Search

There is currently no clear understanding of the molecular participants providing cytotoxic and/or cytostatic activity of neutrophils (Nph) in relation to tumor cells. Cytokines produced by neutrophils are necessary for their paracrine and autocrine interactions with surrounding cells. In order to assess the effect of regulatory neutrophilokines on the morphofunctional state of circulating Nph in benign ovarian tumors and ovarian cancer, the ELISA method was used to assess the level of IL-4, IL-6, IL-10, IL-8, IL-18, IFNγ, MCP-1 and MMP-1in neutrophils, expression of CD11b, CD63, CD16, CD95. Determined the rigidity of the membrane and the ability of neutrophils to form NET. Statistical processing of the obtained data was carried out using the software Statistica 13.0, Jamovi 1.6.5.0. It was found that in ovarian cancer the rigidity of neutrophils depends on the level of IL-10, MCP-1, IL-18, IL-8, IL-4, IFNγ, IL-6 in neutrophils. The method of multiple regression revealed the dependence of the ability to form NETs on the level of IL-4 and IL-6 in Nph. Revealed an inverse correlation between the rigidity of the membrane Nph and their ability to form traps in ovarian cancer. In benign ovarian tumors, a noticeable direct correlation was found between the rigidity of the neutrophil membrane and the adhesive marker CD11b. In ovarian cancer, a correlation was found between the rigidity of the Nph membrane and the CD63 degranulation marker. At benign ovarian tumors, no correlations were found between the number of activated neutrophils and the level of intracellular cytokines in Nph. In ovarian cancer, correlations were found between the number of CD11b+Nph and the level of IL-6, IL-8 in them; between the amount of CD63, CD95 and intracellular IL-8. The amount of CD16+Nph correlated with the level of MMP-1 and IL-8 in Nph. The amount of CD95+Nph correlated with the level of IL-18 in Nph. Thus, the change in the level of neutrophilokines in benign ovarian tumors did not correlate with changes in the ability to NETosis, expression of activation markers, but was accompanied by an increase in the rigidity of the membrane of circulating neutrophils. In ovarian cancer, an increase in IL-8 correlated with a decrease in CD16 expression and an increase in CD63; a decrease in CD16 correlated with an increase in MMP-1. An increase in membrane rigidity in ovarian cancer was associated with changes in all considered neutrophilokines (IL-4, IL-6, IL-8, IL-10, IL-18, MCP-1, IFNγ). The combination of IL-4, IL-6, IL-18 indices, NET number and membrane rigidity of circulating Nph (according to the results of multivariate analysis) can be used for differential diagnosis of ovarian cancer.

Ovarian cancer (OC) is able to develop implantation metastases in the abdominal cavity. Ascites is potentially useful for evaluating cancer features. The aim of the study was to assess the content of stem-like tumor cells and inflammatory mediators in ascites of OC. The prospective study included 11 patients with primary OC having ascites, 8 patients with benign ovarian tumors having ascites and 22 healthy women. In ascitic fluid obtained by laparocentesis, the populations of tumor stem-like cells were determined on a Cytoflex S` flow cytometer (Beckman Coulter, USA) and CytExpert Software using monoclonal antibodies to CD45, CD44 and CD133. The cytokine profiles of ascitic fluid and blood serum (IL-1β, IL-18, IL-4, IL-10 and VEGF) were assessed by ELISA. Stem-like cells were found in all samples. 5 cell populations were evaluated. The number of cells expressing both markers: CD44 + and CD133+, was the lowest. The highest, about 32%, was the number of CD44+ cells. The number of cells CD45-CD44+CD133- in ascites strongly positively correlated with the content of IL-10 in ascites, and the numbers of CD45-CD133+ and CD45-CD44-CD133+ - with the level of VEGF in blood serum. No correlations were found between the numbers of stem-like cells and the disease stage or the level of CA125 in blood. The combination of IL-4 and IL-10 in ascites had the greatest significance in predicting the disease stage. These results suggest a relationship between the levels of VEGF, IL-10, and cancer stem cells in the OC ascites. Stem-like cells in OC ascites are heterogeneous and are present even at an early stage of the disease. It seems promising to study cell populations and cytokine profile of ascites together, to assess the biomarker potential of their combination.

At the present time, there is no common point of view to the role of circulating neutrophils (NP) in emergence and development of neoplasia. It is suggested that due to high functional plasticity, the neutrophils may exhibit both pro- and antitumor activity. In order to study the NP phenotype at different stages of cervical neoplasia (CN), we have evaluated their absolute and relative amounts, myeloperoxidase activity, spontaneous and induced NST-test markers, and the level of intracellular cationic proteins. Spontaneous production of elastase and active forms of matrix metalloproteinases, the levels of IL-2, IL-8, IL-18, IFNy, G-CSF were determined in the NP cell lysates and in blood serum. The formation of extracellular traps (NET) was evaluated using 1-day cultures of Saccharomyces cerevisiae as an inducer. We examined 31 patients with cervical intraepithelial neoplasia (CIN) and 21 primary patients with cervical cancer (CC, Ia stage according to FIGO scale), as well as 25 practically healthy women. We revealed increased spontaneous and inducible oxygen-dependent cytolytic and phagocytic activity and spontaneous production of NET if compared to normal values, along with decreased absolute NP numbers in patients with CIN, thus suggesting the antitumor activity of NP. The levels of “pro-tumor” cytokines (MMP-9, IL-2 and G-CSF) become increased over normal levels as early as at the CIN stage, both for the neutrophils and blood plasma. High levels of regulatory IFNy and neutrophil-priming IL-8 in blood plasma do not presume any use of exogenous NP-activating factors at the stage of cervical dysplasia. At the initial stage of cervical cancer, the absolute NP amounts are significantly increased compared to normal counts. However, despite increased spontaneous oxygen-dependent cytolytic activity, the NPs have a significantly reduced activity of phagocytosis and sharply increased spontaneous production of NET, thus, generally, being characteristic to the “pro-tumorous” NP phenotype. IL-2 levels are elevated, and MMP-9 values are still increased in NP and blood plasma of patients with CC (stage Ia). Hence, the obtained results suggest some changes of NP phenotype to a pro-tumorous pattern during transition from intraepithelial dysplasia to cervical cancer. These results allowed us to design an algorithm for examining women with suspected cervical cancer, including IL-2 measurement in blood serum, and MMP-9 amounts in the NP lysates.

Circulating tumor cells (CTCs) are essential for hematogenous metastasis. In 2003, it was found out that such cells were present in the blood of patients diagnosed with ovarian cancer (OC). It is known that inflammation plays an important role in tumor progression. There are CSCs with a large number of components in the blood, e.g. cytokines that can modulate the metastatic potential of a tumor cell. The aim of the study is to assess the blood serum cytokine status in ovarian cancer patients with different levels of circulating tumor cells. Materials and Methods. Untreated primary patients (n=24) with histologically or cytologically verified ovarian cancer, stage II–IV according to FIGO classification, were the trial subjects. Flow cytometry was used to detect the number of circulating tumor cells in the blood from the patients; the authors also determined IL-6, IL-17A, IL-1β, TGF-α, IL-4, VEGF, TNF-α, HGF, IL-18, IL-10, IL-8 levels. The results were processed using the Statistica Windows software package. Results. The authors determined that TNF-α, HGF, IL-10, IL-18 cytokine level in the blood serum from OC patients significantly increased, and IL-8 level decreased with CTC increase. Conclusion. The obtained results suggest a correlation of CTC level with TNF-α, HGF, IL-10, IL-18, IL-8 cytokine serum level in patients diagnosed with advanced ovarian cancer. At the same time, a sharp and significant increase in TNF-α level accompanied with CTC increase may indicate a change in the phenotypes of TNF-producing cells in OC. Keywords: ovarian cancer, cytokines, circulating tumor cells. Циркулирующие опухолевые клетки (ЦОК) представляют собой основу гематогенного метастазирования. В 2003 г. было продемонстрировано их существование в крови больных раком яичников (РЯ). Известно, что воспаление играет важную роль в прогрессировании опухолей. В крови ЦОК встречаются с большим количеством компонентов, в т.ч. с цитокинами, которые способны модулировать метастатический потенциал опухолевой клетки. Целью данного исследования была оценка цитокинового статуса сыворотки крови больных раком яичников с различным уровнем циркулирующих опухолевых клеток. Материалы и методы. Объектом исследования явились первичные больные с верифицированным (гистологически либо цитологически) раком яичников II–IV степеней по FIGO (n=24) до лечения. В крови пациенток определяли число циркулирующих опухолевых клеток методом проточной цитофлюориметрии и уровни IL-6, IL-17A, IL-1β, TGF-α, IL-4, VEGF, TNF-α, HGF, IL-18, IL-10, IL-8. Обработку результатов проводили с использованием пакета программ Statistica Windows. Результаты. Было установлено, что уровень цитокинов TNF-α, HGF, IL-10, IL-18 в сыворотке крови больных РЯ статистически значимо повышался, а уровень IL-8 снижался с увеличением количества ЦОК. Выводы. Полученные результаты позволяют предполагать корреляцию уровня ЦОК с сывороточным уровнем цитокинов TNF-α, HGF, IL-10, IL-18, IL-8 у больных распространенным РЯ. При этом резкое и значимое возрастание уровня TNF-α при увеличении числа ЦОК может свидетельствовать о смене фенотипов TNF-продуцирующих клеток при РЯ. Ключевые слова: рак яичников, цитокины, циркулирующие опухолевые клетки.

Stem properties allow circulating tumor cells (CTCs) to survive in the bloodstream and initiate cancer progression. We aimed to assess the numbers of stem-like CTCs in patients with ovarian cancer (OC) before treatment and during first-line chemotherapy (CT). Flow cytometry was performed (Cytoflex S (Beckman Coulter, CA, USA)) using antibodies against CD45, epithelial markers EpCAM and cytokeratin (CK) 8,18, mesenchymal vimentin (vim), and stem-like CD44, CD133 and ALDH. This study included 38 stage I–IV OC patients (median age 66 (Q1–Q3 53–70)). The CK+vim- counts were higher (p = 0.012) and the CD133+ALDHhigh counts were lower (p = 0.010) before treatment in the neoadjuvant CT group than in the adjuvant group. The patients with ascites had more CK+vim- cells before treatment (p = 0.009) and less EpCAM-vim+ cells during treatment (p = 0.018) than the patients without ascites. All the CTC counts did not differ significantly in paired samples. Correlations were found between the CK-vim+ and CD133+ALDHhigh (r = 0.505, p = 0.027) and EpCAM-vim+ and ALDHhigh (r = 0.597, p = 0.004) cells before but not during treatment. Multivariate Cox regression analysis showed that progression-free survival was longer with the presence of surgical treatment (HR 0.06 95% CI 0.01–0.48, p = 0.009) and fewer CD133+ALDHveryhigh cells (HR 1.06 95% CI 1.02–1.12, p = 0.010). Thus, CD133+ALDH+ CTCs have the greatest prognostic potential in OC among the phenotypes studied.

Ovarian cancer (OC) is able to develop implantation metastases in the abdominal cavity. Ascites is potentially useful for evaluating cancer features. The aim of the study was to assess the content of stem-like tumor cells and inflammatory mediators in ascites of OC. The prospective study included 11 patients with primary OC having ascites, 8 patients with benign ovarian tumors having ascites and 22 healthy women. In ascitic fluid obtained by laparocentesis, the populations of tumor stem-like cells were determined on a Cytoflex S` flow cytometer (Beckman Coulter, USA) and CytExpert Software using monoclonal antibodies to CD45, CD44 and CD133. The cytokine profiles of ascitic fluid and blood serum (IL-1β, IL-18, IL-4, IL-10 and VEGF) were assessed by ELISA. Stem-like cells were found in all samples. 5 cell populations were evaluated. The number of cells expressing both markers: CD44 + and CD133+, was the lowest. The highest, about 32%, was the number of CD44+ cells. The number of cells CD45-CD44+CD133- in ascites strongly positively correlated with the content of IL-10 in ascites, and the numbers of CD45-CD133+ and CD45-CD44-CD133+ - with the level of VEGF in blood serum. No correlations were found between the numbers of stem-like cells and the disease stage or the level of CA125 in blood. The combination of IL-4 and IL-10 in ascites had the greatest significance in predicting the disease stage. These results suggest a relationship between the levels of VEGF, IL-10, and cancer stem cells in the OC ascites. Stem-like cells in OC ascites are heterogeneous and are present even at an early stage of the disease. It seems promising to study cell populations and cytokine profile of ascites together, to assess the biomarker potential of their combination.

Aim. Study of the relationship of vascular endothelial growth factor expression in tumor cells of ascites and primary tumor in patients with ascitic form of ovarian cancer undergoing neoadjuvant chemotherapy according to the AP scheme (doxorubicin + cisplatin). Methods. The level of vascular endothelial growth factor messenger ribonucleic acid (mRNA) was assessed using PCR-RT in epithelial cells of ascites and primary tumor tissue obtained intraoperatively after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme from 25 patients with a verified diagnosis of ovarian cancer (serous adenocarcinoma) at stage III–IV according to the International Federation of Gynecology and Obstetrics. Processing of the results was performed using the software package Statistica 10. Results. An increase in the expression of vascular endothelial growth factor mRNA in epithelial cells of ascites in 60% of samples was established. In the intraoperatively obtained tumor tissue after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme, vascular endothelial growth factor expression was increased in 73% of the samples. At the same time, the increase of relative expression in the tumor after neoadjuvant chemotherapy did not depend on the degree of therapeutic pathomorphosis but was related to early relapse and increased level of cancer antigen-125 (CA-125). Median of relapse-free survival in vascular endothelial growth factor-positive ovarian cancer was less than 5 months. Conclusion. Neoadjuvant chemotherapy according to the AP scheme increases the expression of vascular endothelial growth factor in the tumor tissue in advanced ovarian cancer; the degree of vascular endothelial growth factor overexpression in ovarian tumor is related to the presence of early relapse and an increase in the serum CA-125 level.

Chemoresistance is one of the main causes of treatment failure in advanced ovarian cancer (OC). In order to identify predictors of chemoresistance, 30 patients with ascitic form of OC, who received neoadjuvant chemotherapy (NACT) according to AP regimen, were examined. In the epithelial cells of ascites and in tumor tissue after NACT, expression of the VEGFa, ABCB1 and ERCC1 genes was assessed by PCR-RT. The effectiveness of NACT was assessed using the criteria of therapeutic pathomorphosis, survival - using the Kaplan-Meier criterion, the relationship of clinical and molecular parameters - using the Mann-Whitney criterion. Results. We established that in 43% of patients the response to NACT was absent (CRS1), the significant response (CRS2) was in 35% of cases, and the complete response (CRS3) was in 21% of cases. VEGFa expression was increased in ascites in 17% of cases, in tumor tissue - in 33% in the CRS1 group. In groups with CRS 2,3, overexpression of VEGFa in ascites was detected in 25% of cases, in tumor tissue in 37.5%. ABCB1 mRNA expression was increased after NACT in 50% of cases in the CRS1 group and in 25% of cases in the CRS 2,3 group. Thus, platinum-based NACT initiates an increase in the expression of VEGFa and ABCB1 in tumor tissue. We established that in case of high expression (ERCC + / VEGF + / ABCB1 +) in ascites, there is a statistically significant increase in relapse-free survival compared with the group with low or absent expression (p = 0.012). The overall survival rate was 41.8 months in patients with overexpression of the studied genes in the tissue of the primary tumor after NACT and 25.3 months in patients with low expression (p = 0.058). Conclusion. The levels of ERCC1, VEGFa and ABCB1 mRNA in ascitic cells before NACT and in the primary tumor after NACT according to the AP scheme can serve as a predictor of the effectiveness of this treatment in ascitic OC.

e17536 Background: During the ovarian carcinogenesis, circulating neutrophils (Nph) phenotype switches to the pro-tumor, with an increase in the Nph C-C motif ligand 2 (CCL2) expression. Proinflammatory interleukin-17A (IL-17A) is able to induce the CCL2 expression in Nph. The study aimed to evaluate the IL-17A and CCL2 levels in serum and circulating Nph in patients with ovarian tumors. Methods: The study included 97 ovarian cancer (OC) patients, 30 patients with benign ovarian tumors (BT) and the control (n=22). The levels of IL-17A (LLC "Cytokine", Russia) and CCL2 (Vector-Best-Volga, Russia) in serum and Nph lysate were assessed by ELISA. The systemic inflammation was assessed by neutrophil to lymphocyte ratio (NLR). Informed voluntary consent was obtained from all women. Statistical processing was performed using one-way ANOVA, Spearman correlations (Statistica 13.0 (TIBCO, USA)). Results: We found an increase in NLR in BT (4.3 ± 1.0) compared with the control (2.2 ± 0.1) (p = 0.020), a decrease in stage I-II OC (2.4 ± 0.2) compared with BT (p = 0.053), and an increase in stage III (4.5 ± 0.9) and IV (4.6 ± 0.5) compared with stage I-II OC (p = 0.054 and p = 0.046, respectively). The cytokines levels are presented in Table. The serum level of IL-17A in BT was higher than in the control (p = 0.010). In stage I-II OC, the serum IL-17A level was decreased in comparison with the BT (p = 0.004). In stage IV OC, the serum IL-17A was higher than in stage I-II (p = 0.015) and the control (p = 0.017). The level of IL-17A in Nph in BT did not differ from the control. In all stages of the OC, the IL-17A Nph level was lower than in the control (p1, 2, 3 = 0.0001). In stage IV OC, the expression of IL-17A in Nph was lower compared with stage III (p = 0.031). The serum and Nph levels of IL-17A were correlated only in stage I-II OC (r = 0.679, p = 0.011). The CCL2 serum levels in BT (p = 0.0002) and OC stage I-II, III, IV were higher compared with the control (p1 = 0.031, p2 = 0.001, p3 = 0.0005, respectively). The Nph CCL2 level was higher in BT than in the control (p = 0.001). In stage I-II OC, the expression of CCL2 in Nph was higher than in the control (p = 0.034), but lower than in the BT (p = 0.003). In OC stages III and IV, the Nph CCL2 level was higher than in the control, but did not differ from that in BT and stages I-II. In stage III OC, the CCL2 level in Nph negatively correlated with the serum IL-17A level (r = -0.405, p = 0.049). Conclusions: The pro-tumor polarization of circulating Nph is not IL-17A-dependent in patients with ovarian tumors. The patterns of the systemic immune response differ in benign ovarian tumors, early, and advanced ovarian cancer.[Table: see text]

e17542 Background: Circulating tumor cells (CTCs) are a potential source of dissemination and relapse in ovarian cancer (OC). Stem cell properties can provide a survival advantage for CTCs. The clinical significance of stem-like CTCs in OC remains to be studied. We aimed to assess the quantities of the stem, epithelial, mesenchymal CTCs and their relationships with the clinical parameters in the OC. Methods: Peripheral blood samples (7.5 ml) were obtained from patients with primary epithelial OC before treatment. CTCs were isolated by flow cytometry (Cytoflex S (Beckman Coulter, USA)) using antibodies to CD45 (BioLegend, USA); CD44 (BioLegend, USA), CD133 (Miltenyi biotec, Germany), ALDH (Stemcell, Canada) to detect the stem markers; EpCAM (BioLegend, USA), cytokeratins 8, 18 (Abcam plc., UK), vimentin (BioLegend, USA) for epithelial and mesenchymal markers. Blood samples from patients with benign ovarian tumors served as a control. Informed voluntary consent was obtained from all the women. Statistical processing included Mann-Whitney U-test, linear regression, Cox proportional hazards model for progression-free survival (PFS) (Statistica 13.0 (TIBCO, USA)). Results: The study included 30 patients, median age 64 (34-76) years. 15 patients had a FIGO stage IV, 12 - stage III, 1 – stage II and 1 – stage I. The content of CTCs populations is presented in the table. The CTCs counts did not differ depending on age, platelet count, and stage 3 or 4. The amount of CD45-CK+Vim- was higher in the presence of ascites (p = 0.035). We found a regression relationship between the serum CA-125 and the number of CD45-CD44+CD133+ (R2= 0.220, p = 0.016); the leukocyte count in blood and CD45-CD44+ALDHhigh (R2= 0.234, p = 0.017); the number of CD45-Vim+ and CD45-CD44+CD133+ALDH+ (R2= 0.305, p = 0.014); CD45-CK-Vim+ and CD45-EpCAM+CK+ (R2= 0.717, p < 0.001). The Cox regression model for PFS included the number of CD45-CD44+CD133+ALDH+ (HR 1.51 95% CI 1.01-2.24 p = 0.043) and the cytoreductive surgery performance (HR 0.09 95% CI 0.01-0.89 p = 0.039) during the first line of treatment. Conclusions: Various populations of circulating tumor cells coexist in ovarian cancer patients. The use of a combination of stem markers in the CTCs detection can increase their prognostic value in OC. This work was supported by the RFBR grant No. 19-315-90011.[Table: see text]

Chemotherapeutic treatment using standard solvents is stopped in every fourth ovarian cancer patient because of significant side effects. The autohemochemotherapy method alters the pharmacokinetics of the drug, and significantly reduces the frequency and severity of side effects. In order to compare the effects of neoadjuvant chemotherapy using autologous liquid tissues or standard solvents on cytokine status and neutrophils in the peripheral blood we evaluated the number of neutrophils and their ability to generate reactive oxygen species, and levels of interleukin-1p, IL-1Ra, IL-6, IL-10, IL-18 and TNF-a in 244 primary stage III-IV ovarian cancer patients during chemotherapy according to the AP scheme (cisplatin 75 mg/m2, doxorubicin 50 mg/m2) with infusion by standard intravenous method or as autohemochemotherapy. We established that the effectiveness of chemotherapy was associated with the redox status of neutrophils and cytokine status of peripheral blood, registered prior to neoadjuvant chemotherapy. For patients with tumor progression, increased absolute and relative number of neutrophils with reduced ability to produce reactive oxygen species is significant. For patients with a lack of tumor response, significant are the high levels of serum IL-10 and IL-18. For patients with complete or partial tumor response, significant is the decrease of IL-6 level, compared to the norm. No significant differences in neutrophil and cytokine status in both modes of infusion of chemodrugs have been established. Thus, serum IL-10, IL-18 and IL-6 may be used to predict tumor response to neoadjuvant chemotherapy according to the AP scheme.

Background The role of neoangiogenesis in progression of solid tumors is crucial [Han J.M., 2017]. Activated neutrophils (Nph) can synthesize vascular endothelial growth factor (VEGF) and interleukine-8 (IL-8) [Merritt W.M., 2008], regulated by nuclear factor-kappa B (NF-κB) [Annunziata C.M., 2010]. The aim of the study was to assess the role of pro-angiogenic factors of circulating Nph in the progression of ovarian cancer (OC). Methods We isolated circulating Nph from the peripheral blood of 97 OC patients (stage I-IV FIGO) 36-76 years old on a double layer of ficoll-urographin. Using ELISA, the VEGF-A (RayBiotech, USA) and IL-8 (Vector-Best-Volga CJSC, Russia) levels were determined in the Nph lysate, and the expression of NF-kB (eBioscience, USA) was determined in the Nph nuclear fraction. The control group included 30 healthy women 23-65 years old. The study was conducted in accordance with the requirements of the IMEPC ULSU ethics commission (protocol №3 from 15.03.2015). The correlation between the parameters was evaluated using the Spearman coefficient, the statistical significance of the differences was evaluated using the nonparametric Mann-Whitney U-test (p ≤ 0.05). Results We detected a statistically significant increase in the level of VEGF in Nph already at the initial stage of the OC compared to the control (469.764±185.028 pg/ml vs 50.483±9.505 pg/ml, p = 0.0053). With more advanced OC stage, the VEGF level in Nph decreased by 2 times, but remained statistically significantly high compared to control. An average correlation was revealed between the level of VEGF in Nph and the stage of OC (p = 0.5196). The level of NF-kB in the nuclear fraction of Nph was statistically significantly increased in I stage OC (0.589±0.129 mg/ml vs 0.166±0.028 mg/ml in the control, p = 0.0253) and decreased statistically significantly with the OC spread. A weak correlation between the level of NF-kB in Nph lyzate and the OC stage was revealed. The level of IL-8 in Nph did not significantly change in OC. An average correlation was found between the NF-kB level and IL-8 level in Nph (p = 0.6914). Conclusion The results obtained indicate an increase in the pro-angiogenic activity of circulating Nph with the progression of OC. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Peripheral blood neutrophils of 123 patients in stage Ia of the endometrial cancer have been assayed. The receptor system and ability of neutrophils to form extracellular traps (NET activity) were assessed by fluorescence microscopy, and spontaneous production of IL-2, IFN-γ, matrix metalloproteinase (MMP), G-CSF was found to exist determined with enzyme-linked immunosorbent assay. Phagocytic and myeloperoxidase activity, the level of cationic proteins, and neutrophil activity were evaluated in an NBT test determining the oxygen-dependent bactericidal action of neutrophils. Topology and rigidity of neutrophil membranes were assessed using scanning probe microscopy. It was established that an increased number of neutrophils was accompanied by changes of their receptor system, aerobic and anaerobic cytotoxicity, enhanced phagocytic activity, and reduced NET activity. The secretory activity of neutrophils changed. An increased level of MMP-1, possibly by enhanced production of reactive oxygen species, reduced IL-2 level, and a drastic increase in G-CSF were observed. The architectonics of neutrophils at stage Ia of endometrial cancer was characterized by altered cell shape and granularity loss. The rigidity of the cell membrane decreased. The changes in neutrophil morphology and persistent hyperactivity suggest the existence of the equilibrium between the immune system and the tumor in stage Ia of endometrial cancer.

Background Long non-coding RNAs (lncRNAs) appear to be one of the key regulators of cellular signaling processes. According to several authors, they play a crucial role in the regulation of the antitumor immune response. Dysregulation of lncRNA profile is associated with carcinogenesis. Alteration of the lncRNA profile in ovarian cancer (OC) tumor tissue was shown. The aim of our study was to identify serum lncRNAs as potential biomarkers in ovarian cancer. Methods The study included 34 patients with verified ovarian cancer II-IV FIGO stage. Blood sampling was performed before treatment and after 3 courses of standard neoadjuvant (paclitaxel 175 mg/m² + carboplatin AUC 6 every 3 weeks) chemotherapy. To isolate free-circulating RNA from serum, the SileksMagNA-Direct magnetic particle kit was used. Analysis of the normalized expression of lncRNA MALAT, HOTAIR and PVT1 was carried out using qPCR method with normalization by 18S. We assessed the correlations between lncRNA levels and the patient age, tumor stage and CA-125 level. For statistical analysis, the ANOVA criterion (Statistika 13.0) was used. Results We have established strong correlations between the HOTAIR plasma level and age (p = 0.120) and CA-125 level (p = 0.082), and weak correlation with a relapse-free period duration (p = 0.167). The level of MALAT strongly correlated with age (p = 0.050) and with a relapse-free period duration (p = 0.014) and weakly correlated with the stage of ovarian cancer (p = 0.140). PVT1 expression did not reliably correlate with any of the studied clinical and biological parameters. Conclusion The revealed relationships between the levels of circulating MALAT and HOTAIR in the blood plasma of patients with OC require further study of these epigenetic regulators in OC carcinogenesis and chemoresistance. Free circulating lncRNA MALAT and HOTAIR, as an element of a liquid biopsy, are potential prognostic and predictive biomarkers for OC. Legal entity responsible for the study The authors. Funding RFBR (project number 19-315-50012). Disclosure All authors have declared no conflicts of interest.

Antiangiogenic therapy requires to search for molecular markers of angiogenesis in ovarian cancer (OC). The diagnostic and prognostic role of VEGFA in OC is controversial today. The aim of the study was to compare the serum level of VEGF, the cytoplasmic protein content and VEGF transcript in the tumor tissue of primary patients with advanced ovarian cancer and to analyze the relationship between these parameters and the response to standard polychemotherapy using the AP scheme. 82 primary patients with advanced OC were incladed in the study. The control group included 30 healthy women. All patients received adjuvant chemotherapy according to the AP scheme after surgery. The serum VEGF-A level was determined by direct immunoassay analysis "Human VEGF ELISA Kit" ("RayBiotech", USA). Analysis of the VEGF transcript in tumor tissue was performed by PCR-RT using TaqMan® Gene Expression Assay (Thermo Scientific) probes, and for the data normalization the 18S and GAPDH referee genes were used. To calculate the IHC score of cytoplasmic expression of VEGF, immunohistochemical reactions were performed in paired samples of OC patients using antibodies (GeneTex, USA). For the genotyping of patients' DNA, test systems for the analysis of functional polymorphisms of the VEGF-A C12143A, G634C (LLC "Sintol", Moscow) were used. The level of VEGF-A in the serum of patients with OC was increased in comparison with the norm and depended on the response to chemotherapy according to the AP scheme. However, the specificity of this indicator is insufficient for use in prognostic purposes. There are no significant differences in the cytoplasmic expression of VEGF-A, depending on the response to chemotherapy. VEGF mRNA level was increased in 58% of patients. There was no correlation between serum VEGF levels and the transcript in the tumor tissue. Genotyping showed that the combination of genotypes of CC / GG reduces the risk of recurrence by 2.6 times. The results obtained make it possible to question the value of studying the serum level of VEGF for evaluating the sensitivity of patients with OС to anti-angiogenic drugs.

The purpose of the study was to investigate the effect of the scheme autohemochemotherapy ATS on redox-dependent processes in red blood cells of tumor-bearing organism at an experimental ovarian cancer.We studied the dynamics of redox-dependent processes in red blood cells of rats with experimental ascitic ovarian tumor during CAP-regimen autohemochemotherapy (cyclophosphamide, doxorubicin and cisplatin). We assessed the indicators of oxidative modification of proteins in erythrocytes - carbonyl derivatives at l = 346 nm, 370 nm, 430 nm and 530 nm, the parameters of lipid peroxidation - malondialdehyde, ketodienes, diene conjugates, Schiff bases; the enzymatic part of antioxidant system - the activity of catalase, glutathione transferase and superoxide dismutase biochemically.The red blood cells of tumor-bearing animals were found having increased the products of lipid peroxidation and oxidative modification of proteins while reducing the activity of antioxidant enzymes, suggesting a state of oxidative and carbonyl stress.We showed that extracorporeal incubation of cytotoxic drugs used in the CAP scheme with autoblood prior to infusion - the method of autohemochemotherapy - either in monochemotherapy, or in CAP-regimen, decreases the levels of lipid peroxidation, oxidative modification of proteins and increases activity of first line antioxidant defense enzymes - catalase and superoxide dismutase in circulating red blood cells. Such dynamics of redox-dependent processes suggests a stabilizing effect of autohemochemotherapy on circulating erythrocytes in a tumor-bearing organism.

Background: Polychemotherapy in ovarian cancer (OC) is the second major component of treatment. However, treatment with cytostatics is stopped in 25% of cases because of significant side effects. It is shown that concentration of certain cytokines and their balance is largely formed in accordance with a genetic polymorphism. Objective: The Objective of the study was to evaluate the cytokine status of blood serum of patients with ovarian cancer with different tumor response to neoadjuvant chemotherapy (NACT). Method: Patients received 2 courses NACT according to the scheme AP. The levels of IL-1β and IL-1Ra, IL-10, TNF- α in blood serum were determined by solid phase ELISA. For molecular genetic studies we selected polymorphic variants in the promoters of the gene represented in dbS`NP NCBI and SNP500 Cancer databases. Results: The sharply declined in patients with ovarian cancer compared with the normal, level of IL-1β correlates with increased levels of IL-1RA. It is found that 75% of patients who had progression of the disease after NACT bear CT genotype of gene IL-1β associated with a low expression of the cytokine, while the TT genotype, providing a high level of the expression of IL-1β gene had met only 25% among patients in this group. At the same time 70% of patients with a complete response are the carriers of the T allele, while a complete response was associated with a higher level of IL-1β than in the progression group. Low secretion of TNF-α in all types of tumor response when testing TNF-α G-308A gene polymorphisms was associated with carriage of GA and AA genotypes, which are associated with low production of this cytokine. Increased compared to the control IL-10 production in patients with ovarian cancer associated with genotype replacement at position 1082 G/A IL-10 gene, which occured in 10% of patients with a complete response and 25% of patients with tumor progression after NACT. Conclusion: All the studied polymorphisms of IL-1β, IL-10 and TNF-α genes in patients with OC are associated with the level of these cytokines and tumor NACT response.

Aim. To develop the model of specific diagnostics of III-IV stages of ovarian cancer. Methods. 404 patients with I-IV stages of primary ovarian cancer according to International Federation of Gynecology and Obstetrics classification were examined. Ultrasonography of genitalia and internal organs, chest X-ray in order to detect possible remote metastases, cytological, histological and immunohistological examination of tumor mass, as well as «lipid peroxidation - antioxidants» system condition of tumor tissue and patients’ serum were examined. Results. Elevated level of a lipid peroxidation product, malondialdehyde, was found in tumor tissue of patients with ovarian cancer, with levels of malondialdehyde growing from clinical stage I to clinical stage III of the disease. Leukocyte counts of peripheral blood statistically significantly decreased at stages I-III and elevated at stage IV. However, aerobic and anaerobic cytotoxicity of neutrophils decreases. An algorithm of supplementary diagnostic tools use to define the stage of ovarian cancer was developed, as well as differentiation coefficient calculation formula. Conclusion. Defining the serum levels of malondialdehyde, catalase and glutathione reductase activity allows to calculate the coefficient for differentiation between stages III and IV of ovarian cancer, and detection of cation proteins level, myeloperoxidase activity and phagocytic index allows to calculate the coefficient for differentiation between stages II and III of ovarian cancer.

We have examined peripheral blood neutrophils from 123 patients with primary endometrial cancer at stage Ia. Receptor system and the ability of neutrophils to form extracellular traps were assessed by fluorescence microscopy, the spontaneous production of cytokines IL-2, IFN-γ, g-CSF, matrix metalloproteinases-1,9,13 by the method of enzyme-linked immunosorbent assay, phagocytic activity, myeloperoxidase activity, the level of cationic proteis activity in NBT-test were evaluated by cytochemical methods, activity of neutrophils in the spontaneous NBT-test was used to evaluate the oxygen-dependent bactericidal action of neutrophils. The topology and the rigidity of the membrane of neutrophils were assessed by scanning probe microscopy. We have shown that the increase in the relative number of neutrophils lead to a change in their receptor system, aerobic and anaerobic cytotoxicity and ability to phagocytosis are enchanced while reducing NET-activity. We have observed a change in the secretory activity of neutrophils, which is characterized by increased level of MMP-1, possibly initiated by enhanced production of reactive oxygen species, by a reduction in the IL-2 level (inductor of cytotoxic activity) and a sharp increase in the level of the G-CSF. Architectonics of neutrophils in the case of endonetrial cancer at stage Ia is characterized by changing the shape and loss of grit. The rigidity of the cell membrane decreased. Changes in the morphology of neutrophils on the background of the continuing hyperactivity suggests that a state of balance between the immune system and the tumor is already in stage Ia endometrial cancer.

The neutrophil is considered as the peculiar monocelled sekretorny gland realizing the effector potential including by secretion of soluble products - cytokines and for today. Influence of a tumor on functional activity of neutrophils depends on type, localization and a stage of its development. In our research dynamics of metabolic and of neutrophils of peripheral blood, the contents in a lysate and serums of blood of cytokines of IL-1β, 1Ra, 2, 6, 10, 18, TNF-α, IFN-γ and is studied when progressing a cervical cancer. Cytokines and metabolism indicators - activity of, determined by an immunofermental method, level of cationic proteins, a share of active cages in the spontaneous NST-test were cytochemical. It is shown that when progressing cervical cancer against increase in total of neutrophils significant decrease in their, aerobic and anaerobic bacterial action, decrease in the IL-1β and IL-1 Ra level, and also IFN-γ takes place at TNF-α increase, increase of production of matrix metalloproteinas-2 on Ib-IIa of a stage of a disease that allows to assume emergence at this stage of cervical cancer of pro-tumoral effect of neutrophils.

There were assessed morphofunctional state of peripheral blood neutrophils and cytokine status in the serum of primary patients with progression of cervical cancer in the dynamics. Against the background of increasing the total number of neutrophils, a significant reduction occurred in the phagocytic activity, aerobic and anaerobic bactericidal activity, an increase of the rigidity of the membrane and the change of cell cytoarchitectonics at Ib-IIa stage of the disease. At the same stage, in neutrophils there were reduced the level of IL-1β and its receptor IL-1Ra as well as the number of IFN-γ. Wherein there was increased the level of pro-inflammatory cytokine TNF-α. Cytokine status of the serum, characterized by sig- nificant boosting of IL-1Ra and IL-18 while lowering TNF-α, IFN-γ and IL-2, was advantageous for tumor progression and was detected at Ib-IIa stage of cervical cancer. The observed changes of cytokine status in the serum and morphofunctional state of neutrophils were discussed in the light of connection with the possibility of potential pro-tumor effect of the latter.

The dynamics of the redox-dependent processes in blood plasma, neutrophils and erythrocytes of the patients with ovary cancer of the IIIrd clinical stage by FIGO after polychemotherapy according to the CAP scheme is considered. In the blood plasma and erythrocytes there were estimated the values of protein oxidative modification: carbonyl derivatives at λ = 346 nm, 370 nm, 430 nm and 530 nm; the lipid peroxidation parameters: malonic dialdehyde, dienic conjugates, ketodiens, shiff's bases; the fermentative chain of the antioxidant system: activities of catalase, glutationtransferase and superoxide dismutase. In the peripheral blood neutrophils there were cytochemically determined the myeloperoxidase activity and the number of the active neutrophils in the spontaneous NBTR-test. After the polychemotherapy there were detected higher levels of the protein oxidative modification products and the products of the lipid peroxidation in the blood plasma and erythrocytes of the patients. Simultaneous increase of the activity of the antioxidant enzymes in the blood plasma could be evident of a high level of the lipid antioxidants peroxidation system functioning, whereas the simultaneous decrease of the activity of the antioxidant enzymes in the erythrocytes was indicative of passible development of oxidative stress in them. After the chemotherapy there was observed a significant and reliable decrease of the total number of the neutrophils. After the second course of the chemotherapy the activity of myeloperoxidase in them in the spontaneous NBTR-test as well decreased. Such a dynamics of the redox-depended processes in various components of the blood in the tumour carrier was characteristic of the tumor biological picture and required the use of differential multicomponent antioxidant therapy in patients with ovary cancer.

Background: The aim of the study was to elaborate a method of specific diagnostics of progressive ovarian cancer. Methods and Findings: We observed 21 parameters in plasma of 300 primary ovarian cancer patients, stage III and IV. Using the least squares method, approximation of the relative frequencies by the Rayleigh distribution density was conducted for each parameter. The likelihood functions were constructed and intervals for each measurement on each stage were determined. Approximation accuracy was identified while checking the hypothesis of correspondence of parameter practical value to the theoretical law of density distribution by Pearson criterion. Conclusions: The obtained results suggest that the level of the oxidative modification of proteins may serve as a reliable indicator for the differential diagnosis of stages III and IV ovarian cancer.

The diagram of differential diagnostics of stages of ovarian cancer is developed. Survey of 404 patients included ultrasonic research of genitals, an internal, radiological research of lungs for the purpose of detection of the remote metastasises, cytologic, histologic and immunohistochemical research of tumor, and also biochemical indexes of peripheral blood and level of an onkomarker of CA125. It is shown that indexes of level malondialdehyde, activities of a catalase, a glutation-reductase of plasma can be used for the differential diagnosis of III and IV stages. The assessment of cationic proteins level, phagocytic number, activity of myeloperoxidase of neutrophils of peripheral blood will allow to calculate coefficient for differentiation of II and III stages of ovarian cancer. The diagram of additional laboratory researches is developed for differentiation of stages of ovarian cancer on FIGO, and also calculation formula of differentiation coefficient.

The possibility of laser initiation of oxidative stress was studied by the example of the tumour tissue of cervix. The laser facility with the operating wavelength 1265 nm that falls within the region of resonance absorption of molecular oxygen was used for initiation. The source of radiation in the experiments was a fibre SRS laser with the repeated cascade conversion of radiation of a 1125-nm ytterbium laser.

The study highlights the effect of different modes of in vivo laser irradiation of mice using a PFL8LA laser with λ = 1560 nm, pulse duration of 1,4•10 -12 s, peak power of 3,72•10 3 W and average output power of 20•10 -3 W on the lipid peroxidation parameters: conjugated dienes, ketodienes and conjugated trienes, malondialdehyde, Schiff bases and the activity of antioxidant enzymes - catalase, glutathione -S-transferase and superoxide dismutase in erythrocytes and plasma of mice. Two groups of mice received a total dose of 3.8 J/cm 2 per group, but the 1st group was irradiated only once, while the 2nd – four times. Significant differences in the parameters of the 1st and 2nd groups indicate different effects of the irradiation modes on redox-dependent processes in red blood cells of mice.

In order to evaluate the role of cytokines in the development of polymodal local and distant effects in patients with stages I-IV cervical cancer, the following parameters were measured: serum concentrations of TNF-α, IFN-γ, IL-1β, IL-6, and IL-10; levels of malondialdehyde, activities of catalase, superoxide dismutase, glutathione reductase, and glutathione-S-reductase in the plasma, erythrocyte, and cervix uteri homogenate. The expression of p53, Bcl-2, VEGF, and Ki-67 in tumor tissue was evaluated. High levels of malondialdehyde in tumor tissue and erythrocyte were paralleled by low levels of superoxide dismutase and catalase and high activities of glutathiones. Medium correlations between Ki-67, Bcl-2, and p53 and the levels of IL-6, IL-10, and TNF-α at stages Ib-IIa were detected. The results indicated that the progress of cervical cancer was associated with the neoplasm integration in the host homeostasis by using the regional and systemic cytokine functions. These effects, amplifying the biological potential of the tumor, were the most significant at stages Ib-IIa.

The cytochemical analysis of the aerobic and anaerobic bactericidal, phagocytic activity, phagocytosis completeness, as well as membrane topology and rigidity has been performed with the Scanning probe microscopy (Solver Pro, NT-MDT, Russia) in neutrophils of the patients with cervical carcinoma treated with the Erbium laser radiation at different intensity. The obtained data reveal a significant effect of the femtosecond laser radiation on neutrophil morphofunctional state in patients with cervical cancer. The efficiency of the femtosecond radiation depends on its intensity and clinical stage of the cervical cancer.

The purpose of the study was to evaluate the morphofunctional state of polymorphonuclear neutrophils and cytokine levels in the lysate of blood neutrophils of ovarian cancer patients. The absolute and the relative number of peripheral blood polymorphonuclear neutrophils in 120 primary ovarian cancer patients (III-IV clinical stage FIGO) and 80 healthy women were determined. The expression of CD11a, 11b, 15 and CD95 in polymorphonuclear neutrophils was assessed by fluorescent microscopy. Cytochemically the myeloperoxidase activity, the content of cationic proteins, the activity in the spontaneous NBT-test and phagocytic activity in polymorphonuclear neutrophils was determined. The amounts of cytokines TNF-α, IL-1β, 1Ra, 6, 18, and G-CSF in serum were measured by the enzyme immunoassay method. Rigidity and topology of native polymorphonuclear neutrophils were assessed by scanning probe microscopy. To detect the differences between the data obtained in the evaluation of ovarian cancer patients and healthy people, a non-parametric U-Mann-Whitney test was used. Reliable difference was considered at p ≤ 0.05. These studies found an increase of the total number of polymorphonuclear neutrophils in peripheral blood, while reducing their cytotoxicity parameters. We observed reduced myeloperoxidase activity and level of cationic proteins. Reducing in the levels of CD15 and CD95 expression on the background of increasing polymorphonuclear neutrophils activity in the spontaneous NBT-test may indicate a decrease in ability to complete phagocytosis. The dynamics of CD11a and CD11b expression in polymorphonuclear neutrophils was multidirectional, and the changes were not statistically significant. Polymorphonuclear neutrophils membrane stiffness decreased in stage IV ovarian cancer. We also observed the levels of IL-1β reduced up to 248.5 ± 9.00 pg / ml in stage III ovarian cancer, up to 230.9 ± 15.04 pg / ml in stage IV ovarian cancer vs 36.8 ± 10.74 pg / ml in control, reduced levels of 1Ra, and IL-18. We found that the level of IL-6 increased at stage III ovarian cancer up to 34.38 ± 9.74 pg / ml vs 7.66 ± 2.28 pg / ml in control, and the levels of TNF-α and G-CSF decreased. Thus, these results suggest a state of secondary immunodeficiency in patients with ovarian cancer at III-IV clinical stages of the disease.

Background: Ovarian cancer (OC) is the most lethal of gynecologic tumors because women generally present with advanced stage disease. Platinum-based chemotherapy play a pivotal role in OC treatment. The aim of the study was to assess the effects of CAP-regimen chemotherapy on blood redox status in patients with ovarian cancer. Materials and Methods: Patients were 132 women with primary OC with FIGO stage III-IV. Patients were examined before treatment, 3 and 14 days after the first and 3 and 14 days after the second course of CAP-regimen chemotherapy. The activity of antioxidant enzymes, the intensity of lipid peroxidation, the level of oxidative modification of proteins (OMP) were evaluated. Results: We have found elevated levels of OMP products in plasma and erythrocytes of patients with ovarian cancer in comparison with donors. Our results suggest activation of lipid peroxidation in plasma and erythrocytes of ovarian cancer patients in comparison with healthy women. Sensitive and specific indicators of oxidative stress are levels of glutathione-S-transferase (sensitivity 80%, specificity 89%), diene conjugates (sensitivity 85,71%, specificity 72,73%) and OMP 430 nm (sensitivity 80%, specificity 90%) in blood plasma and the activity of catalase in erythrocytes (sensitivity 100%, specificity 89%). Conclusion: CAP chemotherapy in patients with ovarian cancer with FIGO stage III-IV induces radical formation and changes the homeostasis of the patient. The lipid peroxidation and antioxidant system in plasma move to a higher level of functioning, and the erythrocytes develop oxidative stress.