Combination of high anti-SKI and low anti-TMED5 antibody levels is preferable prognostic factor in esophageal carcinoma.

Given that esophageal cancer is highly malignant, the discovery of novel prognostic markers is eagerly awaited. We performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified SKI proto-oncogene protein and transmembrane p24 trafficking protein 5 (TMED5) as antigens recognized by serum IgG antibodies in patients with esophageal carcinoma. SKI and TMED5 proteins were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens. The serum anti-SKI antibody (s-SKI-Ab) and anti-TMED5 antibody (s-TMED5-Ab) levels were significantly higher in 192 patients with esophageal carcinoma than in 96 healthy donors. The presence of s-SKI-Abs and s-TMED5-Abs in the patients' sera was confirmed by western blotting. Immunohistochemical staining showed that the TMED5 protein was highly expressed in the cytoplasm and nuclear compartments of the esophageal squamous cell carcinoma tissues, whereas the SKI protein was localized predominantly in the nuclei. Regarding the overall survival in 91 patients who underwent radical surgery, the s-SKI-Ab-positive and s-TMED5-Ab-negative statuses were significantly associated with a favorable prognosis. Additionally, the combination of s-SKI-Ab-positive and s-TMED5-Ab-negative cases showed an even clearer difference in overall survival as compared with that of s-SKI-Ab-negative and s-TMED5-Ab-positive cases. The s-SKI-Ab and s-TMED5-Ab biomarkers are useful for diagnosing esophageal carcinoma and distinguishing between favorable and poor prognoses.

Stage-Specific Alteration and Prognostic Relationship of Serum Fumarate Hydratase Autoantibodies in Gastric Cancer.

The relationship between energy production and cancer is attracting attention. This study aimed to investigate the clinicopathological significance of fumarate hydratase (FH), a tricarboxylic acid cycle enzyme, in gastric cancer using autoantibodies as biomarkers. The study analyzed 116 patients who underwent gastric cancer surgery and 96 healthy controls. Preoperative serum FH autoantibody (s-FH-Ab) titers were analyzed using an immunosorbent assay with an amplified luminescent proximity homogeneous assay. Receiver operating characteristic analysis was used to determine the cutoff s-FH-Ab titer. Clinicopathological factors and prognosis were compared between the high and low s-FH-Ab groups. The s-FH-Ab levels were significantly higher in the gastric cancer group than in the control group (p = 0.01). Levels were elevated even in patients with stage I gastric cancer compared with healthy controls (p = 0.02). A low s-FH-Ab level was significantly associated with distant metastasis (p = 0.01), peritoneal dissemination (p < 0.05), and poor overall survival (p < 0.01). Multivariate analysis revealed that low s-FH-Ab levels were an independent risk factor for poor prognosis (p < 0.01). Therefore, s-FH-Ab levels may be a useful biomarker for early diagnosis and the prediction of prognosis in patients with gastric cancer.

JMJD6 Autoantibodies as a Potential Biomarker for Inflammation-Related Diseases.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima-media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

Analysis of patients with colorectal cancer shows a specific increase in serum anti-ING1 autoantibody levels.

Colorectal cancer (CRC) is the third most prevalent cancer in the world, yet the sensitivity and specificity of biomarkers for CRC diagnosis are insufficient. In the present study, we performed a protein microarray screening method to identify antibody markers for CRC. Inhibitor of growth family 1 (ING1) was identified as a candidate tumor antigen for CRC using protein microarrays (ProtoArray). Subsequent amplified luminescence proximity homogeneous assay-linked immunosorbent assay using recombinant ING1 protein showed that the serum levels of anti-ING1 antibodies were increased not only in patients with CRC but also in those with esophageal cancer (EC), gastric cancer (GC), breast cancer (BrC), and pancreatic cancer (PC) compared with those of healthy donors (HDs). Antibodies against the ING1 amino acids between 239 and 253 were present at significantly higher levels in patients with CRC than in those with EC, GC, BrC, or PC. Anti-ING1 antibody levels were significantly higher in the patients with CRC at any stages than in the HDs. Immunohistochemical staining revealed higher expression of ING1 protein in CRC cells than in the adjacent normal tissues. In luciferase reporter assays using a CRC cell line, ING1 augmented p53-mediated NOXA promoter activity but attenuated p53-stimulated Bax, p21, and PUMA promoter activities. Consequently, serum anti-ING1 antibodies can be used for sensitive and specific diagnoses of CRC.

Serum anti-PCK1 antibody levels are a prognostic factor for patients with diabetes mellitus.

BACKGROUND: Autoantibodies develop in autoimmune diseases, cancer, diabetes mellitus (DM), and atherosclerosis-related diseases. However, autoantibody biomarkers have not been successfully examined for diagnosis and therapy. METHODS: Serological identification of antigens through recombinant cDNA expression cloning (SEREX) was used for primary screening of antigens. The cDNA product was expressed in bacteria and purified. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was used to evaluate antibody levels in serum samples. RESULTS: Phosphoenolpyruvate carboxykinase 1 (PCK1) was recognized as an antigen by serum IgG antibodies in the sera of patients with atherosclerosis. AlphaLISA showed significantly higher serum antibody levels against recombinant PCK1 protein in patients with DM and cardiovascular disease than in healthy donors, but not in those with acute ischemic stroke, transient ischemic attack, or obstructive sleep apnea syndrome. The area under the receiver operating characteristic curve for anti-PCK1 antibodies was 0.7024 for DM. The serum anti-PCK1 antibody levels were associated with age, platelet count, and blood pressure. Anti-PCK1-antibody-positive patients showed significantly lower overall survival than the negative patients. CONCLUSIONS: Serum anti-PCK1 antibody levels were found to be associated with DM. The anti-PCK1 antibody marker is useful for predicting the overall survival of patients with DM.

Low anti-CFL1 antibody with high anti-ACTB antibody is a poor prognostic factor in esophageal squamous cell carcinoma.

BACKGROUND: Cofilin (CFL1, actin-binding protein) and ß-actin (ACTB) are key molecules in the polymerization and depolymerization of actin microfilaments. The levels of these antibodies were analyzed, and the clinicopathological significance in patients with esophageal carcinoma were evaluated. METHODS: The levels of anti-CFL1 and anti-ACTB antibodies were analyzed in serum samples of patients with esophageal carcinoma and of healthy donors. Eighty-seven cases underwent radical surgery and the clinicopathological characteristics and prognosis was examined. RESULTS: Serum anti-CFL1 antibody (s-CFL1-Ab) levels and anti-ACTB antibody (s-ACTB-Ab) levels were significantly higher in patients with esophageal carcinoma than in healthy donors. Following the receiver operating characteristic curve analysis between healthy donors and esophageal carcinoma, the sensitivity and specificity for serum anti-CFL1 antibody (s-CFL1-Ab) were 53.3% and 68.8%. The sensitivity and specificity for serum anti-ACTB antibody (s-ACTB-Ab) were 54.9% and 67.7%, respectively. Univariate and multivariate analysis showed that s-CFL1-Ab and s-ACTB-Ab levels were not associated with sex, age, tumor depth, lymph node metastasis, or anti-p53-antibody levels. s-ACTB-Ab levels but not s-CFL1-Ab levels significantly correlated with squamous cell carcinoma antigen. Neither s-CFL1-Ab nor s-ACTB-Ab levels alone were obviously related to overall survival. However, patients with low s-CFL1-Ab levels and high s-ACTB-Ab levels exhibited significantly more unfavorable prognoses than those with high s-CFL1-Ab and low s-ACTB-Ab levels. CONCLUSIONS: Serum levels of anti-CFL1 and anti-ACTB antibodies were significantly higher in patients with esophageal carcinoma than in healthy donors. A combination of low anti-CFL1 and high anti-ACTB antibodies is a poor prognostic factor in esophageal carcinoma.

Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke.

BACKGROUND: Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. METHODS: Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. RESULTS: The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297-311 of DIDO1, 426-440 of FOXJ2, and 607-621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case-control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. CONCLUSIONS: Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

Association of serum levels of antibodies against ALDOA and FH4 with transient ischemic attack and cerebral infarction.

BACKGROUND: Ischemic stroke, including transient ischemic attack (TIA) and acute-phase cerebral infarction (aCI), is a serious health problem in the aging society. Thus, this study aimed to identify TIA and aCI biomarkers. METHODS: In 19 patients with TIA, candidate antigens recognized by serum IgG autoantibodies were screened using a human aortic endothelial cell cDNA library. Through amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), serum antibody levels against the candidate antigens were examined in healthy donor (HD), TIA, and aCI cohorts (n = 285, 92, and 529). The plasma antibody levels in the Japan Public Health Center-based Prospective Cohort Study (1991-1993) were also examined. RESULTS: The candidate antigens were aldolase A (ALDOA) and fumarate hydratase (FH). In AlphaLISA, patients with TIA or aCI had higher anti-ALDOA antibody (ALDOA-Ab) and anti-FH antibody (FH-Ab) levels than the HDs (P < 0.05). In a multivariate logistic regression analysis, the ALDOA-Ab (odds ratio [OR]: 2.46, P = 0.0050) and FH-Ab (OR: 2.49, P = 0.0037) levels were independent predictors of TIA. According to the case-control study, the ALDOA-Ab (OR: 2.50, P < 0.01) and FH-Ab (OR: 2.60, P < 0.01) levels were associated with aCI risk. In a correlation analysis, both ALDOA-Abs and FH-Abs were well associated with hypertension, coronary heart disease, and habitual smoking. These antibody levels also correlated well with maximum intima-media thickness, which reflects atherosclerotic stenosis. CONCLUSIONS: ALDOA-Abs and FH-Abs can be novel potential biomarkers for predicting atherosclerotic TIA and aCI.

Serum anti-LRPAP1 is a common biomarker for digestive organ cancers and atherosclerotic diseases.

Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti-human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S-transferase-fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis-related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well-known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking.

Investigation of novel biomarkers for predicting the clinical course in patients with ulcerative colitis.

BACKGROUND: The clinical course of ulcerative colitis (UC) is characterized by repeated episodes of relapse and remission. We hypothesized that biomarkers that help distinguish refractory UC patients who are in remission using strong anti-immunotherapy could contribute in preventing the overuse of corticosteroids for treatment. Here, we clarified novel autoantibodies for UC patients in remission as clinical indicators to distinguish between refractory and non-refractory UC. METHODS: Antigen proteins recognized by serum antibodies of patients with UC in remission were screened using the protein array method. To validate the results, AlphaLISA was used to analyze the serum antibody titers with candidate protein antigens. Serum samples from 101 healthy controls, 121 patients with UC, and 39 patients with Crohn's disease were analyzed. RESULTS: Of 66 candidate protein antigens screened by ProtoArray™, six were selected for this study. The serum titers of anti-poly ADP-ribose glycohydrolase (PARG), anti-transcription elongation factor A protein-like 1, and anti-proline-rich 13 (PRR13) antibodies were significantly higher in patients with UC than in healthy controls. Anti-PARG and anti-PRR13 antibody titers were significantly higher in patients with refractory UC than in patients with non-refractory UC. There were no significant differences in any antibody titer between the active and remission phases. CONCLUSIONS: The serum titers of anti-PARG, anti-transcription elongation factor A protein-like 1, and anti-PRR13 antibodies were elevated in patients with UC. Anti-PARG and anti-PRR13 antibody titers may be novel clinical indicators for detecting refractory UC in patients in remission.

Immune-complex level of cofilin-1 in sera is associated with cancer progression and poor prognosis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. To improve its outcome, reliable biomarkers are urgently needed. In this study, we aimed to elucidate the key molecules involved in PDAC progression using proteomics approaches. First, we undertook 2-D electrophoresis to identify the proteins overexpressed in PDAC tissues. Following the analysis of agarose gel spots, cofilin-1 was identified and verified as a candidate protein commonly upregulated in PDAC tissues. In immunohistochemistry, cofilin-1 was strongly expressed in the cytoplasm of PDAC cells. Samples were divided into two groups based on the level of cofilin-1 expression. The high expression group showed significantly higher incidence of hematogenous dissemination in relapsed patients than the low expression group (P = 0.0083). In in vitro experiments, knockdown of cofilin-1 significantly decreased chemotaxis in PDAC cell lines. After we confirmed that cofilin-1 was secreted from PDAC cells, we established a detection system for the immune-complex of cofilin-1 in sera. Using this system, we measured the IC levels of cofilin-1 in sera and observed that the IC levels of cofilin-1 in PDAC patients were higher than those in healthy volunteers and patients with pancreatitis (PDAC vs. healthy volunteers, P < 0.0001; PDAC vs. patients with pancreatitis, P < 0.026). Notably, the IC levels of cofilin-1 showed a stepwise increase during PDAC progression (P = 0.0034), and high IC levels of cofilin-1 indicated poor prognosis of patients after surgery (P = 0.039). These results suggest that the IC of cofilin-1 in sera is a potentially attractive serum biomarker for the prognosis of PDAC.

Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α-chain (C4BPA) by quantitative proteomic analysis using tandem mass tags.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC. METHODS: Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins. RESULTS: Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC. CONCLUSIONS: We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Prognostic and diagnostic significance of preoperative Jumonji domain­containing 6 antibodies in colorectal cancer.

Jumonji domain-containing 6 (JMJD6) protein has been reported to be upregulated in different cancer cells; however, to the best of our knowledge, no report has analyzed serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with cancer. Therefore, the present study evaluated the clinical significance of s-JMJD6-Abs in patients with colorectal cancer. Preoperative serum samples were analyzed from 167 patients with colorectal cancer who underwent radical surgery between April 2007 and May 2012. The pathological stages were as follows Stage I (n=47), stage II (n=56), stage III (n=49) and stage IV (n=15). In addition, 96 healthy participants were analyzed as controls. s-JMJD6-Abs were analyzed by amplified luminescent proximity homology assay-linked immunosorbent assay. The cutoff value of s-JMJD6-Abs for detecting colorectal cancer was calculated to be 5,720 using the receiver operating characteristic curve. The positive rate of s-JMJD6-Abs was 37% in patients with colorectal cancer (61 of 167), independent of carcinoembryonic antigen or carbohydrate antigen 19-9 and p53-Abs. Clinicopathological factors and prognosis were compared between the s-JMJD6-Abs-positive group and the s-JMJD6-Abs-negative group. The s-JMJD6-Ab-positive status was significantly associated with older age (P=0.03), but was not associated with other clinicopathological variables. Regarding recurrence-free survival, the s-JMJD6-positive status was a significant poor prognostic factor in both univariate (P=0.02) and multivariate (P<0.01) analyses. Similarly, regarding overall survival, the s-JMJD6-Abs-positive status was a significant poor prognostic factor in both univariate (P=0.03) and multivariate (P=0.01) analyses. In conclusion, preoperative s-JMJD6-Abs was positive in 37% of patients with colorectal cancer and may be considered an independent poor prognostic biomarker.

The combination of positive anti­WDR1 antibodies with negative anti­CFL1 antibodies in serum is a poor prognostic factor for patients with esophageal carcinoma.

WD repeat-containing protein 1 (WDR1) regulates the cofilin 1 (CFL1) activity, promotes cytoskeleton remodeling, and thus, facilitates cell migration and invasion. A previous study reported that autoantibodies against CFL1 and ß-actin were useful biomarkers for diagnosing and predicting the prognosis of patients with esophageal carcinoma. Therefore, the present study aimed to evaluate the serum levels of anti-WDR1 antibodies (s-WDR1-Abs) combined with serum levels of anti-CFL1 antibodies (s-CFL1-Abs) in patients with esophageal carcinoma. Serum samples obtained from 192 patients with esophageal carcinoma and other solid cancers. And s-WDR1-Ab and s-CFL1-Ab titers were analyzed using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Compared with those of healthy donors, the s-WDR1-Ab levels were significantly higher in the 192 patients with esophageal, whereas these were not significantly higher in the samples from patients with gastric, colorectal, lung, or breast cancer. In 91 patients treated with surgery, sex, tumor depth, lymph node metastasis, stage and C-reactive protein levels were significantly associated with overall survival, as determined using the log-rank test, whereas the squamous cell carcinoma antigen, p53 antibody and s-WDR1-Ab levels tended to be associated with a worse prognosis. Although no significant difference was observed in the survival between the positive and negative groups of s-WDR1-Abs or s-CFL1-Abs alone in the Kaplan-Meier test, the patients in the s-WDR1-Ab-positive and s-CFL1-Ab-negative groups exhibited a significantly poorer prognosis in the overall survival analysis. On the whole, the present study demonstrates that the combination of positive anti-WDR1 antibodies with negative anti-CFL1 antibodies in serum may be a poor prognostic factor for patients with esophageal carcinoma.

Serum anti­TSTD2 antibody as a biomarker for atherosclerosis­induced ischemic stroke and chronic kidney disease.

Autoantibodies can be used in the early diagnosis and treatment of atherosclerosis-related diseases. Using ProtoArray® screening of samples from patients with atherosclerosis, the present study identified thiosulfate sulfurtransferase-like domain-containing 2 (TSTD2) as a novel atherosclerosis antigen. The serum TSTD2 antibody levels were then quantified using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay. This demonstrated the levels of TSTD2 antibodies (TSTD2-Abs) to be significantly higher in patients with acute cerebral infarction or chronic kidney disease than in healthy donors. The TSTD2-Ab levels were also found to be higher in males, older adults, smokers, in those who consumed alcohol regularly, and in those with hypertension. Furthermore, Spearman's rank correlation analysis revealed TSTD2-Ab levels to be strongly associated with measures of atherosclerosis severity, including plaque scores, intima-media thickness of the carotid artery and the cardio-ankle vascular index. Thus, TSTD2-Abs may thus be a promising novel biomarker for atherosclerosis-related cerebral infarction and kidney disease.

Utility of atherosclerosis-associated serum antibodies against colony-stimulating factor 2 in predicting the onset of acute ischemic stroke and prognosis of colorectal cancer.

Introduction: Autoantibodies against inflammatory cytokines may be used for the prevention of atherosclerosis. Preclinical studies consider colony-stimulating factor 2 (CSF2) as an essential cytokine with a causal relationship to atherosclerosis and cancer. We examined the serum anti-CSF2 antibody levels in patients with atherosclerosis or solid cancer. Methods: We measured the serum anti-CSF2 antibody levels via amplified luminescent proximity homogeneous assay-linked immunosorbent assay based on the recognition of recombinant glutathione S-transferase-fused CSF2 protein or a CSF2-derived peptide as the antigen. Results: The serum anti-CSF2 antibody (s-CSF2-Ab) levels were significantly higher in patients with acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD) compared with healthy donors (HDs). In addition, the s-CSF2-Ab levels were associated with intima-media thickness and hypertension. The analyzes of samples obtained from a Japan Public Health Center-based prospective study suggested the utility of s-CSF2-Ab as a risk factor for AIS. Furthermore, the s-CSF2-Ab levels were higher in patients with esophageal, colorectal, gastric, and lung cancer than in HDs but not in those with mammary cancer. In addition, the s-CSF2-Ab levels were associated with unfavorable postoperative prognosis in colorectal cancer (CRC). In CRC, the s-CSF2-Ab levels were more closely associated with poor prognosis in patients with p53-Ab-negative CRC despite the lack of significant association of the anti-p53 antibody (p53-Ab) levels with the overall survival. Conclusion: S-CSF2-Ab was useful for the diagnosis of atherosclerosis-related AIS, AMI, DM, and CKD and could discriminate poor prognosis, especially in p53-Ab-negative CRC.

Serum anti-Ku86 is a potential biomarker for early detection of hepatitis C virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41±0.28 vs. 0.18±0.08Abs at 450nm, P<0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor <2cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC.

Increased serum levels of pigment epithelium-derived factor by excessive alcohol consumption-detection and identification by a three-step serum proteome analysis.

BACKGROUND: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip(®) system and the ClinProt™ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins. METHODS: Two serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA). RESULTS: Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 µg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 µg/ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects. CONCLUSION: Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.

Fucosylated C4b-binding protein α-chain, a novel serum biomarker that predicts lymph node metastasis in pancreatic ductal adenocarcinoma.

C4b-binding protein α-chain (C4BPA) was previously identified as a novel serum biomarker for pancreatic ductal adenocarcinoma (PDAC). To apply this biomarker for clinical diagnosis, a lectin ELISA was established to measure serum fucosylated (Fuc)-C4BPA levels in 45 patients with PDAC, 20 patients with chronic pancreatitis (CP) and 50 healthy volunteers (HVs) in one training and three validation sets. The lecithin ELISA developed in the current study exhibited satisfactory within-run (2.6-6.7%) and between-day (1.8-3.6%) coefficient of variations. Serum Fuc-C4BPA levels in patients with PDAC (0.54±0.27 AU/ml) was significantly higher than that in HVs (0.21±0.06 AU/ml; P<0.0001) and patients with CP (0.25±0.03 AU/ml; P<0.0001). Additionally, serum Fuc-C4BPA levels in preoperative patients were significantly decreased compared with postoperative patient sera (P<0.0003). The receiver operating characteristic (ROC) curve analyses revealed that the area under the curve (AUC) of Fuc-C4BPA (0.985) was higher than that of carbohydrate antigen (CA)19-9 (0.843), carcinoembryonic antigen (0.548) and total C4BPA (0.875) (P<0.001). To analyze the clinical significance of Fuc-C4BPA, the ability of Fuc-C4BPA to predict lymph node metastasis was compared with that of CA19-9. The AUC of serum Fuc-C4BPA levels (0.703) was significantly higher than that of serum CA19-9 levels (0.500) in patients with PDAC (P<0.001). The current study established a novel lectin ELISA for measuring serum Fuc-C4BPA levels. Thus, Fuc-C4BPA has potential clinical applications owing to its high diagnostic value in PDAC.

Identification of serum anti-striatin 4 antibodies as a common marker for esophageal cancer and other solid cancers.

Solid cancers have a poor prognosis, and their morbidity and mortality after surgery is high. Even after radical surgery for esophageal cancer, there have been cases of early postoperative death. The present study therefore aimed to explore new tumor markers that can predict the early postoperative prognosis. To identify antibody markers, serological antigens were identified using recombinant cDNA expression cloning (SEREX). The results identified striatin 4 (STRN4) as the antigen recognized by serum IgG antibodies in patients with esophageal cancer. After performing an amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), it was revealed that when compared with healthy donors, serum anti-STRN4 antibody (STRN4-Ab) levels were significantly higher not only in patients with esophageal cancer but also to lesser extent, in those with gastric cancer, colorectal cancer, lung cancer and breast cancer. Compared with STRN4-Ab-negative patients with esophageal cancer, STRN4-Ab-positive patients had a poorer postoperative prognosis at early stages, suggesting that STRN4-Abs may be useful for predicting poor early-stage prognoses of patients with esophageal cancer. The positive diagnosis rates of esophageal cancer using the STRN4-Ab marker and conventional markers, including squamous cell carcinoma antigen and p53 antibody alone, were 26.4, 35.2 and 19.1% respectively; a result that increased up to 59.1% by combining all three markers. Serum STRN4-Ab may serve as a novel marker of esophageal cancer.