used a non‐carcinogenic rat model that closely resembling the natural history of EAC development in human. Using Illumina beadarray platform, we first generated gene expression data from human tissues 28 NE, 72 BE, and 75 EAC as well as from rat tissues 15 NE, 18 BE, and 30 EAC. Statistical analyses on human and rat gene expression data revealed gene expression patterns significantly associated with progression of disease (NE to BE and BE to EAC). Using orthologous genes presented in both microarray platforms, we identified 397 genes are commonly altered as NE progresses to BE and 205 genes including SPP1 were altered as BE progresses to EAC in both species. Systems‐level characterization of gene expression data from human and rodent could become reliable biomarkers for early detection or therapeutic targets for treatment of EAC patients.