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e22516
Background: Soft tissue sarcomas (STS) are aggressive tumors with a high degree of recurrence. Radical resection within healthy tissues allows to reduce the recurrence percentage ...to 25-30% without subsequent therapy. The literary analysis has shown that the study of various biological properties of primary and recurrentsoft tissue tumorsis being conducted. However, currently there is a lack of information to understand the reasons for STS recurrence. The goal of investigation was to reveal the distinctive features of the DNA content and cell distribution in the phases of the cell cycle of recurrent STS. Methods: DNA cytometry in the tumor tissue of 30 primary soft tissue sarcomas and 30 STS recurrences was carried out using the method of flow cytofluorometry. The tumor ploidy and cell distribution in the cell cycle phases were analyzed. Results: A number of differences in the DNA cytometric parameters of primary and recurrent STS have been revealed, they include: an increase in the proportion of aneuploid tumors in case of recurrence, the number of tumors with DNA index within the mitotic cycle, an increase in the proportion of cells in G2+M- phase of diploid and aneuploid tumors and a decrease in S- phase of aneuploid ones. It has been shown that with a G2 differentiation degree, the proportion of cells in G2+M, S- and proliferation index of recurrent STS is significantly lower than the primary parameters. An increase in the proportion of cells in G2+M- phase and a decrease in the rate of proliferation of recurrent STS, depending on the stage, are shown only in case of stage III. Conclusions: The revealed features of DNA content and cell cycle of tumor cells of soft tissue sarcomas will allow to approach to understanding of biological bases of recurrence of this malignant disease.
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e23513
Background: The purpose of the study was to reveal the levels of biochemical parameters for assessing the intensity of bone metabolism in patients with primary and metastatic ...bone tumors. Methods: The study included 29 patients aged 55.9±12.17 years with primary (group 1, n = 12) and metastatic (group 2, n = 17) bone tumors. Group 2 included patients with breast cancer (2a, n = 10) and renal cancer (2b, n = 7) with a history of pathological fractures. Serum levels of the bone turnover marker osteocalcin, TSH, the resorption marker β-Cross Laps (C-terminal telopeptide) (Cobas e411, Japan) and calcium (Vitros 5600, USA) were measured before and after (day 14) organ-preserving treatment. The data were compared with that in non-cancer patients of similar age (n = 15) and evaluated in the Statistika 10.0 program. Results: Osteocalcin levels in group 1 prior to surgery was 1.74 times (p < 0.05) higher than in controls (13.56±1.35 ng/mL) and decreased by 35% (p < 0.05) compared to the initial values. In group 2, changes in osteocalcin levels were multidirectional: group 2a – 24% (p < 0.05) higher than in controls both before and after surgery; group 2b – similar to control values before surgery and decreased by 19% (p < 0.05) after it. Levels of β-Cross Laps in all patients before surgery were within the control range (0.49±0.02 ng/mL). After surgery, the levels in groups 1 and 2a did not change, while group 2b showed an increase in β-Cross Laps by 1.5 times (p < 0.05) and a decrease in calcium by 20% (p < 0.05) from the initial levels (2.33±0.09 mmol/L), which, along with a decrease in osteocalcin, could be the result of specific kidney damage. TSH in groups 1 and 2b was similar to controls, but initial TSH levels in group 2a were increased by 86% (p < 0.05) and did not change after surgery, as well as osteocalcin levels, which in the absence of changes in β-Cross Laps suggested a more favorable prognosis after organ-preserving treatment. Conclusions: Bone tissue remodeling processes are determined by the nature of the initial tumor. Measuring osteocalcin and β-Cross Laps in patients with specific bone lesions may be useful in assessing the risk of fractures.
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e23545
Background: The tumor ability to induce and maintain angiogenesis is one of the main stages of its development. Studies of molecular mechanisms of angiogenesis showed that the ...dynamic balance that ensures the formation and development of new vessels inside the tumor depends on pro- and anti-angiogenic factors. VEGF and the CD34 endothelial cell marker are considered among the main activators of angiogenesis. Our purpose was to study characteristics of angiogenesis in primary and recurrent soft tissue sarcomas. Methods: The study included 30 patients with primary sarcomas (group 1) and 26 patients with recurrent sarcomas (group 2). Sections of tissues embedded in paraffin blocks were studied by immunohistochemistry using the Thermo Scientific 480S automated stainer. The Reveal Polyvalent HRP-DAB Detection System was used for the visualization. The density of blood vessels stained with antibodies against VEGF and CD34 was determined. The statistical analysis of results was performed in the STATISTICA 10.0 program (StatSoftInc., USA). Results: The numbers of blood vessels ranged: CD34 in group 1 – 7-16 blood vessels in one field of view; in group 2 – 2-18 vessels. VEGF in group 1 – 2-20 vessels, with up to 40 vessels in 3 cases (11.5%) only; in group 2 – 5-11 vessels, with up to 20 vessels in 2 cases (7.7%). The average numbers of microcirculatory vessels stained with antibodies against VEGF and CD34 were: CD34 –12.2±1.04 and 8.4±1.4 (p = 0.0247) in groups 1 and 2, respectively; VEGF – 12.8±4.06 and 8.4±11.6 (p = 0.3074) in groups 1 and 2, respectively. Conclusions: The IHC analysis showed the minimal numbers of microcirculatory vessels in one field of view in patients with recurrent soft tissue sarcomas. The value was 1.5 times lower for both CD34 and VEGF, compared to patients with primary tumors. However, only CD34 value was statistically significant (the Mann–Whitney U-test). Probably, a certain decrease in angiogenesis factors in recurrent tumors can be explained by adjuvant chemotherapy suppressing tumor growth.
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e12550
Background: BC is still one of the main causes of death in women due to the tumor recurrence and/or resistance to anticancer therapy. The criteria to assess the effectiveness of ...BC treatment are important. The purpose of the study was to analyze blood levels of steroid and pituitary hormones in BC patients after two chemotherapy cycles. Methods: The study included 42 patients with various biological BC subtypes: luminal A, luminal B and triple-negative BC (TNBC). Levels of estradiol, testosterone, progesterone, prolactin, LH, FSH and cortisol were measured by RIA in the blood of all patients before and after two neoadjuvant chemotherapy cycles. Significance of differences was evaluated by the Student’s t-test. Results: Levels of many hormones were high before the treatment in patients with all BC subtypes. After two chemotherapy cycles, unidirectional changes in the values were found in patients with subsequent remission for more than three years. Levels of estradiol decreased in luminal A BC by 1.7 times (p˂0.05), in luminal B BC – by 11.6 times (p˂0.05), cortisol decreased by 2.4 and 1.7 times (p˂0.05) respectively, and prolactin – on average by three times (p˂0.05). LH levels increased in luminal A and luminal B BC by 1.65 times (p˂0.05). In patients with TNBC, levels of estradiol decreased by 1.8 times, and cortisol – by two times (p˂0.05). Patients with subsequent remission regardless of BC subtypes had unchanged levels of testosterone, progesterone and FSH. Patients with luminal B and TNBC subtypes with progression in 6-12 months did not show significant changes in prolactin and cortisol levels after two chemotherapy cycles, compared with the values before treatment. Conclusions: A decrease in blood levels of prolactin and cortisol after two chemotherapy cycles is an indicator of a long-term remission in patients with breast cancer.
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e15201
Background: A review of scientific literature has shown that IL-2 is most often used for the LAK generation, while the potential of other NK-stimulating interleukin cells remains ...poorly understood. In this study, we investigated the effect of IL-7 and IL-15 on ex vivo LAC generation. Methods: A fraction enriched in NK cells was isolated by magnetic cell sorting with the NK Cell Isolation Kit (#130-092-657, Miltenyi Biotec, Germany) from PBMC in 11 patients with stage II-III breast cancer without treatment. Cells were introduced into a 6-well 3x10
5
plate in RPMI medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA). Cytokines 40 ng/ml were added to the wells in 6 variants: 1) IL-15; 2) IL-2; 3) IL-7; 4) IL-15+IL-7; 5) IL-15+IL-7+IL-2; 6) control without cytokines. Cells were cultured at 5.0% CO
2
and 37°C. Cells were counted with a hemocytometer daily for 5 days and on days 8, 9 and 10 of cultivation. Results: The number of NK cells in control samples gradually decreased: by 2 times on day 5 and by 3 times on day 10. On day 5, the number of NK cells was 1.5 times higher than in the control when cultured with IL-2, and 1.4 times higher when cultured with IL-7+IL-15. After 9 days, a statistically significant increase in the number of cells, compared to the control sample, was observed with the addition of IL-2 (1.6 times); IL-15 and IL-7+IL-15 (1.5 times). On day 10, significant differences from the control were found in most samples: the number of cells was higher in samples cultured with IL-2 and IL-7+IL-15 (1.9 times) and with IL-15 and IL-2+IL-7+IL-15 (1.7 times). IL-7 alone led to a gradual decrease in the number of cells, and on days 8, 9 and 10 it was lower than in the control samples. Conclusions: In general, the introduction of cytokines into the samples enriched with NK cells contributed to the preservation of this subpopulation on days 5-10 of cultivation. However, the use of IL-7 and IL-15, both alone and in combination, did not lead to a significant increase in LAK compared to the use of IL-2.
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e15220
Background: The purpose of the study was to evaluate the effect of IL-2 and INF-γ on the proliferation and phenotype of lymphocytes in patients with breast cancer (BC) after ...T-regs removal from the pool in vitro. Methods: Lymphocytes were isolated from the blood of 11 patients with stage II BC, T-regs were removed by immunomagnetic separation, and samples were cultured for 6 days with cytokines: IL-2 - 0.1/1 μg, INF-γ - 10 IU and their combinations. Results: The level of NK cells in the samples with IL-2 was maintained throughout the experiment, with IFN-γ - gradually decreased, and in the control (samples without stimulation) it decreased sharply. In experimental samples (IL-2, IL-2+IFN-γ), the amount of NKT remained at the initial level by day 6, and in the control it decreased by 2 times (p < 0.05). The number of CD335+ NK cells with high cytotoxicity against target cells increased after 3 days in the test samples with IL-2 by 5.2 times, and after 6 days - by 11.6 times in comparison with the control (p < 0.05). On day 6, the level of double-positive T cells increased by 1.8 times in co-culturing with IL-2+INF-γ (1 μg/10 IU), (p < 0.05). The percentage of double-negative T cells increased in the dynamics of cultivation from days 1 to 6, in some cases statistically significantly (IL-2 - 1 μg: by 2 times; IL-2+INF-γ - 0.1 μg/10 IU: by 1.8 times; p < 0.05). The percentage of CD4+CD38+ cells on day 6 of incubation with IL-2 at both doses increased by 1.8-2 times compared with the control, and CD8+CD38+ by 4-6 times. The percentage of CD4+HLA-DR+ cells after 6 days of cultivation with IL-2 increased by 6 times, and CD8+HLA-DR+ by 8-9 times. Cultivation with INF-γ reduced the stimulating effect, and in some cases it did not appear. The combined effect of IL-2 and INF-γ on day 6 led to an increase in the proportion of CD4+CD25+CD127dim cells by 1.8 times in comparison with the control (p < 0.05), and when exposed to INF-γ only, the number of these cells remained at the control levels. Conclusions: INF-γ is not an effective inducer of activation of immune effector cells, while the effect of IL-2, both alone and in combination with INF-γ, leads to a significant increase in the proportion of cytotoxic cells, as well as to activation of the T cell unit, including CD4 + CD25 + CD127dim.
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12005
Background: Numerous pharmacogenetic studies have led to the identification of genetic polymorphisms associated not only with the development of cardiovascular disease, but also ...increase the risk of complications due to the use of anthracycline drugs, widely used in the treatment of cancer. The purpose of this study was to study the frequency of rs4673 and rs28714259 and possible associations with the risk of cardiovascular changes in patients with breast cancer during anthracycline therapy (anthracycline-mediated cardiotoxicity — AMC). Methods: The study included 256 Caucasian patients (median age - 55 years) with a diagnosis of breast cancer without diagnosed cardiovascular changes, who were treated with anthracyclines at the National Medical Research Center of Oncology in 2019-2020. For genotyping of rs4673 and rs28714259, DNA was extracted from blood using DNA-sorb-B (AmpliSens, Russia) and HRM-PCR was performed on a CFX96 amplifier (Bio-Rad, USA). The presence of polymorphisms was confirmed by Sanger sequencing on a Genetic Analyzer 3500 (ABI, USA). Results: During the follow-up period 21 (8.2%) patients were diagnosed with signs of subacute (changes developed within several weeks after the last course of therapy) or early chronic AMC (changes developed within a year after completion of anthracycline therapy). In the group of patients without AOC the allelic frequency of rs4673 (c.214T > C CYBA) was 0.38, the frequency of genotypes C/C – 0.4, C/T – 0.43, and T/T – 0.17. In the same group, the frequency of the A allele rs28714259 was 0.07, the frequency of the G/G genotypes – 0.87, G/A – 0.13, and A/A – 0. The prevalence of genotypes T/T rs4673 and allele G rs28714259 in a cohort of Russian patients differed from the European population (p = 0.014 and p = 0.05, respectively). The risk of cardiovascular changes on the background of anthracycline therapy increased in the presence of the rs4673 polymorphic allele by 6.49 times, in the case of the G/A and A/A rs28714259 genotypes - by 3.27 times. The results of the ROC-analysis suggested high quality of the tests based on the dominant models rs4673 and rs28714259 (AUC was 71.9% and 76.3% correspondingly). Conclusions: In this study the prognostic efficiency of the genetic markers rs4673 and rs28714259 was shown for the prompt detection of the risks of AMC development in the management of cancer patients. However, population characteristics should be taken into consideration for risk assessment.
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e13060
Background: Monotherapy with low doses of cytokines does not provide significant therapeutic results, while treatment with high doses leads to a number of side effects, for ...example, cytokine release syndrome, etc. Therefore, it is necessary to study the effect of cytokines on immune cells which are involved in the regulation of pro- and antitumor immune response. Thus, the aim of this study was to analyze the dynamics in the proliferation of the total fraction of lymphocytes after expansion and exposure to γc-cytokines: IL-2, IL-7 and IL-15 and their combinations in vitro in patients with diagnosed breast cancer. Methods: The study included 10 patients with locally advanced stage I-III breast cancer. Peripheral blood mononuclear cells (PBMC, ficoll-hypaque) were used as material. After density separation the cells were cultured at a dose of 500 thousand cells / ml in 6-well plates (Biofil, China) in RPMI 1640 (Gibco, USA) supplemented with 10% FBS (HyClone, USA) at 37° C, 5% CO2. Expansion of lymphocytes was performed by kit with anti-biotin magnetic particles MACSiBead to CD2, CD3 and CD28 (Miltenyi Biotec, Germany), according to the manufacturer's protocol. Stimulation of lymphocyte proliferation was performed on days 4, 8 and 12 by introducing recombinant cytokines - IL-2, IL-7, IL-15 (Miltenyi Biotec, Germany) and their combinations. Cytokines were introduced at a concentration of 40 ng / ml each in the following variants: IL-2; IL-2 / IL-15; IL-2 / IL-15 / IL-7; IL-15; IL-15 / IL-7. Plates with cell suspension were cultured at 37 ° C, 5% CO2 for 15 days. Results: The data obtained on the 11th day of incubation demonstrated statistically significant differences in cell viability in samples with the addition of interleukin IL-7 / IL-15 combination (778%) compared to control samples by 1.5 times (p < 0.05). The proportion of lymphocytes in samples with the addition of only IL-15 (702%) and IL-7 / IL-15 (756%) combination was 1.47 and 1.6 times higher, respectively, compared with the control (p < 0.05) at the 12th day of co-cultivation. Conclusions: Stimulation of lymphocyte proliferation by the IL-7 / IL-15 combination results in a high frequency of viable cell production. Despite the only culture stage, the results of the study may be important for optimizing the use of γc-cytokines in the treatment of patients with breast cancer.
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e22517
Background: Soft tissue sarcomas (STS) are relatively rare malignant tumors of mesenchymal origin, constituting about 1% of all solid human tumors. About 11% of STS cannot be ...classified. The relapses of STS are difficult to predict. So, the studying of molecular origin of STS remains to be an actual issue. We analyzed copy number variations (CNVs) of apoptosis-regulating genes and MKI67 gene in primary and recurrent STS. Methods: 32 patients with primary STS and 26 patients with relapses were investigated. Relative CNVs of bax, bcl2, casp3, casp8, casp9, p53, mdm2 and M kI67 were detected with the use of RT–qPCR in fresh frozen tumor and normal tissues obtained while surgical access. Relative Copy Quantitation (RCQ) was accounted with GAPDH and ACTB as reference genes. Results: The frequencies of CNVs of the studied genes in groups of primary and recurrent STS are presented in the table below. A significant change only in the group of primary STS sarcomas was observed for CNV of MDM2, whereas the amplification of the CASP3 and MKI67 genes was characteristic of recurrent sarcomas. CNVs of BAX and CASP8 were significantly more frequent in primary STS. Increased amplification of MDM2 gene, was observed only for liposarcomas among all the others STS. The literature describes amplification of chromosome 12q13-15 where MDM2 gene is localized as a diagnostic feature of liposarcoma which is consistent with our observations Todorov SS, Kit OI Modern understanding of the morphogenetic features of liposarcomas // Archives of Pathology. 2012. T. 74. №6. p.59-61. Conclusions: The study showed the potential role of CNV of the BAX, CASP3, CASP8 and MKI67 genes to develop an approach to predicting the course of STS and the effectiveness of the MDM2 gene amplification as an additional marker for liposarcoma molecular classification.Table: see text
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e22504
Background: Positive effect of the monoclonal antibody denosumab in cancer treatment has been reported in recent years. Since surgical treatment is considered the main method of ...treatment for giant-cell tumors of the bone (GCTB), the purpose of our study was to improve treatment outcomes using denosumab. Methods: The study included 10 patients with verified GCTB (5 men, 5 women, mean age 36±3.14 years). Tumors were located in the upper third of the tibia (3 patients), the lower third of the tibia (3), iliac bone (1), heel bone - 1, the lower third of the femur - 1, the lower third of the humerus - 1.Patients received 2 cycles of neoadjuvant denosumab treatment (120 mg subcutaneously once every 4 weeks) with further tumor resection. The bone defect was reconstructed with bone grafting in marginal resections and endoprosthetics in segmental resections. The effect of neoadjuvant therapy was assessed morphologically by optical light microscopy; histological and histochemical methods were used. Results: Neoadjuvant treatment reduced pain syndrome and restored the support ability. X-ray and spiral X-ray CT showed sclerotic sites in the lytic foci. Consolidation of pathological fractures in the tumor area was registered, as well as fibrosis of the tumor focus. The tissue on the sections of removed material appeared dryish and whitish, it replaced the medullary canal. Microscopic examination of tumors revealed areas of extensive sclerosis foci, and a slight lymphohistiocytic infiltration in some cases. Osteoclasts and osteoblasts were absent in the histological preparations. Conclusions: Neoadjuvant denosumab in GCTB with the following surgery has a pronounced positive effect. This was confirmed by the morphological picture of the operative material demonstrating the development of extensive foci of loose and dense fibrous connective tissue with a small amount of lymphohistiocytic elements.
