Along with advances in precision oncology, checkpoint inhibitors and targeted therapies have substantially improved outcomes for cancer patients. However, many patients still demonstrate a limited response to these therapies due to many biological factors, including genetic heterogeneity, unique molecular profiles, and the complex features of the tumor microenvironment (TME). Therefore, the selection of personalized effective treatment requires a comprehensive source of therapy response biomarkers, enabling precision medicine strategies for therapy selection. Here, we present a first-in-class automated biomarker analysis database, Astraea, that comprehensively describes genomic, transcriptomic, and TME biomarkers across a wide array of cancers. Automated daily literature reviews of the therapeutic efficacy of biomarkers provided the foundation of Astraea. To date, the database contains a total of 4,116 published biomarkers associated with genomic events, the TME, and targeted proteomic, transcriptomic, and gene signatures. To ensure accuracy of the final inclusion of biomarkers in the database, a multi-step quality control process was implemented that includes an automatic validation step and manual review. After selection, each biomarker is organized into a unique profile in the database which includes assay specifics, the biomarker-associated cancer type, therapy, primary study design, and statistical analysis. Data available from The Cancer Genome Atlas (TCGA) was then used to aggregate interrelated biomarkers into 25 biologically meaningful clusters, with the most prominent clusters identified as components of the TME (i.e., cytotoxic T cells, B cells, fibroblasts) and proliferation rate signatures. The aggregation enabled an easier interpretation and understanding of potentially actionable molecular findings as well as insight into unique neoplastic drivers. To apply Astraea in a clinical setting, we then developed a platform to match therapies to patients based on 1) identified biomarkers prioritized according to level of evidence, including both number of associated publications, statistical strength of individual studies, and cohort size and 2) therapies scored according to supporting biomarkers and associated relevance (resistance/response). By providing comprehensive, up-to-date biomarker identification and matching through utilization of a large automated multi-platform database, this technique aids in the identification and application of biomarkers unique to each patient. Taken together, our results show that Astraea, accompanied by a multi-step personalized cancer therapy-matching platform, could improve precision medicine strategies and help optimize therapeutic decisions. Citation Format: Azamat Gafurov, Ivan Mamichev, Elena V. Vasileva, Georgy D. Sagaradze, Maria S. Shitova, Grigorii Nos, Nikita Kotlov, Jessica H. Brown, Alexander Bagaev, Nathan Fowler. Astraea: A first-in-class biomarker database integrating genomic, transcriptomic, and tumor microenvironment properties for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 205.

The present review contains data on microtubular protein ßIII-tubulin (TUBB3): its structure, functions, role in tumor progression, expression in normal cells and in neural and epithelial tumors of different origins. Basic working principles of microtubular system and links between TUBB3 and related β-tubulins are also briefly reviewed. We analyzed the clinical potential of TUBB3 as prognostic marker of tumor aggressiveness and drug-resistance and suggested that locally disseminated tumor cells might be found by comparison of TUBB3 expression in normal and tumor tissue of each patient. Finally, we conclude that screening for TUBB3 and other tumor markers in morphologically normal tissue adjacent to the tumor is essential for accurate diagnostics.

Platinum-based drugs are widely used in clinical practice. Their efficacy varies widely among patients, which may be due to a disruption of the expression of ERCC1 - excision repair protein. In the prospective study a strictly quantitative analysis of the ERCC1 protein expression was carried out in surgical biopsy specimens of serous ovarian cancer by flow cytometry. ERCC1 expression was revealed in 100% of cases, but the expression level of the marker (the percentage of cells expressing ERCC1) varied significantly between tumors: the minimum was 40%, the maximum - 77%, the median - 64.5%; the average - 64.3+9.0%. The analysis of the Kaplan-Mayer curves showed an inverse relationship between the expression of ERCC1 and the duration of the relapse-free period during a 40-month follow-up after the first-line platinum-taxane chemotherapy (p

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.

Tamoxifen is a first targeted drug that continues to be the gold standard in treatment of estrogen receptor positive breast cancer for almost 50 years. The current review is an update of the paper published in 2012. We provide the new data on the tamoxifen targets that are the key points of signaling cascades activating cellular proliferation, which determines aggressiveness of disease and chemotherapy resistance or sensitivity. Some inspiring clinical cases dealing with tamoxifen efficiency in treatment of different tumors are discussed. Additionally, the review includes data on antiviral, antibacterial, antifungal and antiparasitic activity of tamoxifen

The proportion of diploid cells in the G0/G1 cell cycle phases was estimated by flow cytometry in 60 samples of stage III serous ovarian cancer tissue. The tumor tissue shows considerable heterogeneity with regard to the content of this tissue fraction, which ranged from 27 to 95% with a median of 73%. Statistically significant differences in the size of this fraction were identified by comparing tumor subgroups sensitive and resistant to first-line platinum-taxane chemotherapy. Predictive significance of the G0/G1 fraction was concluded and quantitative evaluation of this fraction is recommended for clinical use.

Background. Cell cultures used as a models in studies of epithelial tumors, are obtained not only from solid tumors, but also from extracellular fluids. It is known that dissemination of ovarian cancer in the peritoneum and further growth of tumor cells in ascetic liquid is accompanied with the activation of epithelial-mesenchymal transition, and, therefore, cell cultures derived from extracellular fluids can have a distinct molecular phenotype from primary tumors. Objective: evaluation the “persistence” of epithelial phenotype in breast and ovarian cancer cell cultures. Materials and methods. The cells obtained from pleural fluid (MCF-7, T-47D), colostrum (HBL-100), solid tumors (BT-474, HCC1937) of patients with breast cancer and ascitic fluid (SCOV- 3) of patients with ovarian cancer. The expression of cytokeratins and vimentin was evaluated using a quantitative immunofluorescence method associated with flow cytometry. Results. Vimentin expression in cells derived from extracellular fluids was not changed (line HBL-100), slightly decreased (SCOV-3 cells), or even was lost (MCF-7 and T-47D cells). HCC1937 cells obtained from solid tumor with expected low expression of vimentin acquired a molecular phenotype with a high expression of this mesenchymal marker. In breast cancer cells BT-474 derived from solid tumor a “persistence” of epithelial phenotype was discovered. Conclusion. Quantitative assessment of the de novo expression of mesenchymal protein vimentin showed that the tumor phenotype within the organizm is not always realized in cells adapted to growth in culture, and is not always «strictly» epithelial, and this evidence must be considered with different kinds of molecular studies of epithelial cells in vitro.

Drug-sensitive MCF7 cells and drug-resistant MCF7/ADR cells are shown to differ with regard to the vimentin and P-gp expression levels, which is indicative of the coexistence of two drug resistance mechanisms in MCF7/ADR cells. The mesenchymal phenotype of epithelial cells makes a much greater contribution to the resistance mechanisms than the ABC transporter’s overexpression, since the difference in the vimentin expression levels in drug-sensitive and drug-resistant cultures is significantly higher than the difference in the P-gp expression.

ABSTRACT Introduction Predictive markers of platinum-based chemotherapy efficacy in patients with ovarian cancer have been widely discussed in recent years. Oestrogen receptors (ER) are involved in regulation of proliferative factors responsible for tumour growth, so they could predict the effectiveness of anticancer chemotherapy. The aim of the study was to determine the predictive value of ER-beta expression in a cohort of ovarian cancer patients treated with platinum and taxanes. The level of ER-beta expression was correlated with progression-free survival and also with a number of disease relapses during 40 months of monitoring. Material and methods The quantitative immunofluorescence flow cytometry analysis was performed to detect ER-beta in 34 serous ovarian cancer surgical specimens. All the patients were treated with the first line platinum and taxane-based regimen. The primary anti-ER-beta (ab14C8) and the secondary antibodies (DyLight650, ab98729) were used for the analysis. The level of ER-beta expression was calculated by Kolmogorov-Smirnov statistical test as the ratio (%) of specifically fluorescent cells to the number of cells incubated only with secondary antibodies. Association of ER-beta expression levels with progression-free survival was analysed using the Kaplan-Meier method and log-rank tests. Results and discussions ER-beta were revealed in all tumour specimens tested and the median value of the expression level was 41,5%. Patients were dichotomized in groups with low and high level of ER-beta expression, below- and above-median value respectively. Significant differences were shown between these groups. First, median of progression-free survival was larger in the high-level expression group as compared to the low-level expression group – 25,5 vs 8 months. Second, the disease rate during 40 months of monitoring was lover in the high-level expression group as compared the low-level expression group – 6 vs 15 respectively. Conclusion A quantitative index of the ER-beta level expression in tumour tissue predicts the efficacy of the first line platinum and taxane-based chemotherapy in ovarian cancer patients. Progression-free survival was about 3.0 times longer and the number of the disease relapses during 40 months of monitoring was 1.5 times less in patients with high level of ER-beta expression. The study was supported in part by RSF 17-75-10212.

Introduction Epithelial-mesenchymal transition (EMT) plays key roles in all stages of cancer progression from primary tumour growth, invasion, and metastasis to resistance to chemotherapy. Earlier we have shown that EMT phenotype in ovarian cancer tumours associated with the increase in the metastatic potential and resistance to anticancer therapy; EMT markers were more often discovered in ascites as compared to solid cancers. The main goal of the present work was the analysis of EMT phenotype during cancer cell adaptation to the growth in vitro. Material and methods Cell lines were purchased from ATCC and were cultured in vitro according to the manufacturer’s recommendations. Level, intensity and integral index (product of level and intensity) of de novo expression vimentin (EMT marker) was estimated in cell cultures originated from solid tumours and extracellular fluids by immunofluorescence assay associated with flow cytometry. Primary anti-vimentin antibodies (CRM312A, Biocare) and secondary DL650 conjugated antibodies (ab98510) were used. Results and discussions Significant differences were revealed between BT-474 and HCC1937 cell culture lines originated from solid breast cancer. In BT-474 breast cancer cells expression level of vimentin was more than 2 times lower (29% vs 71%), and well as intensity – 3 times (2.1 vs 6.4), and the index – 6 times (0.7 vs 4.8) lower compared to HCC1937 breast cancer cells. At the same time, vimentin expression was low in MCF-7 and T-47D luminal type breast cancer cells originated from pleural fluid: level – 8% and 20%, intensity – 1.1 and 1.3, and index – 0.2 and 0.4. On the contrary, in ovarian cancer SCOV-3 and breast cancer HBL-100 cells, originated from extracellular tumours (ascites and colostrum), expression level of vimentin was high: level – 51% and 75%, intensity – 3.6 and 6.8, index – 1.9 and 5.1. Conclusion Vimentin, one of key EMT markers, in original epithelial tumour does not predict molecular epithelial phenotype of the cells during their adaptation to the growth in vitro. It may be lost, increased or persisted at the same level as in vivo. The results showed the plasticity of epithelial tumour cell phenotype and high level of EMT in some epithelial cell culture what must be taken into account in molecular studies of epithelial tumour cells in vitro. The study was supported by RFBR #18-015-00422, 16-04-00347.

In the present research, we compared two datasets obtained from the quantitative estimation of ERCC1 protein using two monoclonal antibodies—8F1 and FL29. Quantitative evaluation of immunofluorescence parameters was performed by flow cytometry on ovarian cancer biopsy specimens (n = 53). Agreement between two different assays was estimated by correlation analyses and Bland–Altman plots. It was shown that, despite of large spread of differences etween two measurements obtained by 8F1 and FL297, both clones may be used for estimation of ERCC1 expression level (i.e., the number of positively stained cells) in tumor.

Background: Estrogen receptors beta (ERβ) are highly expressed in different normal and neoplastic tissues that, until recently, has been considered to be ER-negative based on ERα expression evaluation. Among the genes, regulated via estrogen signaling there is one, coding microtubule protein beta-III tubulin (TUBB3). TUBB3 expression is found in many solid tumors and is linked to poor prognosis and resistance to taxanes. Since it is little known about mechanisms behind TUBB3 expression in non-small cell lung cancer (NSCLC), we decided to find out if there is a correlation between ER and TUBB3 expression in this type of cancer. Methods: 104 surgical samples of NSCLC were converted to single-cell suspension, stained with primary anti-ERα (abSP-1), anti-ERβ (ab14C8), anti-TUBB3 (ab7751) antibodies and secondary fluorescent antibodies. Immunofluorescent estimation was performed using flow cytometry. Expression level was determined as the ratio (%) of specifically fluorescent cells to the number of cells stained with secondary antibodies. Spearman rank correlation was used to test the association between variables. Results: Both ER were revealed in all NSCLC specimens. Mean expression level of ERβ was significantly higher compared with ERα (46,6 ± 17,0% vs 23,2 ± 14,2%, respectively). Mean TUBB3 expression level was 43,1±15,7%. In all the tumors investigated only weak correlation observed between and ER status and TUBB3 expression level (rs = 0,3 and rs = 0,4 for ERα and ERβ, respectively). In the group of squamous cell cancer specimens (n = 68) the association was strong (rs = 0,5 and rs = 0,5 for ERα and ERβ, respectively). In the group of adenocarcinoma specimens (n = 36) the correlation between ERα and TUBB3 was very weak (rs = 0,3) and there was no correlation between ERβ and TUBB3. Conclusions: 1. Strong correlation between TUBB3 and ER expression was found only in squamous cell cancer tissue. 2. The dominant type of estrogen receptors is ERβ. 3. In clinical terms high ERβ expression means that in case of resistance to standart platinum/taxane duplets, patients with high tumor ERβ expression may benefit from antiestrogen therapy. Supported by RFBR grants (№№15-04-06991-а, 16-34-01049-mol-a) and grant of the President of RF МК-7709.2016.7. Legal entity responsible for the study: N.N. Blokhin Russian Cancer Research Center. Funding: Russian Foundation for Basic Research Grants, grant of the President of Russian Federation. Disclosure: All authors have declared no conflicts of interest.

Background. Beta-III tubulin (TUBB3) is a tumor-specific isoform of the microtubule protein beta-tubulin. TUBB3 is considered to be a marker of adverse prognosis and tumor resistance to therapy with taxanes and Vinka alkaloids. Association between TUBB3 expression and histological type of the tumor has not been studied properly yet. Objective. The expression level of TUBB3 in non-small cell lung cancer biopsy specimens has been measured on 2 groups of patients with adenocarcinoma and squamous cell carcinoma. Materials and methods. The samples with adenocarcinoma (n = 43) and squamous cell carcinoma (n = 39) were converted to suspension, filtered, fixed with 4 % formaldehyde, stained with monoclonal antibodies for TUBB3 and DyLight 650-conjugated secondary antibodies to mouse IgG and analyzed by flow-cytometry. The method was developed in N.N. Blokhin Russian Cancer Research Center. Results. The average level of TUBB3 expression in the adenocarcinoma group is higher than in the squamous cell carcinoma group (33.1 ± 12.4 % of cells expressing the marker in adenocarcinoma vs. 26.0 ± 13.6 % in squamous cell carcinoma; differences were statistically significant). The average level of TUBB3 expression in adenocarcinoma is 29.7 ± 8.1 % in female and is 34.9 ± 13.9 % in male (differences statistically insignificant). Since the group of patients with adenocarcinoma was presented by both men and women, while the group of patients with squamous cell carcinoma was only men, from the analysis was excluded all female patients. Differences between the groups remained statistically significant. Conclusion. TUBB3 expression in tumor tissue does not depend on the gender, at least among patients with adenocarcinoma of the lung, and at the same time, the level of TUBB3 in adenocarcinoma tissue is higher in comparison with squamous cell carcinoma tissue.