Performance evaluation and characterization of pre-analytical effects by sample types and storage conditions on tumor marker measurement by the point-of-care test platform IchromaTM II.

Tumor markers should be measured regularly and accurately to prevent, diagnose, and monitor cancers efficiently. We aimed to characterize the pre-analytical factors effecting on the analytical performance of point-of-care test (POCT) platform IchromaTM II (Boditech Med Inc., Gangwon-do, Korea) for alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) and evaluate their consequences in clinical practice. Based on comprehensive evaluation for the analytical performance of IchromaTM II including precision, linearity, and method comparison performed according to CLSI guidelines, pre-analytical factors of sample types and conditions were extensively analyzed. A total of five sample types [serum, plasma (PL) and whole blood (WB) from EDTA tube, PL and WB from sodium heparin tube] from 40 patients were used for comparing among specimen types. Additionally, stability was assessed up to 21 h at room temperature, refrigerated for 8 days, and frozen for 16 weeks by using 4 levels of pooled patient samples which were measured in triplicate. Precision, linearity and correlation with central laboratory analyzers observed in all three tumor markers were within acceptable criteria. However, variable degrees of percent deviations were observed according to sample type and storage conditions. Only EDTA PL samples presented clinically acceptable percentage biases for all three tumor markers when stored at room temperature or refrigerated condition. Positive bias of CEA and PSA in storage duration until 16 weeks were observed when stored in frozen condition. While IchromaTM II showed an adequate analytical performance as a POCT platform with simple operating procedures for the measurement of tumor markers, clinical laboratories should be aware of stability issues when different types of blood specimens are practically utilized.

Clinical implication by differential analytical performances of serum free light chain quantitation analysis using fully automated analyzers.

OBJECTIVES: Free light chain (FLC) is used for the diagnosis and prediction with regard to the progression risk of plasma cell disorders and Freelite reagent using the SPAplus analyzer (The Binding Site) has been one of the widely used option. However, N Latex FLC reagent with the Atellica CH 930 analyzer (Siemens Healthineers) has shown the advantages of automation and high throughput. We aimed to evaluated clinical implication by differential analytical performances of two assays. METHODS: A total of 322 serum samples were collected from 193 patients requested for FLC analysis including 131 multiple myeloma patients. The precision, linearity, dilution recovery of N Latex FLC assay was evaluated. We compared the two assays and analyzed the monomer-dimer pattern for discrepant results. RESULTS: The precision, linearity, and dilution recovery performance was appropriate for the routine use in clinical laboratories. Despite the good correlation within normal range, proportional bias up-to 170% was observed in samples with high concentrations especially for lambda. The higher value samples with N Latex FLC assay contained more monomer forms than controls. All opposite changes of FLC burden by the N Latex FLC assay proved to present concordant dynamic changes when assessed by serum protein electrophoresis. CONCLUSIONS: Clinical laboratories should be aware of the inter-assay variability of FLC quantitative measurements using different platforms, especially for high concentrations of both kappa and lambda measurements, possibly due to monomer/dimer ratio diversity. Clinical interpretations for multiple myeloma disease status might not be dramatically affected only when the same assay is utilized during follow-up periods.

Associations of LDL Cholesterol, Non-HDL Cholesterol, and Apolipoprotein B With Cardiovascular Disease Occurrence in Adults: Korean Genome and Epidemiology Study.

Background: Despite the superiority of non-HDL cholesterol (non-HDL-C) and apolipoprotein B (ApoB) as lipid markers for atherosclerotic cardiovascular disease (ASCVD), these are only suitable as secondary markers. We compared LDL cholesterol (LDL-C), non-HDL-C, and ApoB concentrations with respect to the occurrence of cardiovascular disease in adults enrolled in the Korean Genome and Epidemiology Study (KoGES). Methods: We used information on age; sex; medical history; family history of ASCVD; current lipid-lowering therapy; current smoking status; and creatinine, total cholesterol, HDL-C, LDL-C, triglyceride, and ApoB concentrations from 5,872 KoGES participants without ASCVD. New ASCVD development was monitored during the 8-year follow-up period. Adjusted hazard ratios (aHRs) for ASCVD of LDL-C, non-HDL-C, and ApoB concentrations were calculated based on the multivariate Cox regression analyses. The participants were also grouped as low and high according to the median values for each lipid marker, and calculated aHRs of each group combined by two lipid makers. Results: ApoB showed the highest aHR per 1-SD for ASCVD (1.26; 95% confidence interval [CI], 1.11-1.43), followed by non-HDL-C (1.25; 95% CI, 1.11-1.41) and LDL-C (1.20; 95% CI, 1.06-1.37). The group with low LDL-C and high ApoB concentrations had a significantly higher aHR for ASCVD (1.61; 95% CI, 1.05-2.48) compared to the reference group values (low LDL-C and low ApoB concentrations). The aHR for the group with high LDL-C and low ApoB concentrations was not significant (1.30; 95% CI, 0.79-2.16). Conclusions: ApoB, non-HDL-C, and LDL-C are independent risk factors for ASCVD. Increases in the aHR per 1-SD for ASCVD were more strongly affected by ApoB, followed by non-HDL-C and LDL-C. Participants with low LDL-C and high ApoB concentrations showed increased ASCVD risk. For individuals with ASCVD risk factors, even those presenting normal LDL-C concentrations, measuring ApoB concentrations can provide useful information for better evaluation of ASCVD risk.

Performance Evaluation of the DxC 700 AU Chemistry Analyzer in Hemoglobin A1c Measurement

Background: Accurate measurement of glycated hemoglobin (HbA1c) is crucial for a diabetes diagnosis and subsequent patient management. The detection method and presence of variant Hb can interfere with HbA1c measurements. We evaluated the HbA1c-measuring performance of the DxC 700 AU (Beckman Coulter, Brea, CA, USA) immunoassay-based device in comparison with another immunoassay device and the reference method. Methods: A total of 120 normal and 14 variant Hb samples were analyzed using the Cobas c 513 (Roche Diagnostics, Mannheim, Germany) and DxC 700 AU analyzers. Variant Hb samples were also analyzed using the reference method, along with 20 normal samples. The accuracy, precision, linearity, and carryover were determined. Results: DxC 700 AU results strongly correlated with those of Cobas c 513 and exhibited accuracy in comparison with the reference method. The within-run, between-run, between-day, and total imprecision (%CV) values for the low- and high-concentration control materials were below 2%. The results of DxC 700 AU were linear over a wide HbA1c range (3.39%-18.30%). Although DxC 700 AU performed well in the presence of variant Hb, the HbA1c concentration was underestimated in the presence of fetal Hb. The possibility of interference from a high HbH proportion could not be ruled out. Conclusions: The overall analytical performance of DxC 700 AU was acceptable. The device is accurate, precise, and linear over a wide HbA1c concentration range. Although DxC 700 AU results highly correlated with those of Cobas c 513, caution should be exercised in cases of high HbF and HbH concentrations.

The role of Jagged1 as a dynamic switch of cancer cell plasticity in PDAC assembloids.

Background: Pancreatic ductal adenocarcinoma (PDAC), which commonly relapses due to chemotherapy resistance, has a poor 5-year survival rate (< 10%). The ability of PDAC to dynamically switch between cancer-initiating cell (CIC) and non-CIC states, which is influenced by both internal and external events, has been suggested as a reason for the low drug efficacy. However, cancer cell plasticity using patient-derived PDAC organoids remains poorly understood. Methods: First, we successfully differentiated CICs, which were the main components of PDAC organoids, toward epithelial ductal carcinomas. We further established PDAC assembloids of organoid-derived differentiated ductal cancer cells with endothelial cells (ECs) and autologous immune cells. To investigate the mechanism for PDAC plasticity, we performed single-cell RNA sequencing analysis after culturing the assembloids for 7 days. Results: In the PDAC assembloids, the ECs and immune cells acted as tumor-supporting cells and induced plasticity in the differentiated ductal carcinomas. We also observed that the transcriptome dynamics during PDAC re-programming were related to the WNT/beta-catenin pathway and apoptotic process. Interestingly, we found that WNT5B in the ECs was highly expressed by trans interaction with a JAG1. Furthermore, JAG1 was highly expressed on PDAC during differentiation, and NOTCH1/NOTCH2 were expressed on the ECs at the same time. The WNT5B expression level correlated positively with those of JAG1, NOTCH1, and NOTCH2, and high JAG1 expression correlated with poor survival. Additionally, we experimentally demonstrated that neutralizing JAG1 inhibited cancer cell plasticity. Conclusions: Our results indicate that JAG1 on PDAC plays a critical role in cancer cell plasticity and maintenance of tumor heterogeneity.

BRAK and APRIL as novel biomarkers for ovarian tumors.

Aims: To evaluate BRAK and APRIL in serum samples from healthy patients and an ovarian tumor group and analyze their effective value as biomarkers. Materials & methods: BRAK and APRIL were measured in 197 serum samples including 34 healthy controls, 48 patients with benign ovarian cysts and 115 patients with ovarian cancer, and the best statistical cut-off values were calculated. Then, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value for selected cut-off points were assessed. Results: The healthy control group had statistically significant higher BRAK and lower APRIL than the ovarian tumor group. BRAK was excellent for differentiating healthy patients from patients with ovarian tumors, showing area under the receiver operating characteristic curve 0.983, 98.16% sensitivity and 100% specificity. When BRAK was combined with APRIL and CA-125, it also played a role in distinguishing benign cysts from malignancies with area under the curve 0.864, 81.74% sensitivity and 79.17% specificity. Conclusions: BRAK and APRIL are good candidates for ovarian tumor biomarkers.

Comparison of blood collection tubes for 29 biochemical analytes in pediatric patients with central venous catheters.

BACKGROUND: Pediatric cancer patients undergoing chemotherapy or radiation therapy generally require a central venous catheter (CVC). However, serum drawn from CVCs has several drawbacks for use in routine chemistry tests. Biochemical analytes were evaluated using heparin plasma instead of serum to maintain turnaround time and to prevent problems caused by micro-clot formation or delayed clotting time. METHODS: Venous blood samples from 52 pediatric oncology patients with chemoports or Hickman catheters were collected in serum separating tubes (SSTs) and lithium heparin tubes (LHTs). A total of 29 parameters were analyzed on a Cobas c702 (Roche Diagnostics, Mannheim, Germany). Passing-Bablok regression and Bland-Altman difference plots were used for statistical analyses. RESULTS: When the mean value of each analyte measured from LHT was compared with those from SST, percentage bias was within the desirable bias limit in most of the analytes. However, albumin, potassium, and inorganic phosphorus showed a negative constant bias of -3.0%, -5.3%, and -1.6%, respectively, and total protein showed a positive constant bias of + 3.8%. CONCLUSIONS: The use of LHTs for sample collection from pediatric patients with CVCs could be helpful for routine chemistry analyses. The results of potassium and total protein should be interpreted with consideration of the difference between serum and plasma samples.

Association of circulating metabolites with incident type 2 diabetes in an obese population from a national cohort.

AIMS: Obesity is the most common risk factor for type 2 diabetes. However, not all obese individuals develop diabetes. In the era of precision medicine, metabolomics may reveal the fundamental metabolic status of an individual. Our aim was to assess the association of metabolites with incident type 2 diabetes in obese individuals using Korean Genome and Epidemiology Cohort Study. METHODS: Using 12 years of metabolomic data from 2,580 individuals, we performed a metabolomic study to define metabolically healthy obesity in an obese population (n = 704) with incident type 2 diabetes. Cox proportional hazards regression model and survival analysis were performed adjusted for the traditional risk factors of type 2 diabetes. RESULTS: Our study revealed that spermine, acyl-alkyl phosphatidylcholines (C34:3, C36:3, C42:1), hydroxy sphingomyelin (C22:2, C14:1), and sphingomyelin (C16:0) were associated with incident type 2 diabetes in obese individuals after the adjustment for risk factors and correction of multiple comparisons by Bonferroni method. Five metabolites (except hydroxy sphingomyelin C14:1 and sphingomyelin C16:0) were also significantly associated with incident type 2 diabetes in lean individuals. CONCLUSIONS: This study highlights the need for defining metabolically healthy obesity based on serum metabolites and elucidates potential biomarkers for type 2 diabetes in an obese population.

Age-group-specific reference intervals for anti-Müllerian hormone and its diagnostic performance for polycystic ovary syndrome in a Korean population.

BACKGROUND: We established age-group-specific reference intervals for serum anti-Müllerian hormone (AMH) levels in a Korean population and investigated the effectiveness of AMH assay for polycystic ovary syndrome (PCOS) diagnosis. METHODS: We analyzed serum levels of AMH, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) from 1540 Korean women. Subjects were divided into three groups: healthy, benign gynecologic diseases, and PCOS. Age-group-specific reference intervals and AMH diagnostic performance were estimated. RESULTS: The PCOS group had a median AMH level of 7.0 µg/L, which was higher than for the healthy (1.8 µg/L) and the benign gynecologic diseases (2.7 µg/L) groups. The upper 97.5% reference limits for age groups 12-20 years, 21-34 years, and 35-46 years were 13.2 µg/L, 15.8 µg/L, and 6.6 µg/L, respectively. The area under the curve (AUC) values to estimate AMH ability to discriminate PCOS from healthy women for each age group were 0.741, 0.785, and 0.789, respectively. AUCs for LH/FSH were 0.719, 0.672, and 0.590. CONCLUSIONS: The better diagnostic ability of AMH over LH/FSH in women of late childbearing ages indicates that age and other clinical characteristics should be considered when interpreting these test results.

Cancer-initiating cells in human pancreatic cancer organoids are maintained by interactions with endothelial cells.

Pancreatic ductal adenocarcinoma (PDAC) shows poor prognosis and high malignancy due to the presence of cancer-initiating cells (CICs) and characteristics of the tumor microenvironment (TME). Organoids are useful for studying PDAC, and establishing organoids is dependent on stem cell growth factors, including Wnt signaling. Herein, using a conventional organoid culture system, we demonstrated that CD44(+)CD24(+) and CD44(+)CD24(+)EpCAM(+) CICs were enriched >65% in a PDAC patient-derived organoid. CICs expressing CD44 formed lumen structures by gathering into circles. Additionally, organoid-derived CD44(-) cancer cells were capable of organoid re-formation and could be re-programed as CD44-expressing CICs in the organoid culture system. To mimic a TME absent artificial stem cell growth factors, a PDAC organoid with vascular niche was established. CICs in the PDAC tumor organoid were maintained by paracrine effects and direct interactions with endothelial cells. Interestingly, CD44(+) cells in PDAC tumor tissue were detected primarily in the vascular niche. Inhibiting both Wnt and Notch signaling in endothelial cells suppressed organoid formation and the maintenance of CD24(+)CD44(+) CICs. Collectively, our results suggest that PDAC patient-derived organoids maintain CICs by interacting with endothelial cells via Wnt and Notch pathways.

Identification of Serum Biomarkers for Diagnosis of Endometriosis Using Multiplex Immunoassays.

Endometriosis is a common gynecologic disorder characterized by chronic pelvic pain, dysmenorrhea, and infertility. Although this condition places significant financial burden on the healthcare system and negatively affects patient's quality of life, the pathophysiology of the disease remains unclear, and noninvasive diagnostic methods are insufficient. The object of this study was to identify potential biomarkers for endometriosis from peripheral blood. We hypothesized that serum biomarkers modified in endometriosis patients would be detected by multiplex cytokine panel, and identification of a combination of these biomarkers would improve diagnostic power. A total of 141 women, aged 15-52 years with regular menstruation, participated in this study. Twenty-one serum cytokines were detected using the commercially available MILLIPLEX MAP Human Cytokine/Chemokine Kit Panel IV. Among these cytokines, breast- and kidney-expressed chemokine (BRAK)/chemokine (C-X-C motif) ligand 14 (CXCL14) was significantly decreased, and proliferation-inducing ligand (APRIL)/tumor necrosis factor ligand superfamily member 13 (TNFSF13) was significantly increased in endometriosis group. APRIL/TNFSF13 and BRAK/CXCL14 alone or in combination, however, failed to show adequate sensitivity or specificity for the diagnosis of endometriosis. Combination of APRIL/TNFSF13 and BRAK/CXCL14 with serum CA-125 levels yielded significantly higher sensitivity (71.2%) for detecting endometriosis without compromising specificity (80.8%) than CA-125 alone in a logistic regression model (P = 0.050). In conclusion, we identified a biomarker combination that detects endometriosis better than CA125 alone. Therefore, we conclude that multiplex cytokine panel is an efficient method for detecting endometriosis, and analysis of additional cytokine panels may lead to identification of a novel biomarker combination with superior diagnostic power.

A Disintegrin and Metalloproteinase 8 as a Potential Blood Biomarker for Early Diagnosis of Gastric Cancer.

PURPOSE: We aimed to evaluate the clinical significance of a disintegrin and metalloproteinase 8 (ADAM 8) as a potential blood biomarker for gastric cancer (GC). MATERIALS AND METHODS: Blood ADAM 8 was measured by ELISA. Cytokines/chemokines [interleukin-23 (IL-23), stromal cell-derived factor 1α/CXC chemokine ligand 12 (SDF-1α/CXCL12), interleukin-8 (IL-8), and soluble CD40 ligand (sCD40L)] were measured by chemiluminescent immunoassay. They were compared among five groups; normal/gastritis, high-risk, early GC (EGC), advanced GC (AGC) without distant metastasis, and AGC with distant metastasis by one-way analysis of variance in both training (n=80) and validation dataset (n=241). Clinicopathological features of GC and GC-associated cytokines were evaluated for their correlations with blood ADAM 8. To evaluate the diagnostic accuracy to predict GC, receiver operating characteristic (ROC) curve and logistic regression were used. RESULTS: Blood ADAM 8 significantly increased along GC carcinogenesis in both training (ANOVA, p<0.001) and validation dataset (p<0.001). It was significantly higher in EGC compared to high-risk (post-hoc Bonferroni, p=0.041) and normal (p<0.001). It was also higher in AGC compared with high-risk (p<0.001) and normal (p<0.001) groups. However, no significant difference was found between cancer groups. Blood ADAM 8 was correlated with N-stage (Spearman's correlation, γs=0.320, p=0.011), but not with T-stage or M-stage. Pearson's correlations showed blood ADAM 8 was closely correlated with pre-inflammatory cytokines, IL-23 (p=0.036) and SDF-1α/CXCL12 (p=0.037); however, it was not correlated with pro-angiogenic cytokine IL-8 (p=0.313), and sCD40L (p=0.702). ROC curve and logistic regression demonstrated that blood ADAM 8 showed higher diagnostic accuracy (sensitivity, 73.7%; specificity, 86.2%) than CEA (sensitivity, 23.1%; specificity, 91.4%). Combination of ADAM 8 and CEA further increased the diagnostic accuracy to predict GC (sensitivity, 81.8%; specificity, 84.0%). CONCLUSION: Blood ADAM 8 is a promising biomarker for early detection of GC.

High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression.

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high-mobility group box-1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC-associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34-positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1-treated or HMGB1-TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti-HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin-8 (IL-8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1-TF were significantly reversed by IL-8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL-8 mediation, and combined targeting of HMGB1 and IL-8 can control tumor angiogenesis in GC.

Identificaiton of Novel Immunogenic Human Papillomavirus Type 16 E7-Specific Epitopes Restricted to HLA-A*33;03 for Cervical Cancer Immunotherapy.

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a 5¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E749₋63 (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E749₋63 showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E749₋63, E750₋59 (AHYNIVTFCC), and E752₋61 (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E750₋59- and E752₋61-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E750₋59 and E752₋61 as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.

Functional Recovery after the Transplantation of Neurally Differentiated Mesenchymal Stem Cells Derived from Bone Marrow in a Rat Model of Spinal Cord Injury.

This study was designed to investigate functional recovery after the transplantation of mesenchymal stem cells (MSCs) or neurally differentiated MSCs (NMSCs) derived from bone marrow in a rat model of spinal cord injury (SCI). Sprague-Dawley rats were subjected to incomplete SCI using an NYU impactor to create a free drop contusion at the T9 level. The SCI rats were then classified into three groups; MSCs, NMSCs, and phosphate-buffered saline (PBS)-treated groups. The cells or PBS were administrated 1 week after SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scores were measured at 1-week intervals for 9 weeks. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were also recorded 8 weeks after transplantation. While transplantation of MSCs led to a clear tendency of motor recovery, NMSC-treated rats had significantly improved BBB scores and showed significantly shortened initial latency, N1 latency, and P1 latency of the SSEPs compared to PBS controls. In addition, 5-bromo-2-deoxyuridine (BrdU)-prelabeled MSCs costained for BrdU and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) were found rostrally and caudally 5 mm each from the epicenter of the necrotic cavity 4 weeks after transplantation. These results suggest that neurally differentiated cells might be an effective therapeutic source for functional recovery after SCI.

CD4+ T Cell Tolerance to Tissue-Restricted Self Antigens Is Mediated by Antigen-Specific Regulatory T Cells Rather Than Deletion.

Deletion of self-antigen-specific T cells during thymic development provides protection from autoimmunity. However, it is unclear how efficiently this occurs for tissue-restricted self antigens, or how immune tolerance is maintained for self-antigen-specific T cells that routinely escape deletion. Here we show that endogenous CD4+ T cells with specificity for a set of tissue-restricted self antigens were not deleted at all. For pancreatic self antigen, this resulted in an absence of steady-state tolerance, while for the lung and intestine, tolerance was maintained by the enhanced presence of thymically-derived antigen-specific Foxp3+ regulatory T (Treg) cells. Unlike deletional tolerance, Treg cell-mediated tolerance was broken by successive antigen challenges. These findings reveal that for some tissue-restricted self antigens, tolerance relies entirely on nondeletional mechanisms that are less durable than T cell deletion. This might explain why autoimmunity is often tissue-specific, and it offers a rationale for cancer vaccine strategies targeting tissue-restricted tumor antigens.

HbA1c for diagnosis and prognosis of gestational diabetes mellitus.

AIMS: HbA1c is a widely used marker in diagnosing type 2 diabetes mellitus (DM), but its clinical utility in diagnosing gestational diabetes mellitus (GDM) is not established. Here, we evaluated the clinical usefulness of HbA1c in diagnosing GDM and predicting the risk of future type 2 DM development among GDM patients. METHODS: This retrospective, cross-sectional study included 321 subjects who underwent 100-g oral glucose tolerance tests (OGTT) during pregnancy. HbA1c and other variables were analyzed to evaluate their diagnostic performance for GDM. To evaluate the clinical usefulness of HbA1c in predicting future type 2 DM development, we classified GDM subjects who had more than 3 months of follow-up data into two subgroups: those who developed postpartum type 2 DM (PDM) and those who did not. RESULTS: HbA1c was significantly higher in the GDM group than in the normal control group. With the 100-g OGTT as reference, HbA1c showed 91.3% sensitivity and 62% specificity at a cut-off value of 5.05% (32 mmol/mol) for GDM diagnosis. At a cut-off value of 5.25% (34 mmol/mol), sensitivity was 73.6% and specificity was 77.2%. HbA1c levels during pregnancy were higher in those with PDM than in those without PDM (5.91 [41 mmol/mol] vs. 5.44% [36 mmol/mol], p<0.001). The prognostic value of HbA1c for PDM was evaluated by ROC curve analysis, with sensitivity of 78.6% and specificity of 72.5% at a cut-off value of 5.55% (37 mmol/mol). CONCLUSIONS: HbA1c showed high sensitivity with relatively low specificity for diagnosis of GDM in pregnant women and was a potential predictor of PDM. HbA1c may be able to be used as a simple and less invasive alternative screening test for OGTT in GDM patients.

Combined targeting of high-mobility group box-1 and interleukin-8 to control micrometastasis potential in gastric cancer.

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial-to-mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT-promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high-mobility group box-1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin-8 (IL-8) at early-stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV-SPORT6-HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU-1 and KATOIII), N87-xenograft BALB/c nude mice and human paired serum-tissue GC samples. HMGB1-induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL-8 were evaluated in N87-xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1-induced IL-8 overexpression contributed the HMGB1-induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL-8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL-8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL-8 as a promising diagnostic and therapeutic target to control GC micrometastasis.

Serum ENA78/CXCL5, SDF-1/CXCL12, and their combinations as potential biomarkers for prediction of the presence and distant metastasis of primary gastric cancer.

BACKGROUND: Chemokines play important roles in cancer development and progression. Epithelial-derived neutrophil-activating peptide-78 (ENA78/CXCL5) and stromal cell-derived factor (SDF-1/CXCL12) supposedly contribute to gastric cancer (GC) development and progression. This study aims to evaluate serum levels of ENA78/CXCL5 and SDF-1/CXCL12 along the GC carcinogenesis, and analyze their clinical significance, and diagnostic potentials through human serum samples. METHODS: A total of 300 subjects were enrolled in this study. Serum levels of ENA78/CXCL5 and SDF-1/CXCL12, measured by chemiluminescent immunoassay, were compared among 4 disease groups; normal, high-risk (intestinal metaplasia and adenoma), early GC (EGC), and advanced GC (AGC) groups in both training (n=25 per group) and validation dataset (n=70, 30, 50, 50, respectively) by ANOVA test (post hoc Bonferroni). Correlations between serum ENA78/CXCL5 or SDF-1/CXCL12 levels and clinicopathological parameters of GC patients were evaluated (Spearman's correlation; γs). To validate the diagnostic accuracy, receiver operating characteristic (ROC) curve and logistic regression analysis was performed. RESULTS: Serum ENA78/CXCL5 and SDF-1/CXCL12 levels were significantly higher in AGC groups than EGC, high-risk and normal groups in both training and validation dataset (Bonferroni, from p<0.01 to p<0.001). Clinicopathologically, serum ENA78/CXCL5 was correlated with T-stage (γs=0.231, p=0.021) and distant metastasis (γs=0.357, p<0.001), while serum SDF-1/CXCL12 was correlated with lymph node (γs=0.220, p=0.029) and distant (γs=0.425, p<0.001) metastasis. ROC curve and logistic regression demonstrated that serum ENA78/CXCL5 and SDF-1/CXCL12 showed higher diagnostic accuracy compared with carcinoembryonic antigen (CEA) in predicting GC. Serum ENA78/CXCL5 could predict both the presence of GC and distant metastasis, while serum SDF-1/CXCL12 could mainly predict its distant metastasis. All combination of serum ENA78/CXCL5, SDF-1/CXCL12, and CEA achieved 92.8% specificity at 75.0% sensitivity to predict distant metastasis of GC. CONCLUSIONS: Combinations of initial serum ENA78/CXCL5, SDF-1/CXCL12, and CEA before any treatment for GC can produce valuable serum biomarker panels to predict the presence and distant metastasis of GC.