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e13088
Background: The development of breast cancer (BC) and ovarian cancer (OC), as well as their chemoresistance may be due to the presence of mutations in many genes, but most often ...in BRCA1/2 and CHEK2. Early diagnosis and subsequent preventive interventions improve the survival of patients with mutations in the BRCA1/2 genes. Genotyping of the gene inactivation events predicts a high sensitivity of BC and OC to platinum-containing cytostatics. Our purpose was to study the spectrum of germline mutations in the BRCA1/2 and CHEK2 genes in BC and/or OC patients in the South of Russia. Methods: The study included 622 patients of 10 nationalities with BC and/or OC from the South of Russia. Genomic DNA was isolated from peripheral blood leukocytes; mutations were detected by Real-Time PCR in the BRCA1 (185delAG, 300T > C, 2080delA, 4154delA, 5382insC, 3819delGTAAA, 3875delGTCT), BRCA2 (6174delT), CHEK2 (1100delC, IVS2+1G > A, 470T > C) genes. Results: Mutations in the BRCA1/2 gene were found in 13.5% of cases, in CHEK2 – 6.2%. In the BRCA1/2 gene, the frequency of 5382insC mutation was 76.2%; 4153 delA – 9.5%; 300T > G – 7.1%; 2080delA – 3.6%; 185delAG, 3875delGTCT, 6174delT - 1.2% each. In the CHEK2 gene: 470Т > C – 73.1%; IVS2+1G > A – 23.1%; 1100delC – 3.8%. Two patients showed a combination of 5382insC mutation in the BRCA1 gene and 470T > C mutation in the CHEK2 gene. The observed prevalence of mutations in BRCA1 was generally consistent with that for European countries (p = 0.062). Identification of 470T > C mutation in CHEK2 in two cases confirmed the final diagnosis of Li-Fraumeni syndrome (OMIM: 609265); however, we failed to establish a family history of cancer in one patient. A high-risk cancer group was formed for prophylactic purposes based on the data of genotyping for major mutations in the BRCA1/2 and CHEK2 genes in patients with BC/OC and their healthy relatives. Conclusions: The frequency of BRCA1/2 and CHEK2 mutations in the studied multinational population of Caucasian patients with BC and/or OC was 17.2%. The 5382insC BRCA1/2 (76.2%) and 470Т > C CHEK2 (73.1%) mutations were the most frequent which corresponded to the occurrence of these SNPs in populations of European countries.
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e13040
Background: The kallikrein-kinin system is a complex multifunctional signaling cascade. Studies confirm its role in the development of cancer, as well as a role of some of its ...proteins with pro-inflammatory properties. Criteria for assessing the risk of breast cancer (BC) progression are required. The purpose of the study was to reveal the effect of blood levels of plasminogen, plasmin, precallikrein, and kallikrein in patients with triple-negative BC on the disease course. Methods: The study included 162 patients with triple-negative BC divided into 2 groups: group 1 – 58 patients with an early progression within 6 to 12 months after combination treatment; group 2 – 104 patients without progression during 2 years after combination treatment. Blood levels of plasminogen, plasmin, prekallikrein, and kallikrein were measured by ELISA before anticancer therapy and after 8 chemotherapy cycles. The levels in the blood plasma of healthy donors were reference values. Results were statistically processed using the Statistika 6.0 program with the Student’s T-test. Results: In patients with an early progression, plasminogen levels before the treatment were decreased by 1.7 times (p≤0.05), while plasmin was elevated by 1.9 times (p≤0.05). Kallikrein was increased by 1.9 times (p≤0.05), and prekallikrein was similar to the values in healthy donors. In patients with prolonged remission, plasminogen levels before the treatment were decreased on average by 2 times (p≤0.05). Plasmin was elevated by 2.3 times (p≤0.05), and kallikrein – by 1.8 times (p≤0.05). During this period of the study, prekallikrein levels decreased by 2 times (p≤0.05), compared with healthy donors. After chemotherapy, prekallikrein levels in the group with an early progression decreased by 1.4 times (p≤0.05), compared to the values before treatment, and kallikrein increased on the average by 1.3 times (p≤0.05). In patients with prolonged remission, no changes in the studied indices were registered. Conclusions: Patients with an early progression of BC demonstrated decreased levels of prekallikrein and increased kallikrein after the treatment, which allowed predicting resistance to chemotherapy and further tumor progression.
e13042
Background: The purpose of this study was to analyze the prognostic value of blood levels of fibrinogen (F) in bone metastases from breast cancer (BC). Methods: The study included 160 patients ...with nodular BC pT2-3N0-1M0-1, including 48 patients in a prospective group (PG) and 112 patients in a retrospective group (RG). All patients underwent initial clinical and laboratory examinations, including determination of alkaline phosphatase (AP) activity (Cobas Integra 4000 plus, Switzerland) and parameters of coagulation hemostasis (STA Compact, France). Bone scintigraphy was performed for complaints of bone pain and/or in cases of elevated AP activity, regardless of SXCT data. Results were considered statistically significant at p < 0.05. Results: Patients in PG were divided into two subgroups depending on F levels: in 31 (64.6%) patients F = 3.67±0.5 g/L, insignificantly exceeding the norm (2.8±0.3 g/L); in 17 (35.4%) patients F = 6.9±0.3 g/L, being 1.9 and 2.5 times (p < 0.05) higher than in subgroup 1 and the norm, respectively. AP activity exceeded the norm by 26% and 40%, respectively (p < 0.1). Bone metastases were detected before treatment in 24 (21.4%) women of RG with pain syndrome; all these patients showed increased blood levels of F, on average up to 7.86±0.8 g/L, similarly to patients in subgroup 2 of PG. F levels in other 88 (78.6%) patients in RG were 3.4±0.6 g/L. The nature and severity of AP changes in RG patients and in both PG subgroups were similar. Initially elevated F levels in 17 PG patients remained unchanged after antitumor treatment (6.2±0.6 g/L). AP activity values were stable after treatment being only 36% and 28% (p < 0.1) higher than the norm, respectively. None of the patients with high F levels complained about bone pain. Unscheduled bone scintigraphy was appointed to 17 patients of PG without clinical symptoms or x-ray (SXCT) signs of pathology but with initially high F which did not decline after treatment. Bone scintigraphy showed metastases to the spine and pelvic bones in 12 of 17 (70.5%) patients 1 month after treatment, subsequently confirmed by MRI results. Conclusions: Initial levels of F within 6.9±0.6 g/L which do not decline after the special treatment allow predicting unfavorable BC course and the presence of metastases to the spine and/or pelvic bones without clinical signs of progression. The proposed method allows diagnosis of advanced tumor process in the absence of clinical signs of the disease with an accuracy of 87.8%, and allows timely corrections to the treatment plan.
e13072
Background: Breast cancer (BC) is the most common cancer in women worldwide. The purpose of the study was to analyze immediate and long-term results of treatment in patients with locally ...advanced primarily inoperable HER2-negative breast cancer depending on the time of progression. Methods: The study included 104 women with locally advanced primarily inoperable HER2-negative breast cancer who developed an early disease progression within 6 to 12 months after standard combination treatment. The blood levels of circulating tumor cells (CTCs) were measured in all patients during the progression stage. Results: An analysis of the overall (OS) and event-free (EFS) survival showed that median EFS in patients with luminal B subtype was 9 months, in patients with triple-negative (TNBC) subtype 8 months. EFS at 6 months was 87.5% in luminal B subtype and 79.4% in TNBC, p = 0.37985. Median OS in patients with luminal B subtype was 25 months, in patients with TNBC 26 months. One-year OS was 100% in luminal B subtype and 93.9% in TNBC, p = 0.138. 2-year OS was 54.2% in luminal B subtype and 55.9% in TNBC, p = 0.697. 3-year OS was 37.5% in luminal B subtype and 41.2% in TNBC, p = 0.639. CTCs were detected in all patients, with higher levels in patients with luminal B subtype than in TNBC patients. HER+ CTCs were found in 30% of patients with luminal B subtype but not registered in patients with TNBC. Such results were obtained for the first time. Conclusions: The absence of significant differences demonstrated the aggressive disease course in patients with luminal BC with identified primary hormone resistance, similar to the TNBC course. The results should be taken into account when predicting the course of the disease and developing further treatment tactics.
e17515
Background: The development of deep postovariectomy disorders in young women with cervical and breast cancers is of extreme concern. They are associated with the suppression of the ...hypothalamic-pituitary-gonadal axis of regulation, estrogen involution, a sharp depression of the psycho-emotional state, and social distancing. In fact, these postovariectomy syndrome (POES) events are associated with low-reactivity stress syndrome. Our purpose included the transition of dominant stress into archetypes of anti-stress adaptive reactions under the influence of programmable exponential dose regimens of xenon-oxygen therapy (XOT) in the early period after the removal of the female reproductive organs. Methods: 123 patients of reproductive age diagnosed with cervical cancer pT
1
B
2
N
0
M
0
and 24 patients with hormone-positive breast cancer pT
2
N
1
M
0
and concomitant gynecological pathology underwent hysterectomy with oophorectomy and developed POES. All patients received a cycle (5 procedures) of low-dose inhalation XOT. The therapy consisted in a gradual increase in the percentage of xenon in the inhaled xenon-oxygen mixture with a reciprocal decrease in exposure time, an exponential regimen in the concentration range from 12–14% to 20–24% and exposure from 25 to 10 minutes. Blood levels of FSH, LH, estradiol, progesterone, and testosterone were measured by RIA (Immunotech, Czech Republic) before and after XOT. The psycho-emotional status was assessed by the generally accepted quality of life scales for cancer patients MOS-SF-36 and ESAS, the type of adaptive reactions was identified by Garkavi using the analysis of Schilling's leukogram, the severity of POES was determined by the menopausal index (MMI). Results: The POES manifestation and the dominance of acute stress were due to the inversion of hormonal metabolism - a decrease in the level of estradiol 487.3±52.4 and progesterone 1.8±0.1 relative to the initial levels 1017.9±83.4 nmol/L and 11.8±0.7 nmol/L, respectively, with an increase in FSH by 5.8 times, and LH by 2.4 times (p < 0.05). An accompanying XOT resulted in a significant MMI decrease (p < 0.05), training and calm activation reactions prevailed in 80% cases with harmonious changes in the regulation of hormonal processes and a clear regression of the acute estrogen deficiency (elevation of estradiol levels to 751.4±61.4 nmol/L and a decrease in FSH and LH by 1.5 and 2.1 times, respectively (p < 0.05)). MOS-SF-36 and ESAS assessment showed a significant decrease in pathological symptoms. Conclusions: XOT in the early postoperative period in cancer patients of the reproductive age with POES normalized their hormonal status, corrected functional disorders and improved their quality of life.
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e12553
Background: The purpose of the study was to reveal characteristics of Cyclin D1 and β-Catenin expression in subtypes of triple negative breast cancer (TNBC). Methods: The study ...included 60 patients diagnosed with verified TNBC (T1N1M0/T2N0M0, ER-/PR-/Her2-). Immunohistochemical staining with CK5/6, EGFR, Cyclin D1, β-Catenin antibodies was performed. Basal-like (BL) subtype was identified when > 25% cells were CK5/6+ and/or EGFR+ (any staining). The expression of β-Catenin was assessed by its location: the membrane, cytoplasm, nucleus. Cells with negative staining on the membrane and cytoplasmic staining were counted and calculated as a percentage of the total number of tumor cells. Nuclear expression of β-Catenin was considered positive with at least 1 positively stained cell in the field of view at x200 magnification. Parametric statistical methods were used. Significance of the difference between the two means was assessed by the Student's t-test. Results: A number of TNBC samples showed Cyclin D1 overexpression. The loss of β-Catenin on the cell membrane and its abnormal accumulation in the cytoplasm was significantly more frequent in TNBC with Cyclin D1 overexpression. These processes were more pronounced in BL cancers. Loss of membrane expression in BL cancers: 29.6±6.1 and 58±7.4%; accumulation in the cytoplasm: 33.4±5.4 and 63.3±7.2%, with low and high Cyclin D1 respectively. Loss of membrane expression in non-BL cancers: 17.6±3.8 and 33.3±4.3%; accumulation in the cytoplasm: 27.3±6.3 and 49.5±8.2%, with low and high Cyclin D1 respectively. Only BL TNBC demonstrated β-catenin translocation to the nucleus (up to 20 stained cells in the field of view): in 33.3% tumors with Cyclin D1 overexpression and in 12% of tumors with its low expression. Conclusions: Levels of expression of β-catenin and Cyclin D1 in TNBC may have predictive value, and the choice of these biomarkers as targets will improve the treatment with new-generation medications.
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e12552
Background: The purpose of the study was to analyze Ki-67 and Cyclin D1 expression in subtypes of triple negative breast cancer (TNBC) with different androgen profiles. Methods: ...Tissues of 60 patients with verified TNBC (T1N1M0/T2N0M0, ER-/PR-/Her2-) were studied. Immunohistochemical staining was performed with antibodies to androgen receptors (AR), CK5/6, Ki-67, Cyclin D1, and EGFR. Basal-like (BL) subtype was identified when > 25% cells were CK5/6+ and/or EGFR+ (any staining). Tumors with ≥10% positively stained cells were considered AR+. Parametric statistical methods were used. Significance of the difference between the two means was assessed by the Student's t-test. Results: BL TNBC showed significantly higher Ki-67 expression (81.9±3.1%), compared to other subtypes (70.8±3.1%), p < 0.05. A number of TNBC samples demonstrated Cyclin D1 overexpression ( > 30% stained cells) with no correlation with Ki-67 expression (r = 0.12). High Cyclin D1 levels were less common in BL TNBC (32.4% tumors), compared to other subtypes (43.5% tumors), but its average values were significantly higher (89.9±4.7% vs. 66.5±6.6%, p < 0.05). AR+ tumors were observed in 11 cases (18.3%). Levels of Ki-67 were similar in TNBC with different AR profiles. The percentage of BL tumors was similar in AR+ and AR- cancers (63.6% and 61.3%, respectively). Expression of Cyclin D1 was more frequent in AR+ TNBC (45.5%), vs. 34.7% in АR-. Average levels of Cyclin D1 were significantly higher in AR+ tumors (51.8±13.7%), vs. AR- (31.6±5.1%). Conclusions: TNBC differs in the Cyclin D1 leves; some tumors show its overexpression. Inhibition of cyclin-dependent kinases blocks the cell cycle, which may be useful in the TNBC treatment, which currently has no targets for therapy.
e15067
Background: Biogels based on natural components of the extracellular matrix are traditionally used to obtain 3D models of breast cancer (BC) tumor growth. However, such biogels are difficult ...to use in 3D bioprinting due to their poor shape retention and suboptimal curing conditions. Artificial biogels based on gelatin methacrylate and/or alginate have a good printability and can include natural components of the extracellular matrix to improve interaction with breast cancer cells. Nevertheless, for successful reconstruction of the tumor microenvironment, not only the composition but also the microstructure of the resulting models is important. The aim of the study was to investigate the effect of gelatin methacrylate and alginate composed bioink curing method on the microstructure of the resulting 3D construct and the morphology of breast cancer cells enclosed in it. Methods: BT20 breast cancer cells were mixed with GelMA A bioink (Cellink, USA) at a concentration of 10
6
cells/ml. The construct was printed on a BIO X bioprinter (Cellink, USA) at an ink temperature of 26°C, a printing table temperature of 10°C, a pressure of 8 kPa, a print speed of 10 mm/s, and a needle diameter of 22G. Next, the printed constructs were cured chemically by immersion in a 100 mM CaCl
2
solution for 1 min or photo-cured by irradiating with light at a wavelength of 405 nm for 20 seconds from a distance of 5 cm. After washing, the constructs with encapsulated cells were incubated in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA) for two weeks, after which they were fixed in 10% formalin and embedded in paraffin blocks. The sections were H&E stained and photographed at a magnification of 400X, then the area and roundness of the pores were determined using the ImageJ software. Results: During photo-curing of the printed construct, the pore area on the section averaged 1.5±1.07 µm
2
(M±SD, n = 500), which is significantly less than with chemical curing (4.5±0.9 µm
2
, n = 500) (α = 0.05, df = 998). At the same time, the pores had an irregular shape (roundness 2.5±0.6, n = 500), which indicates their communication, in contrast to the chemically cured construct, the pores of which were almost perfectly round (roundness 1.2±0.2 M±SD, n = 500). BT20 culture cells encapsulated in bioink had an elongated process shape, as if squeezing through the fine-mesh structure of the light-cured construct, while in the chemically cured construct they had a rounded shape, not going beyond the boundaries of the pores. Conclusions: When creating 3D tumor growth models of breast cancer using bioinks based on gelatin methacrylate and alginate, photocuring is preferable, as it allows creating a spongy microstructure of communicating pores. Such a structure supports cell migration and helps maintain cell morphology close to that observed in vivo.
e14511
Background: The main effort to improve adoptive cancer immunotherapy is aimed at overcoming the limitations associated with low MHC expression on tumor cells. Reinfusion of patient's ex vivo ...activated NK cells, which eliminate tumor cells, regardless of the MHC molecules presence on their surface, is among promising approaches. Natural killers fate is regulated by a whole set of cytokines, of which IL-18 and interleukins of the IL-2 family (IL-2, IL-15, IL-7, IL-21) are of particular importance. Despite the common structure of their receptor, interleukins of the IL-2 family may exert different influence on NK phenotype when combined with IL-18. The aim of the study was to investigate the effect of IL-18 combinations with interleukins of IL-2 family on activation markers expression on breast cancer patients’ peripheral blood NK cells. Methods: Peripheral blood NK cells were enriched by magnetic cell sorting from PBMC using the NK Cell Isolation Kit (#130-092-657, Miltenyi Biotec, Germany). Next, the sorted NK were incubated at 10
6
cells/ml in RPMI 1640 medium (Gibco, USA) with addition of cytokines at a concentration of 10 ng/ml in 5 variants: 1) IL-18; 2) IL-18 + IL-2; 3) IL-18 + IL-7; 4) IL-18 + IL-15; 5) IL-18 + IL-21. Cells were further incubated at 5.0% CO
2
and 37
0
C. After 48 hours of incubation, the expression of CD16, CD56 and CD25 markers on NK was assessed by flow cytometry. Results: A decrease in CD16++CD56+ subpopulation percentage up to 52.5% and 54.9% compared to 62.2% in the control was observed after incubation with IL-18 alone and when it was combined with IL-2, respectively. At the same time, the addition of IL-15 and IL-21 caused a further decrease to 38.7% and 39.1%, respectively. Nevertheless the combination of IL-18 and IL-7 led to an increase in CD16++CD56+ subpopulation percentage up to 71.8%. The percentage of CD25-positive NK cells on the whole followed the dynamics of CD16++CD56+ subpopulation with the IL-18 +IL-15 combination causing the maximum decrease (up to 7.6%). The proportion of NK cells with the CD16+CD56++ phenotype, on the contrary, increased after incubation with IL-18 alone and its combination with IL-2 from 34.5% in control to 46.9% and 44.3%, respectively. Interleukin 18 combinations with IL-15 and IL-21 exhibited even more prominent effect causing increase in CD16+CD56++ proportion to 59.6% and 60.4%, respectively. At the same time, incubation with IL-18+IL-7 caused only a slight decrease in the percentage of the CD16++CD56+ NK subpopulation (up to 27.5%). Conclusions: Thus, IL-18 stimulated the generation of the cytokine-producing NK fraction while suppressing the cytolytic fraction. Also the antagonistic effect of IL-7 with IL-18 and the synergistic effect of IL-15 and IL-21 with IL-18 were revealed on NK cells in vitro.
e14539
Background: Promising oncolytic viruses are evaluated, as a rule, by their direct cytotoxic effect on cancer cells. However, the positive effect of cancer virotherapy may be associated with an ...effect on the immune system, in particular, on the expression of PD-1 and PD-L1 on lymphocytes. The aim of the study was to evaluate the effect of unclassified non-pathogenic strains of rotaviruses RVK100 and RVK228 on the expression of PD-1 and PD-L1 on peripheral blood T-cells of breast cancer patients. Methods: PBMC were cultured at 10
6
cells/ml cell density in RPMI 1640 medium (Gibco, USA) without the addition of serum at 37°C in an atmosphere containing 5.0% CO
2
. Four experimental variants were established - 1) negative control without the addition of viruses, 2) positive control of activation with phytohemagglutinin (PHA) addition, 3) addition of RVK100 strain in concentration 10
7
particles/ml, 4) addition of RVK228 strain in concentration 10
7
particles/ml. After 24 and 72 hours of incubation, the expression of PD-1 (CD279) and PD-L1 (CD274) on T cells was determined by flow cytometry. Results: After 24 hours of cultivation, an increase in PD-1 expression on CD4+ cells was observed in samples with addition of PHA - 40.5%, RVK100 - 42.3% and RVK228 - 37.5% compared with the control (18.1%). A similar, although less pronounced increase in PD-1 expression, was observed on CD8+ cells: PHA - 41.7%, RVK100 - 46.4%, RVK228 - 42.6% compared with 27.7% in the control. Expression of PD-L1 on CD4+ cells increased to 67.0% and 58.6% in samples with addition of RVK100 and RVK228 strains, respectively, while under the influence of PHA it increased to 75.1% compared with the control (44.8%). A similar trend was also found on CD8+ cells (control - 46.2%, RVK100 - 63.4%, RVK228 - 58.4%, PHA - 52.8%). After 72 hours, an increase in PD-1 expression on CD4+ cells was observed only in the control (up to 41.2%), while in the experimental variant with RVK100 addition there was a 2-fold decrease (up to 21.6%) and no significant changes were found in samples with addition of PHA and RVK228 compared to 24 h. At the same time, cultivation with RVK100 caused a decrease in PD-1 expression on CD8+ cells by 2.7 times (up to 17.4%) compared with 24 h, without significant changes in other samples (control - 33.1%, PHA - 48.1%, RVK228 - 38.4%). The expression of PD-L1 on CD4+ cells generally remained unchanged compared to 24 h, while proportion of CD8+CD279+ cells increased in all variants and reached 67-79% the in experimental and control samples. Conclusions: Both strains, like the nonspecific T-mitogen PHA, stimulated the expression of immune checkpoint receptors PD-1 and PD-L1 on T-helpers and CTLs after 24 hours of cultivation. After 72 hours of cultivation, RVK100, in contrast to RVK228, was able to reduce the expression of PD-1 on these cells.
