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Background: Cytokines and chemokines are known to be involved in tumor growth and progression of disease. Sialyl LewisX (sLeX), a ligand for adhesion molecule E-selectin, is known to affect inflammatory processes and an elevated level is associated with tumor metastasis. Therefore, we assessed serum levels of sLeX and cytokines/chemokines in patients with non-invasive ductal carcinoma in situ (DCIS), early invasive breast cancer (EBC), or metastatic breast cancer (MBC). Patients and Methods: Sera from 250 patients (26 DCIS, 157 EBC, 67 MBC) and 43 healthy donors (HD) were assayed for sLeX using an immunoassay kit (CSLEX; Nittobo Medical Co. Ltd., Japan) and a panel of cytokines and chemokines using a multiplex assay kit. Differences in serum markers between patients and HD, and among patient groups were determined using the Kruskal-Wallis and Mann-Whitney tests. Spearman's correlation determined the non-parametric correlation between the serum levels of sLeX and the inflammatory mediators. The receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analyses were used to determine the sensitivity and specificity of a given cut-off value for a particular serum marker. Results: The median sLeX level tended to increase with the stage of disease: MBC > EBC > DCIS albeit without significant differences among the disease stages. Among MBC patients, patients with sLeX below 1.75 U/mL had significantly improved overall survival (OS, mean survival 11.1 vs. 33.7 months, P = 0.002) and progression-free survival (PFS, mean survival 9.7 vs. 20.9 months, p = 0.042). The Hazard Ratio of high sLeX for OS was 5.5 (95% CI 1.6 to 18.9, p = 0.007) and 2.3 for PFS (95% CI 1.0 to 5.2, P = 0.048). EBC and MBC patients have significantly higher serum levels of IL-1, IL-1RA, IL-6, IL-8, MCP-1, MCP-3, and MIP-1βthan those of hD. In addition, there were positive correlations between the serum levels of sLeX and cytokines IL-1β, IL-1RA, IL-2, IL-8, MIP-1β, and MCP-3. The AUC for sLeX was 0.598 (P = 0.016), and a cut-off of 3.13 pg/mL distinguished hormone receptor (HR)-positive from HR-negative patients ({chi} 2 = 4.0, P = 0.045). Likewise, the AUC for TNF-α was 0.620 (P = 0.003), and a cut-off 7.18 pg/mL distinguished HR-positive from HR-negative patients ({chi} 2 = 12.6, P < 0.001). Using a cut-off value established by ROC curves, few MBC patients (9 of 66, 13.6%) had a serum IL-2 level > 7.1 pg/mL compared to 57 of 185 (30.8%) non-MBC patients (χ 2 = 7.4, P = 0.007), suggesting that metastatic disease may be associated with immune suppression related to low serum IL-2. Conversely, 31 of 66 (47%) MBC patients had a serum MCP-1 level > 750 pg/mL vs. 37 of 185 non-MBC patients (20%) ({chi} 2 = 23.8, P < 0.0001), suggesting that a high level of MCP-1 may play an important role in metastasis. Conclusion: Serum levels of sLeX were able to distinguish HR-positive from HR-negative patients and predict overall survival in metastatic patients. Serum sLeX and some inflammatory mediators tended to increase with the severity of disease, and together may facilitate local invasion of tumor cells. Furthermore, serum levels of MCP-1 and IL-2 may have prognostic value in breast cancer patients.χ [ABSTRACT FROM AUTHOR]

Purpose: The carbohydrate sialyl Lewis X (sLe X ) mediates cell adhesion, is critical in the normal function of immune cells, and is frequently over-expressed on cancer cells. We assessed the association, differential levels, and prognostic value of sLe X and inflammatory cytokines/chemokines in breast cancer sera., Methods: We retrospectively measured sLe X and a panel of cytokines/chemokines in the sera of 26 non-invasive ductal carcinoma in situ (DCIS), 154 invasive non-metastatic breast cancer (non-MBC), 63 metastatic breast cancer (MBC) patients, and 43 healthy controls. Differences in sLe X and inflammatory cytokines among and between patient groups and healthy controls were assessed with nonparametric tests and we performed survival analysis for the prognostic potential of sLe X using a cut-off of 8 U/mL as previously defined., Results: Median serum sLe X was significantly higher than controls for invasive breast cancer patients (MBC and non-MBC) but not DCIS. In univariate analysis, we confirmed patients with serum sLe X  > 8 U/mL have a significantly shorter progression-free survival (PFS) (P = 0.0074) and overall survival (OS (P = 0.0003). Similarly, patients with high serum MCP-1 and IP-10 had shorter OS (P = 0.001 and P < 0.001, respectively) and PFS (P = 0.010 and P < 0.001, respectively). sLe X , MCP-1 and IP-10 remained significant in multivariate survival analysis., Conclusion: Elevated serum sLe X was associated with invasive cancer but not DCIS. High serum sLe X levels were associated with inflammatory mediators and may play a role in facilitating local invasion of breast tumor. Furthermore, serum MCP-1, IP-10 and sLe X may have prognostic value in breast cancer.

Purpose: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9 kDa peptide identified as a fragment of the fibrinogen alpha C chain (FIC5.9) was the most promising. To move FIC5.9 toward potential diagnostic use, we developed an enzyme immunoassay that enables measurement of serum FIC5.9 levels., Experimental Design: Two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotope-labeled dilution mass spectrometry (SID-MS) using the ClinProt™ system., Results: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=-0.011, r(2) =0.908) and the performance of the ELISA was satisfactory in terms of recovery (98.5-103.0%) and within-run (1.4-4.7%) and between-day (2.8-8.4%) reproducibility. The assay was capable of detecting changes in FIC5.9 during abstinence from drinking in patients with alcohol dependency (p<0.0001)., Conclusions and Clinical Relevance: The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum FIC5.9 levels in various pathological conditions, including alcoholism., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.

Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.

Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.