Background. The cytoskeletal protein β-tubulin class III (Tubb3) is associated with tumor resistance to taxanes and vinca alkaloids, as well as with the metastatic potential of neoplasm, however, data from immunohistochemical analysis of Tubb3 expression in non-small cell lung cancer (NSCLC) tissue are few and contradictory. Purpose. Characterization of the level and intensity of Tubb3 expression in NSCLC tissue and analysis of the identified parameters correlation with clinically significant characteristics of the disease. Methods. Quantitative assessment of the level and intensity of Tubb3 expression in 120 surgical samples of NSCLC was carried out by immunofluorescence method associated with flow cytometry. Primary rabbit monoclonal antibodies specific to Tubb3 and secondary anti-rabbit antibodies conjugated with fluorescent dye DyLight650 (ab98510, UK) were used. The expression of the marker was assessed by two parameters: the level of expression measured as the percentage of the cells expressing Tubb3 and the intensity of expression in conventional units (CU) represented as the ratio of the geometric mean fluorescence intensity in the experimental and control samples (cells incubated with secondary antibodies only). Results. 1. Tubb3 expression was detected in all NSCLC samples studied. The median level and intensity of Tubb3 expression was 30.5% and 2.0 CU with significant differences (up to 10 times) in the quantitative values of both parameters in different patients. 2. The distribution of the studied tumors in terms of the level and intensity of Tubb3 expression differs from normal (P<0.001), the associative relationship between the assessed parameters is very strong (Spearman's rank correlation coefficient was 0.91; P<0.0001). 3. Statistical analysis did not reveal correlations between the level of Tubb3 expression and the gender and smoking status of the patients, with the degree of tumor differentiation, as well as with the stage of NSCLC. 4. In the group of lung adenocarcinomas, the median level of Tubb3 expression is higher compared with squamous cell lung cancer in male and female patients (P=0.01). Conclusion. High heterogeneity of Tubb3 expression level in NSCLC tissue in the patients and differences in the parameters between the tumors of various histotypes indicate the importance of further correlation analysis of Tubb3 expression level with the patients' life span in order to identify the prognostic value of the marker.

Quantitative assessment of estrogen receptors alpha (ERα) expression was carried out in 115 samples of non-small cell lung cancer (NSCLC) by an immuno uorescent assay and ow cytometry. It has been shown that high level of ERα ≥20% predicts higher aggressiveness of NSCLC than at low level <20%: median survival at 78 mos of follow-up is increased by 1.5 times; the risk of death is reduced by almost 2.0 times (p = 0.04). Time to death was increased by an average of 18 mos in about 20% of patients with low ERα expression. The results validate informative value of immuno uorescence analysis and owcytometry for quantifying the ERα expression and substantiate the prospects of antiestrogen therapy as a new option for NSCLC treatment, in particular, by analogy with breast cancer - in a long-term adjuvant therapy in ERα+ NSCLC patients.

Background. The search for effective combinations of immune checkpoint inhibitors with common cytostatics, targeted cancer drugs and other treatments is a modern trend to improve the effectiveness of immunotherapy.Purpose. Development of a panel of lung cancer cell cultures and cells of normal lung tissue with a characterized molecular phenotype by expression of one of the targets of immunotherapy — programmed cell death ligand 1 (PD-L1).Methods. PD-L1 expression was quantitatively analyzed by immunofluorescence method associated with flow cytometry.Results. A panel of lung cancer cell cultures of different histotypes and cells of normal lung tissue with characterized molecular phenotype was formed according to the expression of the immunotherapy target PD-L1. In terms of PD-L1 expression intensity, cell cultures can be arranged in a series: Calu-1 HFL-1 Calu-6 Wi-26 A-549 H-596 H-211, with a 7-fold difference in the index between Calu-1 and H-211.Conclusion. The panel of cultures of cancer and normal lung cells is recommended for the search and development of effective modifiers of the immunotherapy target PD-L1.

The expression of PD-L1 protein was quanti ed in 56 samples of urothelial bladder cancer (BC). PD-L1 expression was detected in all BC samples, while in non-muscle-invasive bladder cancer (NMIBC) the median value is 1,5 times higher than in muscle-invasive bladder cancer (MIBC) (p = 0,001). High PD-L1 expression level (≥30%) was observed in 20% of MIBC samples and among NMIBC tumors - in 68% of cases, that is 3,4 times more often (p = 0,0004). The last fact justi es the prospect of starting therapy with immune checkpoint inhibitors already at the onset of the disease - at NMIBC.

Introduction. Despite advances in the treatment of melanoma, the results of therapy cannot be considered satisfactory, and the search for new drugs and effective combinations of medicine continues. The drugs are being developed aimed at reducing the metastatic tumor potential – migrastatics. The targets of the drugs can be cytoskeletal proteins of tumor cells – cytokeratin (CK) intermediate filaments and microtubule protein beta-III tubulin (TUBB3). Aim. To estimate of the CK and TUBB3 expression in melanoma cell lines to form an informative in vitro cell model for screening and studying migrastatics. Materials and methods. The molecular phenotype of 21 human melanoma cell lines from the collection of N. N. Blokhin National Medical Research Center of Oncology, and 18 of which were isolated from tumor metastases in the lymph nodes, soft tissues or subcutaneously. The level of TUBB3 expression and de novo expression of CKs in vimentin-expressing cells (CK + Vim) were assessed by an immunofluorescent method and flow cytometry. Results. Beta-III tubulin expression was detected in all cultures studied, de novo expression of CKs was found in 20 / 21 lines. The exception was primary uveal melanoma 92-1, that did not express CK + Vim. Both parameters significantly differed between the cells of the studied panel: CK + Vim co-expression – from 0 to 91 %, TUBB3 – from 18 to 86 %. No correlation was found between the expression level of TUBB3 and CK + Vim (Pearson’s correlation coefficient r = 0.11; p = 0.65). Three groups of the cell lines with different ratio of TUBB3 expression and CK + Vim co-expression were identified: 1) similar level of expression of both markers; 2) the level of co-expression of CK + Vim more or less high than the index for TUBB3; 3) the level of TUBB3 expression more or less high than the index for CK + Vim co-expression. Conclusion. A panel of 21 human melanoma cell lines was formed with quantitatively estimated expression of cytoske-letal proteins responsible for the migration activity of tumor cells – CKs and TUBB3. Groups of the lines with different expression ratio of the markers can be used for screening and preclinical evaluation potential migrastatics that reduce the metastatic potential of melanoma and may reduce resistance to taxanes.

Immunofluorescent method by flow cytometry was used to quantify the expression of the tumor-associated protein βIII-tubulin (TUBB3) in the tissue of urothelial bladder cancer and visually normal mucosa (56 samples in total). The expression of the marker was detected in 100% of cases, and heterogeneity of the TUBB3 expression level both in tumor tissue and in "normal" mucosa was revealed. The level of TUBB3 in the "normal" mucosa did not depend on the distance from the tumor (1 cm or more than 3 cm) and, on average, it was lower than in the tumor tissue (21.8 ± 10.8% and 24.9 ± 13.2% vs 35.2 ± 12.4%; p = 0.04 and 0.005, respectively). An increase of the TUBB3 expression in the tumor and in the "normal" mucosa was revealed in muscle invasive bladder cancer compared to non-muscle invasive bladder cancer. Therefore, in urothelial bladder cancer, the tumor-associated protein TUBB3 is a molecular marker of bladder mucosa involvement in the malignancy process and predicts the risk of tumor muscle invasion, which may influence indications for early cystectomy.

Expression of tumor-associated protein beta-III tubulin (TUBB3) was evaluated quantitatively by immunofluorescence analysis using flow cytometry in lung tissue of intact animals and on day 11 after the Lewis lung carcinoma transplantation as well in lung metastasis tissue and in visually “normal” surrounding tissue. It was shown that the tumor is characterized by a high TUBB3 expression, while also the expression of the marker was detected in both variants of visually normal lung tissue: it was lower than in tumor tissue but significantly higher than in lung tissue of intact animals. The detection of TUBB3 outside the tumor indicates that the tissue appeared to be normal has already involved in malignancy and this supports the hypothesis that tumor-associated protein TUBB3 can be used as a molecular marker of local tumor spread.

The expression level of estrogen receptors (ERα and ERβ) in surgical tumor biopsy specimens from 167 patients with non-small cell lung cancer (NSCLC) was quantified by flow cytometry-based immunofluorescence analysis. ERα and ERβ expression was revealed in all the tumor samples investigated. The level of ERα expression in the tumors varied from 10 to 58% and ERβ — 12% to 80%, indicating significant heterogeneity of estrogen receptor expression in tumors of different patients. The mean ERβ expression level was approximately 2-times higher compared to ERα (42.1±15.3% vs 21.4±11.3%; p<0.001). It allows to consider ER>β as a major estrogen target in NSCLC tissue. The level of ERα and ERβ expression in NSCLC tissue is independent of gender, smoking status, and the histological type of the tumor. Increased ERβ expression was detected only in male patients with lung adenocarcinoma in comparison with squamous cell carcinoma (p=0.02). The assessment of the correlation between ERα and ERβ in both the whole cohort of patients and subgroups with clinically relevant disease parameters revealed that the level of one marker does not predict the other one’s expression. The coefficient of determination, which characterizes how differences in one variable can be explained by the difference in a second variable, was less than 25% in any comparison group. The authors consider that the high level of ERβ expression and ERα coexpression in NSCLC tissue substantiates the clinical perceptiveness of a new treatment option for the disease, namely, adjuvant hormone (antiestrogen) therapy.

The results of epithelial-mesenchymal transition analysis in serous ovarian cancer by the developed method of double immunofluorescent staining and flow cytometry while surgical tumor specimens and paraffin embedded tissue blocks are compared. When estimating de novo expression of the mesenchymal protein vimentin in epithelial tumor cells expressing cytokeratins, the results turn out to be close not only in quantitative parameters but also visually in the nature of cell distribution on the dotted plots. The results obtained indicate that the introduction of the developed new technology for the molecular diagnostics of the epithelial-mesenchymal transition of human tumors into routine laboratory practice is promising.

Objectives. Quantitative assessment of the expression and co-expression levels of estrogen receptors of different types (ERα and ERβ) in malignant tissues of large cohort of patients with non-small cell lung cancer (NSCLC). Materials and methods . Measurement of ERα and ERβ expression levels was carried out on 167 malignant tissue samples by immunofluorescence flow cytometric method. Level of the ERα and ERβ expression was calculated as a ratio of specifically fluorescent cells (%) compared to the control (incubated with the secondary antibody only). Results. The expression of ERα and ERβ was detected in all the NSCLC samples investigated. High variability in the level of the marker expression was revealed and it was shown 1,5–2,0-fold higher ERβ expression in the most tumors than that for ERα. The Spearman rank correlation coefficient of 0,364 (p <0,001) and the Pearson correlation coefficient of 0,401 (p <0,001) indicate statistically significant weak positive correlation between the levels of expression of ERβ and ERα. Conclusion . The level of expression of ERβ as a main subtype of ER in NSCLC tissue could prove useful for selecting patients for antiestrogen therapy

Abstract—A new method of double immunofluorescence analysis by flow cytometry is developed to evaluate the quantitative expression of the mesenchymal marker vimentin in epithelium cells of a solid tumor, which in turn is a mixture of different histogenesis cells. The method has a number of evident advantages compared to the clinically used immunohistochemistry technique for marker investigation: the intralaboratory precision of the results, gentle preanalytical preparation, lack of subjectivity in the interpretation of the results, and integral evaluation of the markers expression in a large number of cells from a large volume of the tumor tissue sample (≥5000 cells from a sample of ≥2 cm in diameter).

Introduction . Beta-III tubulin (TUBB3), the structural protein of microtubules, is not expressed in normal epithelium, but is frequently present in different epithelial tumors. We suggested that the screening for TUBB3 in morphologically normal tissue outside the tumor may be used for the molecular diagnostics of the local spread of cancer, which is particularly significant in case of the tumors with a high rate of local recurrence, like esophageal cancer. Objective . The quantitative assessment of TUBB3 expression in tumor and morphologically normal esophageal tissue adjacent and remote from the tumor. Materials and methods . The expression of TUBB3 in surgical biopsy specimens of tumor and morphologically normal tissue derived from 40patients after radical surgery for esophageal cancer was measured by immunocytometry. Results . TUBB3 expression was detected in 35 out of 40 studied samples of esophageal cancer, as well as in 10 out of 13 samples of morphologically normal esophagus tissue adjacent to the tumor, and in 25 out of 40samples of esophagus tissue most distant from the tumor near the resection edge. It was shown that the expression of TUBB3 in tissue specimens increases as follows: “normal tissue → adjacent normal tissue → tumor”, which basically means the existence of a positive gradient of TUBB3 expression level and intensity towards the tumor. Conclusion . The expression of TUBB3, a protein associated with tumor growth, outside the primary tumor may indicate that the tissue appearing to be normal is already affected by the malignancy. It can be used as an additional factor in tumor staging and post-operative patient management.

The differences in expression of ERCC1 were estimated between tumor specimens embedded into paraffin blocks and surgical biopsy specimens of non-small cell lung cancer as well as breast and ovarian cancers. Concordance or differences not higher than 20% were observed in 73% of the cases. The number of the cases with more significant differences in ERCC1 expression was less than 17%. The results show that ERCC1 detection in surgical biopsy specimens by flow cytometry is the more preferable method due to reduced preanalytical phase of the analysis.

Background. Class III beta-tubulin (TUBB3) is one of the eight beta tubulin isotypes identified in human. It is constitutively expressed in brain and testis but not in lung tissue. TUBB3 is also known to appear in solid tumors, in particular in non-small cell lung cancer (NSCLC), and it is often associated with poor prognosis and resistance to taxanes and Vinka alkaloids. Objective: We have suggested that TUBB3 expression may cover not only the primary tumor but also a wide adjacent area of morphologically normal lung tissue. To check the hypothesis we have measured TUBB3 expression both in the non-small cell lung carcinoma (NSCLC) and remote lung tissue derived from the same lung. 60surgical biopsy specimens of NSCLC and morphologically normal lung tissue of 30patients were investigated. Materials and methods. Biopsy specimens were converted to suspension, fixed in 4 % formaldehyde and analyzed by flow cytometry using monoclonal antibodies to TUBB3. The method was developed in the laboratory of medicinal chemistry research Institute of experimental diagnostics and therapy of tumors (N.N. Blokhin Russian Cancer Research Center) and patented. Results. Fraction of TUBB3-positive cells in the group of adjacent lung tissue specimens was lower compared to NSCLC group but still positive in the majority of adjacent lung tissue specimens (25 of 30) and achieved up to 39 % of cells. Conclusion. We suggest that TUBB3 expression in adjacent morphologically normal lung tissue indicates presence of transformed and potentially malignant cells far from the primary site.

Introduction. The disruption of BRCA1 function leads to the development of several cancers including breast cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer. Recently it was demonstrated that BRCA1 protein as well as another DNA repair protein, ERCC1, could be prognostic markers of the course of the disease and predictive markers of platinum-based chemotherapy efficiency. Nevertheless, the obtained data are controversial. Aim. In current study the comparison of BRCA1 expression in breast cancer with favorable and unfavorable prognosis based on estrogen receptor status (ER+ and ER - ) was carried out to evaluate its potential prognostic significance for subsets of breast cancer with different platinum chemotherapy sensitivity. Materials and methods. The estimation of BRCA1 expression was made in 50 samples of breast cancer tissue (40 ER+ and 10 ER - ) using the quantitative immunofluorescent method coupled with flow cytometry. Tumor cell suspensions were incubated with primary anti-BRCA1 antibodies (Abs) (SD118, Calbiochem) and secondary fluorescent Abs (DyLight650, ab98729). Cell fluorescence was measured at the Flow Cytometer FACSCanto II (Becton Dickinson, USA) using the program FACSDiva 6.0. The level of BRCA1 expression was analyzed using the program FlowJo 10.0 with statistical approach of Kolmogorov-Smirnov. The level of BRCA1 expression was determined as the ratio of specifically fluorescent cells to the total number of cells. Results. 1. The expression of BRCA1 was revealed in all breast cancer samples with mean expression level 36.8 ± 7.6 % and median 37 %. 2. Using the method of Student t-test statistically significant difference in mean value of BRCA1 expression level (p = 0.02) was revealed between ER+ and ER - breast cancer groups (38.0 ± 7.5 % and 32.0 ± 6.3 %, respectively) 3. Comparison of BRCA1 expression level in ER+ and ER - breast cancer groups using Mann - Whitney test also demonstrated statistically significant difference (p = 0.016). 4. These differences in mean value of BRCA1 expression level were especially prominent (p = 0.007) upon the comparison of ER+ and ER- breast cancer groups with BRCA1 expression levels below median (32.4 ± 5.0 % and 26.8 ± 2.9 %, respectively). 5. Comparison of BRCA1 expression levels in ER- breast tumors and the median for all studied breast cancer samples (37 %) demonstrated the correlation of decreased level of BRCA1 expression and negative ER status of the tumor. BRCA1 expression levels below the median were shown for 80 % of breast cancer patients with negative ER status in tumor, and over the median - in 2 0 % of cases, respectively. Conclusion. The results of current studies demonstrated that BRCA1 protein is expressed at different levels in the tumor tissue of all breast cancer patients. Lower level of BRCA1 expression in ER-negative as compared to ER-positive tumors, and established unfavorable prognostic value of ER-negative breast cancer status point to poor prognostic value of lower level of BRCA1 expression.