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Multi-drug resistance (MDR) is a condition when there is broad cross-resistance of cells to various agents which are different in structure and effect. Modern perceptions on mechanisms of MDR development in malignant tumors have been considered, in particular, in tre­ating breast cancer. Physiological functions and contribution to MDR development of ABC-transporter protein families have been described. The role of activation of glutathione system enzymes and apoptosis-regulating proteins in MDR formation has been shown. Key Words: multi-drug resistance, ABC-transporters, glutathione system, apoptosis-controlling genes.

Aim: The aim of this study was to identify genes that are differentially expressed in gastric tumors and to analyze the association of their expression level with tumor clinicopathologic features. Methods: In the present research, we used bioinformatic-driven search to identify miRNA that are down-regulated in gastric tumors and to find their potential targets. Then, the expression levels of some of the target mRNAs were investigated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: As a result of the bioinformatics analysis, fifteen genes were found to be potentially differentially expressed between the tumors and normal gastric tissue. Five of them were chosen for the further analysis (WNT4, FGF12, EFEMP1, CTGF, and HSPG2) due to their important role in cell proliferation and differentiation. Expression levels of these genes were evaluated in our collection of frozen tissue samples of gastric tumor and paired normal stomach epithelia. Increased FGF12 expression was observed in diffuse type of gastric cancer while WNT4 mRNA was found to be down-regulated in intestinal type of gastric cancer. Besides, CTGF gene overexpression was revealed in diffuse type of stomach cancer in comparison with that in intestinal type. Up-regulation of CTGF was also associated with lymph node metastasis. Conclusions: The findings show its expedient to perform further investigations in order to clarify diagnostic and prognostic value of CTGF, FGF12, and WNT4’s in stomach cancer as well as the role of these genes in carcinogenesis.

BackgroundFor single nucleotide polymorphism – SNP (SNP – Single Nucleotide Polymorphism) is characterized by phenomenon of allelic imbalance (AI). The phenomenon of allelic imbalanced (AI) is typical of many genes in different malignancies. Allelic imbalanced may result allelic deletions (loss of one copy of the locus) or amplification of one allele, resulting in only a single allelic variant of SNP is determined in the tumor in the PCR. Phenomenon of AI in tumors of breast cancer (BC) is considered in one or more genes. The phenomenon of AI during chemotherapy (CT) and especially in the longer format of the gene had not previously been studied.Thus, the aim of this work was to study microarray imbalanced allele in mammary tumors during neoadjuvant chemotherapy (NCT).Materials and methodsThe study included 26 breast cancer patients with stage IIA – IIIC. The patients in the neoadjuvant mode received 2–4 courses of chemotherapy regimens FAC or CAX. DNA from 26 paired samples before treatment and operational samples were isolated by dialing QIAampDNA miniKit (Qiagen, Germany). Microarray analysis was performed on DNA chips of high density company Affymetrix (USA) CytoScanTM HD Array, which contains more than 750 thousand SNP. Microarray analysis was performed on SNP genotypes DNA tumor tissue before and after treatment for each patient and recorded as change occurring allelic imbalanced tumor tissue genotype (AA>AB>AB AA BB>AB>AB BB) during therapy.ResultsThe frequency of the AI in breast tumor during NCT was highly variable (within 0.9–66.5%) of the studied SNP (6850 – 497,979 SNP). For each patient, frequency shift genotype (homozygous in heterozygous genotype, and vice versa) was calculated as a percentage of all the shifts. Changes in the wild or mutant heterozygous genotypes (AA or BB>AB) were combined into one group; the second group was the sum of the change in the heterozygous genotype homozygous wild genotype or the mutant (AB>AA or BB). We have found that the direction of the AI was significantly associated effect of NCT. In the group of patients with partial regression, the direction of AI change from homozygous to heterozygous genotype often occurs (AA or BB>AB) (9/14, 64%), whereas patients with no response to the NCT (with stabilization or progression) have the opposite effect. All these patients (12/12 cases) have the direction of the change of AI from heterozygous genotype to homozygous (AA or AB > BB) (p=0.00071). AI during chemotherapy at the level of the marked tendency (Log-rank test, p=0.062) is associated with 5-year metastasis-free survival. Low metastasis-free survival rate is observed in patients with AI in the direction of the change from the heterozygous to homozygous genotype, while 100% survival is noted in patients with change from homozygous to heterozygous genotypes, and this imbalanced t allele is a favorable prognostic factor.ConclusionAllelic imbalanced in breast tumor during NCT phenomenon is massive and may affect up to 67% of SNP. AI may occur in the direction of change from homozygous to heterozygous genotype, and it is associated with a good response to treatment and 100% metastasis-free survival. Apparently, this can be explained by the fact that the change from homozygous genotypes to heterozygous occur due to partial destruction of tumor cells by chemotherapy, resulting in the increase of stromal elements. In contrast, AI in the direction change from heterozygous to homozygous genotype during NCT is associated with no response to chemotherapy due to metastasis and occurring of new mutant clones in the tumor.

BackgroundThe aim of the study was to identify the factors that determine outcome and overall survival of HNC patients of Tomsk region.Materials and methodsClinical data, morphological characteristics of tumors and outcomes were obtained for 91 patients. Data about lifestyle, food preferences, smoking history were obtained from the questionnaire (n=35). All clinical samples were tested by AmpliSens HPV diagnostic kits (Russia) to determine prevalence of 12 high risk HPV types. Statistical analysis was performed using Kaplan–Meier method, Cox regression, Gehan test, Fisher test, Mann–Whitney and Kruskal–Wallis tests.ResultsGender, age, smoking status, alcohol consumption, distance from harmful factors, professional hazards and duration of its exposure as well as tumor criteria such as T, N, G, the presence of keratinization, invasion into the underlying tissues, HPV-infection, chemotherapy and/or radiation therapy and response to the treatment were assessed for HNC patients. It was shown that the two-year survival rate was about 70%, and the five-year survival rate was about 32%. There was strong correlation between decreased overall survival and increased alcohol consumption (p=0.03) as well as regional lymph nodes status (p=0.01). Patients with early tumor stages and N0 lymph node status as well as patients receiving chemotherapy and/or radiotherapy showed trend towards to better survival (p=0.09, p=0.1, p=0.09, respectively). Overall survival of patients with lymph node metastasis was higher in case of early tumor stages (p=0.08) and in patients who had no alcohol consumption history (p=0.06). Cox regression analysis was used to obtain the model describing overall survival of patients. The model with the highest level of significance includes 3 factors-nodal metastases, the presence of keratinization and radiotherapy. It was shown that the risk of death was 4.2 and 2.6-fold higher in case of lymph node metastases and keratinized cancer, and 2.7-fold lower in case of radiotherapy. It was also shown that metastasis occurred more frequently in cases with invasion into the underlying tissue of a primary tumor (p=0.04) and in cases with a low tissue grade (p=0.02). Association of alcohol consumption with questionnaire data was studied. It was shown that men’s preferably smokers consume alcohol more often than other patients (p=0.006, p=0.02, respectively). Our data showed that HPV prevalence was higher in smokers (p=0.04), and in patients with early tumor stages (p=0.07). Furthermore, response to radiotherapy was better in HPV-positive patients compared to HPV-negative cases (p=0.09). Better response to radiotherapy showed the group of patients who received dose higher than 45 Gy (p=0.03) and who had no lymph node metastases(p=0.05).ConclusionOur data showed that metastasis to lymph nodes and alcohol consumption are the main factors that affect mortality in HNC patients of Tomsk region.

BackgroundRisk of metastasis formation is provided by both tumor cell biological characteristics and the microenvironment features within the primary tumor along with local and systemic conditions for metastatic niche formation. The inflammatory infiltration has been shown to strongly impact on tumor progression (Whiteside, 2013). Dronca et al. (2011) showed that immunosuppressive factors in the tumor microenvironment may impair not only local immune responses but also disturb systemic immunity. Zitvogel et al. anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.To study the impact of immune system on clinical response to neoadjuvant chemotherapy and metastasis-free survival in breast cancer patients.Materials and methods350 patients with newly diagnosed invasive breast cancer treated with neoadjuvant chemotherapy (NAC) were enrolled into the study. The procedures were made in accordance with the Helsinki Declaration. Clinical response to chemotherapy, the 5-year metastasis-free survival and all major clinical and morphological parameters were determined. The original method of multidimensional data visualization was applied to present the immune system state as integral entirety in visual image for classification of patients with different risk of metastasis (NovoSpark Corporation, Canada). Copy number aberrations (CNA) of cytokine gene regions in tumor specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). Cytokine gene polymorphism was analyzed. Subpopulations of lymphocytes and macrophages were determined within the primary tumors by IHC.ResultsWe found, that favorable clinical immediate response to preoperative chemotherapy was related to the high levels of IL-1beta, TNF-alpha and IL-10 production by peripheral mononuclear cells before the treatment. This correlation was further confirmed by data from the study on association between cytokine gene functional polymorphism and response to NAC. We used NovoSpark Corporation visualization approach allowing the representation the immune system state as integral unit and to discriminate breast cancer patients with high and low risk of haematogeneic metastasis. When estimated before cancer treatment, 95% of breast cancer patients had risk of metastasis. The neoadjuvant chemotherapy and surgical tumor removal reduced the risk of tumor progression to 62–71%. However, in a year after adjuvant chemo- and radiotherapy, the patient group with high risk of metastases increased to 81% again. Thus, the cancer treatment can change the primarily estimated outcome prognosis in breast cancer patients, and the monitoring of immune system is a promising approach to predict the risk of cancer progression or resistance to the therapy. We have found the connection between the profile of intra-tumor inflammatory elements and chemotherapy efficacy.Cytokine gene expression may be influenced by the chromosome anomalies (CNA – Copy Number Aberration) – deletion and amplification – of cytokine gene loci in tumor cells. We found the close relation between the clinical response to NAC and gain of function of IL-10 and CHI3L1 (YKL40) genes. In contrast, loss of TNF-alpha and IL-17 gene function due to corresponding CNA was associated with good response to NAC. Metastasis-free survival of breast cancer patients was shown to be closely related to CNA.ConclusionThe parameters of the activation of systemic and intra-tumoral immune system by growing tumor and its dissemination have to be validated in order to identify the new prognostic markers for the efficiency of the neoadjuvant chemotherapy.

BackgroundThere are numerous evidences suggesting that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones have private genetic aberrations, including chromosomal abnormalities. The combined effect of genetic instability and differential selective pressures of the microenvironment and chemotherapy can result in the creation of new tumor clones (Navin et al., 2011; Ng et al., 2012). The aim of this study is to show breast tumor clonal evolution during neoadjuvant chemotherapy (NAC) using microarray analysis.Material and methodsBreast cancer patients (n=26) with stage IIA to IIIC (T1-4N0-3M0), were treated with NAC (FAC or CAX regimens). DNA was extracted from 26 samples of tumor tissue derived before or after NAC using QIAamp DNA mini Kit (Qiagen, Germany). Copy Number Aberrations (CNA, deletions and amplifications, or Loss and Gain, respectively) and number of mutant clones were detected in pre- and post-NAC tumor samples using the high density microarray platform Affymetrix (USA) CytoScanTM HD Array. This study was approved by Tomsk Cancer Research Institute review board.ResultsWe have revealed that 19% (5/26) of patients during the NAC showed the decrease in the number of mutant clones and CNA frequency right up to their complete elimination (genetic regression) at one case. In 7 (27%) cases chemotherapy had no any effect on number of mutant clones and the frequency of CNA in tumor. In the tumors of 10 patients the elimination of some mutant clones as well as the formation of new clones with deleted genetic material occurred under the influence of chemotherapy. 6 patients have demonstrated the appearance of new tumor clones with gene amplifications which were associated with the development of metastases in 83% of cases (5/6). All other patients (n=21) who has not acquired the new tumor clones with Gain function mutation after NAC did not manifest distant metastasis in 5-year follow-up (Kaplan–Meier, p=0.00001 Log-rank test).ConclusionThe first time evidence is presented that the formation of new tumor clones may occur during the NAC. Metastasis of breast cancer is associated with the appearance of new clones with DNA amplifications. Detection of these clones allow getting new prognostic factor in breast cancer.The study was supported by the Russian Foundation for Basic Research – Russia (Project 15-04-03091).

The purpose of the study was to access pattern of microRNA expression in tissue of precancerous lesions and larynx cancer.A total of 25 people with a diagnosis of dysplasia II–III (n=10), control group (dysplasia 0, n=15) and larynx cancer patients (n=46) were examined. Fresh frozen biopsies and adjacent normal epithelium were used. The diagnosis was verified by histology. All qRT-PCR data were analyzed using the Pfaffl analysis. Multiplex RT-PCR on miRNA templates were performed as described by Chen et al. (2005). TaqMan miRNA assays (Iyevleva et al., 2012) were used to quantify expression of mature miRNAs of interest (miR-18a, -21, -155, -200a, -200c, -205, -221, -494). Data were analyzed using Welsh t-test with a 5% FDR correction. According to our results, aberrant expression of some miRNAs was showed. There was no significant differences in miRNA expression in a total group (n=25) although a trend towards overexpression of oncogenic miRNA-21 and -155 according to severity of dysplastic changes in a tissue were present. Detailed analysis showed significant overexpression of that miRNAs as well as miRNA-200c and -205 in a group of dysplasia II- III against control group (15 vs 10, p=0.019, p=0.045 and p=0.020, p=0.038, respectively) but these results did not meet 5% FDR correction.Data showed overexpression of the same microRNA (-205, -155, -200 and -21) in larynx cancer patients compared to control group (46 vs 15, p=0.0002, p=0.008, p=0.009, p=0.013, respectively). It should be pointed that microRNA pattern both in larynx cancer and patients with dysplasia II-III was very close showing similarity of these groups at molecular level. Frequency of cases with microRNA-205 overexpression was 2.98 times higher in cancer patients than in those who had no malignant transformation (CI 95%, 1.41–16.26, p=0.007). Data showed that up regulation of microRNA-205 could be a marker of disease progression (OR=4.79). Three other microRNAs did not show any promising results as biomarkers of cancer progression but data obtained has interesting fundamental value.These miRs (-21, -155, -205 and -200c) are known to be regulators of processes that play an emergent role in carcinogenesis and our results obtained in vivo highlight and expand knowledge about some aspects of larynx cell transformation. Data suggest that miRNAs changing its expression according to dysplasia progress and could be important players in complex process of carcinogenesis as well as could be potential markers of disease progression.

BackgroundBreast cancer, particularly invasive carcinoma of no special type (IC NST), demonstrates considerable intratumor morphological heterogeneity. Five types of morphological structures representing different architectural arrangements of tumor cells – tubular, alveolar, trabecular, solid structures, and discrete groups have been described in IC NST. Previous studies reported the contribution of intratumor morphological heterogeneity of IC NST to chemotherapy efficiency and lymph node metastasis (Zavyalova et al., 2013; Denisov et al., 2014); however, its role in distant metastasis remains unidentified. Aim: to study the contribution of intratumor morphological heterogeneity of IC NST to distant metastasis and to identify gene expression features of metastatic behavior of different morphological structures.Materials and methods358 IC NST patients (age range 29–90, mean age 49.8±9.5, T1-4N0-3M0-1) treated with neoadjuvant chemotherapy (NAC) have been enrolled in this study. Chi-square test and Kaplan–Meier analysis were used to estimate the association between the presence of certain morphological structures in breast tumors and the frequency of distant metastasis and metastasis-free survival. qRT-PCR was applied for measurement of the expression levels of genes involved in cell motility (CDH1, CDH2, CDH3, CTNNA1, CTNNB1, ITGA6, ITGAV, ITGB1, ITGB3, ITGB4, SNAIL, MMP14, ROCK2, L1CAM, MMP2, MMP9, PDPN) and pre-metastatic niche formation (TNFa, TGFb, VEGFa, LOX, M-CSF, GM-CSF, HIF1A, SDF2) in different morphological structures isolated from breast tumors (n=4) by laser microdissection.ResultsPatients with alveolar structures in breast tumors more frequently displayed distant metastasis than cases without this morphological variant (71.9% vs. 56.5%; p=0.004). The association between alveolar structures and high frequency of hematogenous metastasis was found only in patients with poor response to NAC (p=0.003), but not in cases with good chemotherapy efficiency (p=0.377). Increased distant metastasis was also shown in patients with trabecular structures as compared to cases without this morphological type (88.3% vs. 70.0%; p=0.0001). Kaplan–Meier analysis demonstrated a significantly higher probability of developing metastasis in patients with alveolar or trabecular structures in breast tumors (p=0.011). No significant association between other morphological structures and distant metastasis was found. Expression analysis showed the presence of cell motility phenotype in all morphological structures. In particular, we found changes in cell adhesion gene expression, which declined in the row: solid–alveolar–trabecular structures–discrete groups of tumor cells (p