Angiogenic potential of circulating blood neutrophils in endometrial cancer

Neutrophils play an important role in carcinogenesis, mediating inflammation, immunosuppression and metastasis. A dual role of neutrophils in regulation of angiogenesis has been shown. Endometrial cancer is the most common malignant neoplasm of the female reproductive system. To assess the cellular angiogenic potential, we determined the levels of inflammatory and angiogenic cytokines (VEGF-A, IL-17A, IL-1β, IL-6) in cell lysate of peripheral blood neutrophils in the patients with endometrial cancer, and with uterine myoma (comparison group). Expression of the nuclear factor-kB was determined. In nuclear fraction of neutrophils. The neutrophil-to-lymphocyte ratio (NLR) was assessed in the studied groups. Statistical processing of the obtained data was carried out using Statistica 10 software. We have not found any significant changes in NLR in endometrial cancer, compared with controls and the uterine myoma groups. Expression of NF-kB and VEGF was increased as compared to the control for all the studied stages of endometrial cancer and in uterine myoma. There was a change in NF-kB level in neutrophils, depending on the tumor differentiation grade. A regression relationship was found between the content of VEGF and NF-kB in neutrophils. We have found increased IL-1β and IL-6 levels in neutrophils of the uterine myoma patients, and at different stages of endometrial cancer compared with control. The IL-1β level was higher in neutrophils of the patients with intermediate and high tumor grade, compared to low-grade cases. IL-17A expression in the neutrophil lysate was significantly reduced in uterine myoma and at different stages of endometrial cancer, as compared with controls. There was a moderate inverse correlation between the contents of VEGF and IL-6 in neutrophils (r = -0.426, p = 0.001); a remarkable inverse relationship between VEGF and IL-17A (r = -0.615, p < 0.001). The combination of IL-6 and IL-17A levels in the neutrophils lysate (according to the results of multivariate analysis) may be used for the differential diagnosis of endometrial cancer and uterine myoma. Thus, the differences in the expression of inflammatory and angiogenic cytokines included in NF-kB-dependent signaling, may point to acquisition of pro-tumor functions by neutrophils during the endometrial cancer progression

IL-4 and its polymorphism (<i>IL4</i>-589C/T) in cervical neoplasia

The transition of cervical neoplasia (CIN) to cervical cancer occurs with the active participation of IL-4, for which both pro- and antitumor effects have been shown with tumors of various localizations. The expression of cytokines is regulated at the transcriptional level in the promoter region of the gene. It has been shown that the genotype IL4 (589C/T) (rs2243250) is associated with the development of gastric and breast cancer. The contribution of IL-4 genotypic variations to the development of CIN has not yet been studied. The aim of the study was to assess the risk of developing cervical neoplasia by the presence polymorphism of IL4 (589C/T) and the level of IL-4. The object of the study was circulating neutrophils, serum and genomic DNA of 36 patients with CIN and 20 women without dysplasia (comparison group). Using ELISA, the level of IL-4 was determined in neutrophil lysate and serum. Phagocytic activity and adhesive ability (CD11b) of neutrophils were assessed. Allele-specific real-time PCR using Taq-Man probes was used to analyze of the IL4 589C/T (rs2243250). Statistical processing was carried out using Statistica 13 and Jamovi 1.6.5.0. As a result of the study, it was found that the level of IL-4 in serum and circulating neutrophils in patients with CIN is significantly higher than in the comparison group. The -589C* allele of the IL4 gene and the TT genotype are more common in the group with CIN (55.5%) than in the control (25%). At the same time, a direct relationship was established between the presence of polymorphism and increased adhesive ability and with indicators of the phagocytic number of circulating neutrophils. Analysis of the incidence of IL4 C589T by the «case-control» method showed that the chances of CIN formation in carriers of the -589C allele and the TT genotype were 3.75 (95% CI: 1.013 - 13.880, Chi-square = 4.161, p = 0.042). The -589C* allele and TT IL4 genotype, neutrophil and serum IL-4 levels are associated with HPV infection. Using a binary logistic regression model, we demonstrated the possibility of using IL-4 levels in circulating neutrophils and IL-4 gene polymorphism (589C/T) for the differential diagnosis of patients with CIN (χ2 = 15.6, p = 0.001). Significant significance for their combination was assessed by ROC-curve analysis (IL-4 in neutrophils; IL4 (-589С*), 75% probability. Thus, the IL4 (589C/T) is associated with the adhesive and phagocytic activity of circulating neutrophils. In HPV-infected patients, IL4 gene polymorphism (589C/T) can serve as a marker for early detection and prognosis of CIN

Influence of proinflammatory cytokines on membrane rigidity and morphofunctional state of circulating neutrophils in ovarian tumors

Priming and activation mediated by cytokines cause transient reactions of actin polymerization in neutrophils (Nph), expansion and softening of cells, changes in receptor status, phagocytic ability, and generation of extracellular traps (NET), thus ultimately determining pro- or antitumor phenotype of Nph. To assess the effects of pro-inflammatory cytokines on membrane rigidity and morphofunctional state of neutrophils in benign tumors and ovarian cancer, the levels of circulating cytokines (IL-2, IL-18, MCP-1, TIMP-1), expression of adhesion markers (CD11b), degranulation (CD63), as well as FcãRIIIb receptors promoting phagocytosis (CD16). Ability of neutrophils to phagocytosis and the formation of NET was determined. Statistical evaluation of the data obtained was carried out using Statistica 13.0, Jamovi 1.6.5.0 software. An increase in membrane rigidity was found in benign and malignant ovarian tumors. In benign ovarian tumors, phagocytic activity and expression of CD11b were enhanced. In ovarian cancer, the number of CD11b+Nph and CD63+Nph were increased. Meaanwhile, at initial cancer stage, the ability to form NET predominates, and phagocytic activity increases with advancing ovarian cancer. Serum MCP-1 levels are elevated in benign tumors and at all stages of ovarian cancer. IL-2 is elevated at early stage and in advanced ovarian cancer. The level of IL-18 and TIMP-1 in the serum of patients with benign ovarian tumor did not differ significantly from the norm. Using multiple regression approach, the dependence of neutrophil membrane rigidity on the levels of circulating IL-2, TIMP-1, MCP-1, IL-18 was revealed in benign ovarian tumors, and a direct correlation was found between the neutrophil membrane rigidity and CD11b expression. Only IL-2 was associated with neutrophil membrane rigidity in ovarian cancer. At the same time, the rigidity of the neutrophil membrane directly correlated with CD16, CD63, expression like as with phagocytic index and inversely correlates with the number of traps. A combination of IL-2, MCP-1 and membrane rigidity of circulating neutrophils (based on multivariate analysis) could be used for differential diagnosis of ovarian cancer. Thus, in a benign ovarian tumor, circulating proinflammatory cytokines are associated with increased rigidity of neutrophil membrane and increase in their adhesion capacity. In ovarian cancer, only IL-2 is associated with altered rigidity of circulating neutrophils. Increase of the latter index is accompanied by elevated phagocytic activity and decreased ability to form NET

Phenotype of circulating neutrophils at different stages of cervical neoplasia

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

Параметры глутатионовой системы и тиоредоксина в плазме крови и асците и полиморфизм гена GSTP1 Ile105Val как факторы резистентности к платиносодержащей химиотерапии у больных раком яичников

Background. Chemotherapy is one of the main types of treatment in ovarian cancer. Standard first-line treatment includes platinum drugs. Every fifth patient develops chemoresistance after platinum-containing first line therapy. Glutathione detoxification systems play an important role in platinum drugs utilization.Purpose. To assess the redox status of blood plasma and ascitic fluid in ovarian cancer patients before and after neoadjuvant platinum-containing chemotherapy (NACT).Materials and methods. We determined the activity of the glutathione system and thioredoxin levels in bloodplasma before and after NACT and in the ascitic fluid before NACT, and the presence of GSTP1 gene polymorphism (Ile105Val (rs1695), Ala114Val (rs1138272) in 30 III–IV FIGO stage ovarian cancer patients. Patients were divided into 3 groups: NR – no relapse in 2 years after last chemotherapy course; R1 – relapse in less than 6 months; R2 – relapse in more than 6 months.Results. We established an increase of the glutathione-transferase activity and a decrease of the GSH level inplasma after chemotherapy in R1 patients, and an opposite dynamic of glutathione-transferase and GSH in the R2 group. Thioredoxin level in plasma of all patients was lower than in the control group; differences in levels between groups were not statistically significant. GSTP1 105Val allele was more frequently present in patients than in the control group, and more frequently in R2 than in R1.Conclusion. The increase in plasma glutathione-transferase and glutathione-reductase levels can be a prognostic marker of early relapse. Thioredoxine dynamics do not correlate with the chemotherapy response. The presence of the GSTP1 105Val allele is a risk factor for ovarian cancer development, but a protective factor against early relapse.Введение. Химиотерапия является одним из основных видов лечения распространенного рака яичников (РЯ). У каждой пятой пациентки развивается химиорезистентность после платиносодержащей терапии первой линии. Система детоксикации глутатиона играет важную роль в утилизации платиновых препаратов из  опухолевых клеток. Цель. Оценить окислительно-восстановительный статус плазмы крови и асцитической  жидкости у больных РЯ до и после неоадъювантной платиносодержащей химиотерапии  (НАХТ).Материалы и методы. Мы определили активность глутатионовой системы и уровень тиоредоксина в плазме крови до и после НАХТ и в асцитической жидкости до НАХТ у 30 пациентов на III–IV стадиях (по FIGO) рака яичников. Пациенты были разделены на три группы: БР – без рецидивов в течение 2 лет после завершения химиотерапии; Р1 – рецидив заболевания в течение 6 мес после завершения химиотерапии первой линии; Р2 – рецидив после 6 мес от момента завершения химиотерапии первой линии.Результаты. Установлено увеличение активности GT и снижение уровня GSH в плазме  после химиотерапии у пациентов с Р1, а также противоположная динамика GT и GSH в  группе Р2. Уровень тиоредоксина в плазме у всех пациентов был ниже, чем в контрольной группе; различия в уровнях между группами не были статистически значимыми. Аллельный вариант 105Val гена GSTP1 выявлялся с более высокой частотой у пациентов с РЯ, чем в контроле, и чаще в группе Р2, чем у Р1.Заключение. Повышение активности GST и GR в плазме больных РЯ может быть  прогностическим маркером раннего рецидива. Динамика тиоредоксина не коррелирует с  ответом на химиотерапию. Присутствие аллеля 105Val в гене GSTP1 является фактором риска развития рака яичников, но защитным фактором против раннего рецидива

ВЗАИМОСВЯЗЬ УРОВНЕЙ ВОСПАЛИТЕЛЬНЫХ ЦИТОКИНОВ КРОВИ И ЧИСЛА ЦИРКУЛИРУЮщИХ ОПУХОЛЕВЫХ КЛЕТОК С ОТВЕТОМ НА СТАНДАРТНУЮ ХИМИОТЕРАПИЮ У БОЛЬНЫХ РАКОМ ЯИЧНИКОВ

Introduction. Serum chemokines are inflammatory mediators, which role is shown in the occurrence and progression of a number of malignant tumors. Produced by white blood cells, stem cells, tumor and endothelial cells, chemokines control their movement and positioning. Chronic inflammation underlies the progression of ovarian cancer (OC ). This increases the likelihood of chemokines stimulating or blocking tumor progression.The aim of the study was to evaluate the relationship between the blood levels of inflammatory cytokines in blood and the number of circulating tumor cells (CTC s) with the response to standard chemotherapy (CT ) in patients with cancer.Material and Methods. In patients with primary OC before and after 2–4 courses of chemotherapy and in patients with benign ovarian tumors (as a control), serum levels of CCL 2, CCL 3, CCL 4, CXCL 8 and CX3CL 1 were evaluated by multiplex xMAP analysis. The amount of CTC s (population CD 45-/ Epcam+/CK+) was determined using a flow cytometer. Patients with ovarian cancer were divided into 3 groups according to the platinum sensitivity criterion of GC JG 4th, and progression-free interval (PFI) was determined. Results. It was found that the levels of CCL 2, CCL 3, CCL 4, CXCL 8, and CX3CL 1 in case of OC did not significantly differ from that in the control, strongly negatively correlated with age (except for the CCL 2 level). CT significantly increased the level of CCL 2 in the group of refractory OC ; of CCL 3 – in the group of sensitive OC , of CCL 4 – in the groups of resistant and sensitive OC , and C XCL 8 level increased in the groups with resistant and sensitive OC and decreased in the group of refractory OC . The number of CTC s in patients with OC was significantly higher than in the control. After CT , a decrease in the amount of CTC s strongly and significantly correlated with a decrease in the level of CX3CL 1 in the groups of refractory andsensitive OC . The maximum PFI occurred with an increase in serum levels of CCL 3, CXCL 8, a decrease in CCL 4 and a constant level of CX3CL 1.Conclusion. Thus, no significant differences in the levels of CCL 2, CCL 3, CCL 4, and IL -8 between patients with OC and control groups were found. The levels of chemokines studied and the amount of CTC s differed in the groups divided by the tumor sensitivity to CT . We observed significant correlations between the amount of CTC s and the level of CX3CL 1 in the group of platinumsensitive OC .Введение. Сывороточные хемокины – медиаторы воспаления, роль которых доказана в возникновении и прогрессировании злокачественных опухолей ряда локализаций.  Продуцируясь лейкоцитами, стволовыми, опухолевыми и эндотелиальными клетками, хемокины контролируют их движение и позиционирование. Хроническое воспаление лежит в основе прогрессирования рака яичников (РЯ), и это повышает вероятность стимулирования или блокирования хемокинами прогрессирования опухоли. Целью исследования было изучение взаимосвязи уровней воспалительных цитокинов крови и числа циркулирующих опухолевых клеток (ЦОК) с ответом на стандартную химиотерапию у больных РЯ. Материал и методы. У пациенток с первичным РЯ до и после 2–4 курсов химиотерапии (ХТ) и пациенток с доброкачественными опухолями яичников (контроль) в сыворотке крови уровни ccl2, ccl3, ccl4, cXcl8 и cX3cl1 оценивали методом мультиплексного анализа xmap. Количество ЦОК (популяция cd45-/ epcam+/cK+) определяли на проточном цитометре. Пациентки с РЯ были разделены на 3 группы по критерию  платиночувствительности согласно gcJg 4th, было определено время до прогрессирования (pFi). Результаты. Установлено, что уровень ccl2, ccl3, ccl4, cXcl8 и cX3cl1 при РЯ значимо не отличался от такового в контроле, отрицательно коррелировал с возрастом (за  исключением уровня ccl2). Химиотерапия значимо повышала уровень ccl2 в группе  рефрактерного РЯ; ccl3 – в группе чувствительного РЯ, ccl4 – в группах резистентного и чувствительного РЯ, cXcl8 – повышала в группах с резистентным и чувствительным РЯ и снижала в группе рефрактерного РЯ. Количество ЦОК у больных с РЯ значимо выше, чем в контроле. После ХТ снижение количества ЦОК достоверно коррелировало со снижением уровня cX3cl1 в группах рефрактерного и чувствительного РЯ. Максимальная продолжительность периода без прогрессирования отмечена при повышении уровней ccl3, cXcl8, снижении уровня ccl4 и неизменном уровне cX3cl1. Выводы. Уровни ccl2, ccl3, ccl4 и il-8 значимо не различаются у больных РЯ и группы контроля. Уровни изученных хемокинов и количество ЦОК изменялись в группах с  различной чувствительностью опухоли к ХТ. При платиночувствительном РЯ наблюдаются значимые корреляции количества ЦОК и уровня cX3cl1 в сыворотке крови до лечения

DIFFERENTIATION- AND ACTIVATION-ASSOCIATED MARKERS OF PERIPHERAL BLOOD LYMPHOCYTES IN THE PATIENTS WITH OVARIAN CANCER IN THE COURSE OF TUMOR PROGRESSION

Expression features of markers associated with differentiation (CD3, CD4, CD8, CD16) and activation (CD25, CD71, CD95 and HLA-DR) were studied on lymphocytes from patients of reproductive and postmenopausal age with ovarian cancer (FIGO clinical stages I to IV). Absolute lymphopenia, which was more pronounced in postmenopause, was found to be increased in the course of tumor progression. Moreover, the numbers of CD8+ cells significantly increased since early stages of disease, along with decreased amounts of CD3+ andCD4+ cells. CD16 expression was elevated at later stages, being sufficiently higher in postmenopausal patients. CD25 cell scores was increased at all clinical stages, and did not significantly differ in reproductive vs menopausal age. CD95+ cell number was increased, and it proved to be maximal at stage III-IV of the disease. The numbers of CD71+ cells was increased at all clinical stages of disease, being significantly higher in reproductive period, as compared with menopause. Expression of HLA-DR molecules on lymphocytes was enhanced at early stages of disease followed by significant decrease at stage IV of disease. Hence, phenotypic features of lymphocytes from the patients with ovarian cancer exhibit dynamic variability at different stages of tumor growth, being also dependent on reproductive vs. postmenopausal period

Comparative characterization of oxidative stress in cervical cancer at different clinical stages of disease

Parameters of oxidative stress on the level of malondialdehyde (MDA), activity of glutathione-reductase, glutathione-transferase, superoxide- dismutase (SOD) and catalase in tumor tissue, plasma and red blood cells of patients with cervical cancer (CC) w as evaluated a t various clinical stages of the disease. It is revealed that an increase in tumor tissue MD A and glutathione enzymes while reducing SOD and catalase, which may indicate the development of oxidative stress in the dynamics of tumor progression. The data obtained suggest the increase of expres- sion of oxidative stress in red blood cells and tumor tissue in CC in the dynamics of tumor progression

A18

Tumor-associated neutrophils (TANs) are the cell population that differs in morphofunctional characteristics from peripheral blood cells (Gregory and Houghton, 2011). The first study to identify the presence of TANs as an independent poor prognostic factor and to include TANs into a prognostic risk model was published in 2006 (Donskov , 2006). Specific signals during cancer progression have been shown to induce the emergence of a pro-tumor phenotype of neutrophils (PMN). Frinlender (2013) points out an anti-tumor phenotype of TANs. Of particular interest is the study of interaction of neutrophils with T cells because the latter are considered to be cytotoxic cells mediating antitumor immunity. The aim of the study was to assess the lymphoid cell infiltration (LCI) and the functional status of TANs in ovarian cancer. LCI was assessed by immunohistochemistry and the levels of mieloproxydase (MPO) and cationic proteins (CP) were evaluated by cytochemical methods in ovarian carcinoma surgical resection specimens. The results were presented as a mean cytochemical coefficient (MCC). The intensity of nitroblue tetrazolium (NBT) test was expressed as a percentage. Obtained results were analyzed by nonparametric statistical methods. The Kruskal–Wallis test was used to evaluate the differences between groups. We established that low intensity of infiltration in general and formation of marginal lymphoid cell ridge in some tumors are typical for malignant ovarian neoplasms. More intensive infiltration was found in the regions of the rapid growth of tumor cells, i.e. invasion zone. In some cases, a group of tumor cells was separated from the main part of the parenchyma and surrounded by lymphoid elements. Conditions are created under which active spread of cancer cells in the parenchyma is associated with intensive lymphoid cells infiltration in the stroma. Total count and density of lymphoid cells can significantly vary: from single lymphoid elements uniformly scattered in the tumor tissue to focal accumulations. In tumor tissue location of lymphoid cells is irregular, so most of the infiltration is in the stroma and among the cancer cells only solitary lymphocytes are found. Lymphoid cells which make up the infiltration include 12.3 ± 1.52% of macrophages, 22.1 ± 2.3% of plasma cells, 62.5 ± 1.1% of lymphocytes and 3.1 ± 0.9% of PMN. Total amount of cells was 224.1 ± 32.8 per 1 mm2 of tumor. Lymphocytes in ovarian carcinomas do not form large clusters; they are disposed among other lymphoid cells in the stroma on the periphery of the tumor. Plasma cells have a classical morphological structure and often locate in the stroma and on the periphery of the tumor. Macrophages are an integral part of the tumor infiltrate. In the tumor they become stretched and angular. Macrophages usually have large dimensions, and they are located in the stroma as well as in the parenchyma among the cancer cells. Segmented leukocytes (presented in the vast majority by neutrophils) are constantly found in the infiltrate of the stroma and the parenchyma, most often they are found near or among massive necrosis. Analysis of TANs functional activity in comparison with the peripheral blood neutrophils has shown that MPO activity elevated and amounted 2.21 ± 0.43 MCC against 1.40 ± 0.07 MCC in the peripheral blood neutrophils, the level of CP also increased and amounted 1.51 ± 0.39 MCC against 0.83 ± 0.43 MCC. TANs activity in NBT-test was not significantly different from that of the peripheral blood neutrophils. TANs compose the minor part of primary ovarian tumor infiltrate. Indicators of aerobic and anaerobic bactericidal activity of TANs significantly exceed that of the peripheral blood neutrophils in patients with ovarian cancer. This work was conducted in the framework of implementing the state assignment of the Ministry of Education and Science of the Russian Federation No. 1163 – Russia