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e15514
Background: The standard treatment of squamous cell carcinoma of the tongue and the mucosa of the bottom of the oral cavity (SCCTOM) is chemotherapy (CT) in combination with ...cetuximab (mC, anti-EGFR antibody). However, not all patients manage to achieve positive results. A common cause of resistance to anti-EGFR antibodies is the constitutive activation of mediators of the underlying signaling pathways. Therefore, the aim of the study was to analyze mutations in the KRAS using the Digital Droplet PCR method in patients with SCCTOM on the background of CT in combination with targeted therapy or standard CT. Methods: The study used the QX200 system (Bio-Rad) and the ddPCRtm KRAS Screening Multiplex Kit (7 mutations). The study included 60 patients with T3-4N0-1M0 SCCTOM. The main group consisted of 30 patients (cisplatin, 5-fluorouracil+cetuximab). The control group consisted of 30 patients (CT). For research, cfDNA from blood plasma was used. Analysis of the data was performed using QuantaSoft v1.7.4. Results: The frequency of the KRAS mutant type (mtKRAS) is 43% before treatment and 32% after treatment (n = 60). In the group of patients CT+mC the incidence of mtKRAS before treatment was 40%, after treatment - 20%. In the group of patients CT without mC the incidence of mtKRAS before treatment was 47%, after treatment - 43%. Decrease of 20% (p = 0.00089) in the frequency of mtKRAS after CT+mC was found, while it was 23% (p = 0.00009) lower than the frequency of mtKRAS after CT without mC. After treatment, in the CT+mC group of patients, the number of mtKRAS DNA copies increased 1.5 times (p = 0.048). Analysis of clinical response to therapy allowed us to divide the main and control groups of patients into 2: sensitive and resistant to therapy. After treatment, a change in the mtKRAS ratio was not found in the subgroup sensitive to mC compared with the period before treatment, while the frequency of mtKRAS was reduced by 3 times (p = 0.009). In the mC-resistant subgroup, the mtKRAS ratio increased 1.9 times compared to the period before treatment (p = 0.009), while the frequency of mtKRAS increased 3.5 times compared with the mC-sensitive group (p = 0.0045). Conclusions: Prior to treatment, patients resistant to CT+mC were characterized by an increased occurrence of mutations in the KRAS and an apparently large number of tumor cells carrying mutations. The use of mCs caused a change in the direction of the clonal evolution of tumor cells - leading to an increase in the number of cells carrying mtKRAS (conditions were created for the elimination of cells with mutation in EGFR and KRAS wild type).
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e15063
Background: The study of the possible antitumor activity of secondary plant metabolites either in the form of individual agents or in combination with clinically used drugs is ...considered as a promising direction in the therapy of malignant tumors. The aim of this study was an estimation of antitumor activity of secondary plant metabolites in the in vitro experiment on the HeLa cell line. Methods: Secondary metabolites were extracted from plant raw materials and were isolated by preparative chromatography. Determination of their composition was carried out using HPLC; obtained compound structures were identified by NMR. We selected 4 secondary metabolites from Petasites hybridus for testing: (2,4-dihydroxy-2,5-dimethylfuran-3 (2H), 5-(hydroxymethyl) furan-2-carbaldehyde, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, Corynan), and 1 secondary metabolite from Berberis vulgaris : a berberine chloride (BBR, C
20
H
18
NO
4
+
Cl
-
is a derivative of 5,6-dihydrobenzoa,g isoquinolinium). HeLa CCL2 cultivation was carried out under standard conditions in the MEM medium. When reaching the 75-80% confluence level we replaced nutrient medium with the introduction of secondary metabolites (concentration 4 and 12 μg/ml) and cultured for 24 and 72 hours. Cell survival was determined on a NanoEnTek JuliFl counter (Korea) in the presence of 0.4% trypan blue. Apoptosis was assessed on a flow cytometer BD FACSCanto II using FITC Annexin V Apoptosis Detection Kit I. Post-exposure copy number and expression were assessed by Real-time PCR with a panel of genes CASP9, CASP8, CASP3, TP53, MDM2, BAX, BCL2, CDK1, BRCA1, BRCA 2, RB1. Results: All obtained data were normalized by negative control. When we used 4 μg/ml berberine solution with 72-hour exposition, the proapoptogenic effect was maximal, causing the death of 67.2% of HeLa cells (29.3% early apoptosis, 37.2% late apoptosis, 0.7% necrosis). Within 24 hours, berberine at the same concentration caused a 2-fold increase in TP53 expression relative to MDM2. An increase in its concentration to 12 μg/ml and exposure for up to 72 hours led to a 31-fold increase in TP53/MDM2. The terpenoid 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde at a concentration of 12 μg/ml after 72 hours of cultivation caused a 6.5-fold increase in the TP53/MDM2 ratio. The corynan alkaloid (12 μg/ml) at an exposure of 72 hours increased the BAX/BCL ratio by 2.4 times. There were no statistically significant differences in the expression and copy number of the remaining genes studied. Conclusions: Berberine, corynan, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde showed high promise in the HeLa cell line in vitro, since they surpassed the antitumor activity of other metabolites of Berberis vulgaris and Petasites hybridus.
Abstract only
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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e18564
Background: IGF-1 and IGF-2 are powerful mitogenic factors with anti-apoptotic effect. Their overproduction leads to neoangiogenesis and metastasis. Monitoring the level of these ...factors can serve as a potential predictive tool. The purpose of the study was to study blood levels of IGF-1 and IGF-2 in patients with squamous cell carcinoma (SCC) of the oral cavity depending of the efficacy of chemotherapy (CT) and cetuximab. Methods: The study included 50 patients with squamous cell carcinoma of the oral cavity T3-4N0-1M0, stage III-IV. The main group (n = 30) received 2 CT cycles with cetuximab: cisplatin 100 mg/m
2
, intravenously, day 1, 5-fluorouracil 1000 mg/m
2
/day, intravenously, 96-hour continuous infusion, in combination with cetuximab 400 mg/m2 on day 1 in a loading dose, then 250 mg/m2 on days 8 and 15. The control group (n = 20) received 2 CT cycles: intravenous cisplatin 100 mg/m
2
on day 1, intravenous 5-fluorouracil 1000 mg/m
2
/day, 96-hour continuous infusion on days 1-4. Results were compared with the values in 20 non-cancer donors. Levels of IGF-1 (mcg/L) and IGF-2 (ng/mL) were measured in the blood serum by ELISA using standard test systems (Mediagnost, Germany). Statistical analysis of results was performed using the Statistica 6.0 program (Stat-Soft, 2001). Results: Before treatment, levels of IGF-1 and IGF-2 in patients were lower than in donors by 53.5% and 20.3%, respectively. In patients with partial regression after CT, IGF-1 levels significantly increased by 33% (p < 0,05), compared to the values before treatment, while IGF-2 was both within donor and initial values and the IGF-1/IGF-2 ratio was similar to the initial level but 33.3% lower than in donors. The patients in the main group with partial regression normalized IGF-1 levels increasing it compared to initial values – by 87%. IGF-2 levels did not differ statistically significantly from the initial values and were 32.5% lower than in donors. The IGF-1/IGF-2 ratio was 58% higher than before treatment. Conclusions: CT and cetuximab normalized blood levels of IGF-1 in oral cancer patients with partial tumor regression.
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e18563
Background: Cetuximab is a monoclonal antibody directed against the EGFR receptor, able to block the EGFR signaling pathway and also indirectly affect the secretion of some ...growth factors. The search for biomarkers responsible for cetuximab resistance is an urgent task. The purpose of the study was to analyze VEGF-A and TGF-β levels in tumor tissue bioptates in patients with squamous cell carcinoma of the oral cavity receiving chemotherapy (CT) and cetuximab. Methods: The study included 30 patients with HNSCC St III-IV, T3-4N0-1M0. All patients received 2 cycles: cisplatin 100 mg/m
2
, intravenously, day 1, 5-fluorouracil 1000 mg/m
2
/day, intravenously, 96-hour continuous infusion, in combination with targeted therapy cetuximab 400 mg/m2 on day 1 in a loading dose, then 250 mg/m2 on days 8 and 15). Patients were divided into two groups: with response to cetuximab and CT (partial regression and stabilization) n = 17 and progression considered (resistance to the treatment) n = 13. Levels of VEGF-A and TGF-β were determined in tumor tissue bioptates by ELISA using standard test systems (Bender Med System, Austria). Statistical processing of results was performed using the Statistica 6.0 program (Stat-Soft, 2001). Results: CT and cetuximab in patients with resistance did not result in statistically significant changes in levels of VEGF-A, TGF-β and the VEGF-A/TGF-β ratio in tumor tissue bioptates, compared to the initial values. The studied markers in tumor tissue bioptates in patients with response to CT were statistically significantly different from the initial values: VEGF-A was decreased by 1.46 times, TGF-β by 2.96 times, while the VEGF-A/TGF-β ratio was twice elevated (p < 0.05). Conclusions: The results on levels of VEGF-A and TGF-β and the VEGF-A/TGF-β ratio are of a prognostic value and can be used for evaluating the efficacy of cetuximab and CT in patients with HNSCC.
