Soluble B7-H3 in Colorectal Cancer.

We present the results of comparative ELISA of the concentration of soluble form of immunity checkpoint B7-H3 (sB7-H3) in the serum of patients with colorectal cancer (CRC) at different stages before treatment and healthy control donors. The analysis revealed a statistically significant difference between the median levels of sB7-H3 in the blood serum of CRC patients (19.66 ng/ml) and healthy donors (16.76 ng/ml) (p=0.0025). ROC analysis showed 62.9% sensitivity and 56.7% specificity for CRC patients (cut-off 17.62 ng/ml; p=0.0028). An association of sB7-H3 levels with tumor progression was revealed. We demonstrated that sB7-H3 levels were significantly lower in patients with regional metastases than in patients without metastases (p=0.039) and that sB7-H3 concentration tends to decrease at the late stages of the disease. Thus, high serum level of sB7-H3 in CRC patients can be a favorable prognostic factor in future.

Immunosuppression Factors PD-1, PD-L1, and IDO1 and Colorectal Cancer.

Due to the low efficiency of immunotherapy for colorectal cancer (CRC), it is extremely promising and relevant to study the mechanisms of immunosuppression. In this work, a comprehensive study of the expression of soluble and tissue forms of PD-1 and PD-L1 in blood serum and tumors of patients with CRC, as well as IDO1 in tumors was performed for the first time. The diagnostic and prognostic significance of the studied parameters was determined. A statistically significant decrease in the number of soluble forms of PD-1 and PD-L1 in the blood serum and the association of the number of PD-L1+ cells in the stroma of tumors with the CRC stage were established. The absence of correlations between soluble and tissue forms of the studied proteins was shown, indicating the presence of independent mechanisms of immunosuppression in CRC, which may explain the ineffectiveness of immunotherapy for this type of tumor.

Analysis of Anti-Glycan IgG and IgM Antibodies in Colorectal Cancer.

We propose an approach that allows simultaneous determination of the levels of M and G isotypes of antibodies to the panel of glycans using microarrays. The level of IgG antibodies to 3'-O-su-Lea glycan detects patients with colorectal cancer with a sensitivity of 69.77% and specificity of 62.75%. The percentage of correctly classified colorectal cancer patients with the use of a combination of two markers IgM antibodies to glycans 3'-sialyl-TF and 3'-O-su-Lea is as high as 74.23%. The levels of IgM antibodies to 3'-O-su-Lea glycan differ significantly in patients with and without regional metastases. The levels of some of antiglycan IgM or IgG antibodies differed significantly in patients with tumors of different location and differentiation.

Development of hydrogel biochip for in vitro allergy diagnostics.

A hydrogel biochip was developed for the simultaneous quantitative determination of sIgE for 21 allergens and total IgE in human serum. The biochips are manufactured by photoinduced copolymerization of different molecules (allergens and antibodies) with gel-forming monomers resulting in the formation of three-dimensional hydrogel elements (1nl gel drops). After incubation of the biochip with the serum, the results are visualized using fluorescently labeled anti-IgE antibodies. Using biochips, serum samples from allergic patients and healthy donors were analyzed and good correlation with the results obtained using commercial EIA test systems of generally recognized quality (Dr. Fooke Laboratorien GmbH, Germany) was observed.

[Immunoassay of nine serological tumor markers on hydrogel-based microchip].

A prototype of test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), prostate-specific antigen, total (PSAtot) and free (PSAfree) forms, neuron-specific enolase (NSE). The biochip-based assay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patient's blood serum: two-steps sandwich-immunoassay, was proposed. The main analytical characteristics of the method were obtained. The results permit to consider the prototype of the test-system as a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients group, application of modern statistical methods of data-processing in medical researchers, i.e. ROC-analysis and logistic regression, indicted that the simultaneous assay of nine tumor markers on biochips showed much more diagnostic significance (area under the ROC-curve (AUC) reached 0.84) than traditional assay of 2 tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended both for the estimation of people's health, e.g., for standard medical examination, and for tracking of tumoral process in postsurgical period or after specific tumor treatment.