DNA Copy Number Aberrations and Expression of ABC Transporter Genes in Breast Tumour: Correlation with the Effect of Neoadjuvant Chemotherapy and Prognosis of the Disease.

One of the important reasons for the ineffectiveness of chemotherapy in breast cancer (BC) is considered to be the formation of a multidrug resistance phenotype in tumour cells, which is caused by the expression of energy-dependent ABC transporters. The aim of this work was to assess chromosomal aberrations and the level of transcripts of all 49 known ABC transporter genes in breast tumours. MATERIALS AND METHODS: The study included 129 patients with breast cancer. A microarray study of all tumour samples was carried out on microchips. RESULTS: This study established that the presence of a deletion in genes ABCB1, ABCB4, ABCB8, ABCC7, ABCC11, ABCC12, ABCF2, and ABCG4 is associated with an objective response to treatment (p ≤ 0.05). A decrease in the expression of genes was associated with a good response to chemotherapy, whereas an increase in expression caused the progression and stabilization of the tumour. Analysis of metastatic-free survival rates showed that the presence of ABCB1/4 and ABCC1/6 deletions was associated with 100% survival (log-rank test p = 0.01 and p = 0.03). CONCLUSIONS: The study showed that the aberrant state of ABC transporter genes, as well as a decrease in the expression of these genes, is a predictor of the effectiveness of therapeutic treatment and a potential prognostic marker of metastatic survival.

Stem gene expression in breast tumors during chemotherapy: Connection with the main clinical and morphological factors and the disease outcome.

Introduction: In this research, we studied how the expression of 14 stem genes (TERT; OCT3; SMO; MYC; SNAI2; MOB3B; KLF4; BMI1; VIM; FLT3; LAT; SMAD2; LMNB2; KLF1), as well as the TGF-ß1 cytokine gene and its TGFBR1 receptor in breast tumors before and after NAC is associated with clinical and morphological parameters and the disease outcome. Materials and Methods: The study included 82 patients with the morphologically verified diagnosis of T1-4N0-3M0 breast cancer (stages IIA - IIIB). The material was paired biopsy samples of tumor and surgical material for each patient. The stem genes expression was analyzed via qPCR. Results: As a result, we found that increased level of stem genes expression in breast tumors is associated with lymphogenic metastasis, young age, small tumor size, expression of estrogen and progesterone receptors, and the luminal B molecular subtype. NAC stimulates the expression of 7 out of 16 stem genes. Patients who further developed hematogenic metastases have twice as many hyperexpressed stem genes in their tumors before the treatment and after NAC than patients with no hematogenic metastases. The expression level of three genes - OCT3, LAT, and LMNB2 - in a residual tumor allows us to predict metastasis-free survival of patients with breast cancer of various molecular subtypes with a 79% accuracy. Conclusion: Thus, stem genes hyperexpression is associated with tumor progression.

Predictive and Prognostic Significance of mRNA Expression and DNA Copies Aberrations of ERCC1, RRM1, TOP1, TOP2A, TUBB3, TYMS, and GSTP1 Genes in Patients with Breast Cancer.

Increasingly, many researchers are focusing on the sensitivity in breast tumors (BC) to certain chemotherapy drugs and have personalized their research based on the assessment of this sensitivity. One such personalized approach is to assess the chemotherapy's gene expression, as well as aberrations in the number of DNA copies-deletions and amplifications with the ability to have a significant effect on the gene's activity. Thus, the aim of this work was to study the predictive and prognostic significance of the expression and chromosomal aberrations of eight chemosensitivity genes in breast cancer patients. MATERIAL AND METHODS: The study involved 97 patients with luminal B breast cancer IIB-IIIB stages. DNA and RNA were isolated from samples of tumor tissue before and after treatment. Microarray analysis was performed for all samples on high-density microarrays (DNA chips) of Affymetrix (USA) CytoScanTM HD Array and Clariom™ S Assay, human. Detection of expression level of seven chemosensitivity genes-RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, and GSTP1-was performed using PCR real-time (RT-qPCR). RESULTS: The expression of the RRM1 (AC scheme), TOP2α, TYMS, and TUBB3 genes in patients with an objective response to treatment (complete and partial regression) is higher than in patients with stabilization and progression (p < 0.05). According to our results, the presence of a high level of GSTP1 in a tumor biopsy is associated with the low efficiency of the NAC CP scheme (p = 0.05). The presence of RRM1 deletion is associated with complete and partial regression, as for the TOP1 and TUBB3 genes (p < 0.05). Higher rates of metastatic survival are associated with a high level of expression and amplification of the GSTP1 gene (log-rank test p = 0.02 and p = 0.05). CONCLUSION: Thus, a complex assessment of the chemotherapy's gene expression is important not only for understanding the heterogeneity and molecular biology of breast cancer but also to obtain a more accurate disease prognosis.

Five-year survival in luminal breast cancer patients: relation with intratumoral activity of proteasomes.

Background: The purpose of the study was to analyze the relationship between the caspase-like (CL) and chymotrypsin-like (ChTL) activities of proteasomes and the 5-year overall and metastasis-free survival rates in patients with luminal breast cancer. Methods: The study included 117 patients with primary operable invasive breast cancer (T1-2N0-1M0). Tissue samples from breast cancer patients were obtained as a result of the radical mastectomy or breast conserving surgery, which was a first line of therapy. The ChTL and CL proteasomes activities in the tumor tissue and in the surrounding adjacent breast tissues were assessed using the fluorometric method. The coefficients of ChTL (cChTL) and CL (cCL) proteasomes activities were also determined. The coefficients were calculated as the ratio of the corresponding proteasomes activity in the tumor tissue to the surrounding adjacent breast tissues. Within 5 years of follow-up, hematogenous metastases occurred in 14% of patients with luminal A breast cancer, in 31% of patients with luminal B human epidermal growth factor receptor-2 (HER-2) negative and in 23% of patients with luminal B HER-2 positive breast cancers. The study protocol was approved by the Local Ethics Committee of the Cancer Research Institute of Tomsk National Research Medical Center. Written informed consent was obtained from all patients. Results: An increase in the ChTL and CL proteasomes activities was shown in all studied molecular subtypes of breast cancer compared to adjacent tissues. It was found that the cChTL of >35.9 U/mg protein and the cCL of >2.21 in breast cancer patients were associated with the development of distant metastases. In patients with luminal A breast cancer, the 5-year metastasis-free survival rates were associated only with the value of cCL of proteasomes (log-rank test: P=0.008). In patients with luminal B HER-2 negative breast cancer, the 5-year metastasis-free survival rates were associated with the levels of ChTL and cCL proteasomes activities (log-rank test: P=0.02 and P=0.04, respectively). Conclusions: The data obtained on the correlation of 5-year metastasis-free survival rates with the level of proteasomes activities indicate the possibility of their use as additional prognostic criteria for breast cancer.

Cancer Stem Cells and Somatic Stem Cells as Potential New Drug Targets, Prognosis Markers, and Therapy Efficacy Predictors in Breast Cancer Treatment.

New drug targets, markers of disease prognosis, and more efficient treatment options are an unmet clinical need in breast cancer (BC). We have conducted a pilot study including patients with luminal B stage breast cancer IIA-IIIB. The presence and frequency of various populations of cancer stem cells (CSC) and somatic stem cells were assessed in the blood, breast tumor tissue, and normal breast tissue. Our results suggest that patients with BC can be divided into two distinct groups based on the frequency of aldehyde dehydrogenase positive cells (ALDH1+ cells) in the blood (ALDH1hi and ALDH1low). In the ALDH1hi cells group, the tumor is dominated by epithelial tumor cells CD44+CD24low, CD326+CD44+CD24-, and CD326-CD49f+, while in the ALDH1low cells group, CSCs of mesenchymal origin and epithelial tumor cells (CD227+CD44+CD24- and CD44+CD24-CD49f+) are predominant. In vitro CSCs of the ALDH1low cells group expressing CD326 showed high resistance to cytostatics, CD227+ CSCs of the ALDH1hi cells group are sensitive to cytostatics. Epithelial precursors of a healthy mammary gland were revealed in normal breast tissue of patients with BC from both groups. The cells were associated with a positive effect of chemotherapy and remission in BC patients. Thus, dynamic control of their presence in blood and assessment of the sensitivity of CSCs to cytostatics in vitro can improve the effectiveness of chemotherapy in BC.

Amplifications of Stemness Gene Loci-New Markers for the Determination of the Need for Neoadjuvant Chemotherapy for Patients with Breast Cancer. A Prospective Study.

In this prospective study, a new strategy for the prescription of neoadjuvant chemotherapy (NAC) was prospectively tested and depended on the presence of stemness gene amplifications in the tumor before treatment, which in our early studies showed a connection with metastasis. The study included 92 patients with grade IIA-IIIB luminal B breast cancer. Patients underwent a biopsy before treatment, and with the use of a CytoScan HD Array microarray (Affymetrix, Santa Clara, CA, USA), the presence of stemness gene amplifications (3q, 5p, 6p, 7q, 8q, 13q, 9p, 9q, 10p, 10q21.1, 16p, 18chr, 19p) in the tumor was determined. In group 1 (n = 41), in the presence of two or more amplifications, patients were prescribed a personalized NAC regimen. In group 2 (n = 21), if there was no amplification of stemness genes in the tumor, then patients were not prescribed NAC, and treatment began with surgery. Group 3 (n = 30) served as a historical control. The frequency of an objective response to NAC in groups 1 and 3 was 79%. Nonmetastatic survival was found in 100% of patients in group 2, who did not undergo NAC. In patients in group 1, the frequency of metastasis was 10% (4/41). At the same time, in patients in group 3, who received NAC, the rate of metastasis was 47% (14/30). The differences between group 1 and group 3 and between group 2 and group 3 were statistically significant, both by Fisher's criterion and a log-rank test. The appointment of NAC was most feasible in patients with clones with stemness gene amplifications in the primary tumor, while in the absence of amplifications, preoperative chemotherapy led to a sharp decrease in metastasis-free survival. This strategy of NAC prescription allowed us to achieve 93% metastatic survival in patients with breast cancer.

Amplifications of stemness genes and the capacity of breast tumors for metastasis.

INTRODUCTION: The phenomenon of non-CSC (cancer stem cell) to CSC plasticity has been previously described in multiple studies and occurs during the ectopic expression of stemness genes such as OCT3, SOX2, KLF4, MYC, NOTCH1, and NANOG. In our opinion, acquiring the ability to ectopically express stemness genes, selected by bioinformatics analysis and, accordingly, non-CSC to CSC plasticity, is due to amplification of genes at the following locations: 3q, 5p, 6p, 7q, 8q, 13q, 9p, 9q, 10p, 10q21.1, 16p, 18chr, 19p. This paper demonstrates the significance of stemness gene amplifications leading to metastasis and stem-like cancer cell activity. MATERIALS AND METHODS: In our studies, stemness gene amplifications were determined using the CytoScan HD Array. We studied the association of changes in stemness gene amplifications in tumors with metastasis treated with neoadjuvant chemotherapy (NAC) in 50 patients with breast cancer. We used qPCR to evaluate the expression of 13 stemness genes in tumors before and after NAC in 98 patients with breast cancer. Using primary cultures from the breast tumor of patient St23784/17 with stemness gene amplifications (SOX2, MYC, KLF4, NOTCH1, NODAL) and patient Ti41749/17 without stemness gene amplifications in the tumor, we studied the expression of stemness genes, proliferative tumor stem-cell activity, mammosphere formation, and expression of the CD44 tumor stem cell marker. RESULTS: The occurrence of amplifications at regions of stemness gene localization during NAC (22% cases) in residual tumors was associated with a very high metastasis rate (91% cases). Eliminating tumor clones with stemness gene amplifications using NAC (42% cases) led to 100% metastasis-free survival. In patients who developed hematogenic metastases after treatment, the expression of 7/13 stemness genes in the residual tumor after NAC was statistically higher than in patients without metastases. Primary cultures of EpCam+ tumor cells from patients with stemness gene amplifications revealed high proliferative activity. After the 3rd passage, the number of tumor cells increased 30-fold. Due to IL-6, this cell population showed a 2.5-fold increase in the EpCam+CD44hiCD24-/low and 2-fold decrease in the EpCam+CD44lowCD24- subpopulations of tumor stem cells; the formation of mammospheres was also observed. Primary cultures of EpCam+ tumor cells from the patient with no stemness gene amplifications had relatively low proliferative activity. IL-6 caused a 2.3-fold increase in the EpCam+CD44lowCD24- and 2-fold decrease in the EpCam+CD44hiCD24-/low subpopulations of tumor stem cells with no induction of mammospheres. CONCLUSIONS: The results of this study show that stemness gene amplifications in tumor cells are associated with metastasis and determine their potential stem property activation and non-CSC to CSC plasticity with the formation of EpCam+CD44hiCD24-/low cells, active proliferation, mammosphere formation, and metastasis.

DNA methylation markers panel can improve prediction of response to neoadjuvant chemotherapy in luminal B breast cancer.

Despite the advantages of neoadjuvant chemotherapy (NACT), associated toxicity is a serious complication that renders monitoring of the patients' response to NACT highly important. Thus, prediction of tumor response to treatment is imperative to avoid exposure of potential non-responders to deleterious complications. We have performed genome-wide analysis of DNA methylation by XmaI-RRBS and selected CpG dinucleotides differential methylation of which discriminates luminal B breast cancer samples with different sensitivity to NACT. With this data, we have developed multiplex methylation sensitive restriction enzyme PCR (MSRE-PCR) protocol for determining the methylation status of 10 genes (SLC9A3, C1QL2, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1, SKOR2 and GRIK1) that distinguish BC samples with different NACT response. Analysis of these 10 markers by MSRE-PCR in biopsy samples allowed us to reveal three top informative combinations of markers, (1) IRF4 and C1QL2; (2) IRF4, C1QL2, and ADCY8; (3) IRF4, C1QL2, and DPYS, with the areas under ROC curves (AUCs) of 0.75, 0.78 and 0.74, respectively. A classifier based on IRF4 and C1QL2 better meets the diagnostic panel simplicity requirements, as it consists of only two markers. Diagnostic accuracy of the panel of these two markers is 0.75, with the sensitivity of 75% and specificity of 75%.

Neoadjuvant Chemotherapy Induces the Appearance of New Copy Number Aberrations in Breast Tumor and is Associated with Metastasis.

BACKGROUND: In this study, we examined the CNA-genetic landscape (CNA - copy number aberration) of breast cancer prior to and following neoadjuvant chemotherapy (NAC) and correlated changes in the tumor landscape with chemotherapy efficiency as well as metastasis-free survival. OBJECTIVE: Breast cancer patients (n = 30) with luminal B molecular subtypes were treated with anthracycline- based therapy. METHODS: To study CNAs in breast tumors, microarray analysis was performed. RESULTS: Three effects of NAC on tumor CNA landscape were identified: 1 - the number of CNAbearing tumor clones decreased following NAC; 2 - there were no alterations in the number of CNAcontaining clones after NAC; 3 - the treatment with NAC increased the number of CNA-bearing clones (new clones appeared). All NAC-treated patients who had new tumor clones with amplification (20%) had a 100% likelihood of metastasis formation. In these cases, NAC contributed to the emergence of potential metastatic clones. Our study identified the following loci - 5p, 6p, 7q, 8q, 9p, 10p, 10q22.1, 13q, 16p, 18Chr and 19p - that were amplified during the treatment with NAC and maybe the markers of potential metastatic clones. In other patients who showed total or partial elimination of CNA-bearing cell clones, no new amplification clones were observed after NAC, and no evidence of metastases was found with follow-up for 5 years (р = 0.00000). CONCLUSION: Our data suggest that the main therapeutic result from NAC is the elimination of potential metastatic clones present in the tumor before treatment. The results showed the necessity of an intelligent approach to NAC to avoid metastasis stimulation.

Amplification of Stem Genes: New Potential Metastatic Makers in Patients with an Early Form of Breast Cancer.

BACKGROUND: According to our previous studies, the presence of amplifications of stem genes can lead to their ectopic expression and this is associated with an increased activity of tumor stem cells in these patients. This leads to a high aggressiveness of the tumor and the development of metastatic disease. The aim was to evaluate the prognostic significance of the presence of amplifications of stem genes and their expression in patients with early breast cancer (BC). METHODS: The study included 28 patients with T1NxM0 BC. We used surgical specimens, including formalin-fixed paraffin-embedded archive materials, for 8 patients. A microarray analysis was performed on high-density DNA chips from CytoScanHDArray to assess the status of copy number aberration (CNA) of stem genes locus. Gene expression was assessed using RT-qPCR. RESULTS: CNA analysis of the studied tumors of patients without chemotherapy showed that 17/18 patients without metastases did not have two or more amplifications of chromosomal regions. Ten patients had visceral metastases. In 9/10 of these patients in the primary tumor there were two or more amplifications of the stem genes locus. Two or more amplifications of stem genes locus were found in 12 patients with stage I. Hematogenous metastases did not develop in all patients. Comparison of metastasis-free survival rates in groups of patients with 1 or without amplifications and with two or more amplifications showed statistically significant differences (P = 0.01). CONCLUSION: Our studies have shown that the presence of clones with two or more amplifications of stem gene in patients with BC T1NxM0 has a significant prognostic value and determines an unfavorable prognosis for distant metastasis.

Impact of estrogen receptor α on the tamoxifen response and prognosis in luminal-A-like and luminal-B-like breast cancer.

The luminal-A-like and luminal-B-like breast cancer groups have distinct biological features that lead to differences in the treatment response and clinical outcome. The aim of this study was to examine the value of the distribution pattern of ERα expression, ESR1 SNPs as well as ESR1 mRNA expression in predicting tamoxifen response and survival in patients with luminal-A-like and luminal-B-like breast cancer. A total of 135 patients with both subtypes were stratified into two groups depending on the tamoxifen response: tamoxifen-resistant patients (TR) and tamoxifen-sensitive patients (TS). ESR1 mRNA expression was measured by real-time quantitative reverse transcription-PCR. Three polymorphisms of ESR1 (rs2077647, rs2228480 and rs1801132) were genotyped using a TaqMan assay. The distribution pattern of ERα expression was analyzed immunohistochemically using the visual assessment of staining. The primary endpoint was progression-free survival (PFS). There was a significant decrease in ESR1 mRNA expression level in the TR group when compared to the TS group among patients with luminal-B-like subtype (P = 0.038). ESR1 2014AA mutant genotype of rs2228480 was more prevalent in the TR patients with luminal-B-like subtype than the TS group (P = 0.045). In the luminal-A-like group, tamoxifen-resistant tumors were more frequently heterogeneous for ERα expression than tamoxifen-sensitive tumors (P = 0.016). Multivariate analysis showed a strong association of lymph node status and the distribution pattern of ERα expression with tamoxifen responsiveness in this cohort of patients. In addition, a luminal-A-like patients with the heterogeneous ERα expression had a significantly shorter PFS time than those with the homogeneous ERα (P = 0.013). These results indicate that the heterogeneous expression of ERα is an accurate predictor of tamoxifen response and survival in luminal-A-like breast cancer patients. ESR1 rs2228480 may act as a marker with a high prognostic potential in luminal-B-like tumors.

Genome-wide methylotyping resolves breast cancer epigenetic heterogeneity and suggests novel therapeutic perspectives.

Aim: To provide a breast cancer (BC) methylotype classification by genome-wide CpG islands bisulfite DNA sequencing. Materials & methods: XmaI-reduced representation bisulfite sequencing DNA methylation sequencing method was used to profile DNA methylation of 110 BC samples and 6 normal breast samples. Intrinsic DNA methylation BC subtypes were elicited by unsupervised hierarchical cluster analysis, and cluster-specific differentially methylated genes were identified. Results & conclusion: Overall, six distinct BC methylotypes were identified. BC cell lines constitute a separate group extremely highly methylated at the CpG islands. In turn, primary BC samples segregate into two major subtypes, highly and moderately methylated. Highly and moderately methylated superclusters, each incorporate three distinct epigenomic BC clusters with specific features, suggesting novel perspectives for personalized therapy.

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy.

In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PURPOSE: High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. METHODS: Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. RESULTS: We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. CONCLUSIONS: Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer.

The biological properties of circulating tumor cells (CTCs), and their dynamics during neoadjuvant chemotherapy are important, both for disease progression prediction and therapeutic target determination, with the aim of preventing disease progression. The aim of our study was to estimate of different CTC subsets in breast cancer during the NACT (neoadjuvant chemotherapy). The prospective study includes 27 patients with invasive breast cancer, T2-4N0-3M0, aged 32 to 60 years. Venous heparinized blood samples, taken before and after biopsy, after each courses of chemotherapy (on days 3-7), and before surgical intervention, served as the material for this study. Different subsets of circulating tumor cells were determined on the basis of the expression of EpCAM, CD45, CD44, CD24, and N-Cadherin using flow cytometry. As the result of this study, it has been observed that significant changes in the quantity of the different subsets of circulating tumor cells in patients' blood were observed after carrying out the 3rd course of NACT. NACT causes significant changes in the quantity of six CTC subsets, with various combinations of stemness and epithelial-mesenchymal transition (EMT) properties.

[Targeting of the AKT/m-TOR Pathway: Biomarkers of Resistance to Cancer Therapy--
AKT/m-TOR Pathway and Resistance to Cancer Therapy].

Resistance to cancer therapy continues to be a major limitation for the successful treatment of cancer. There are many published studies on therapy resistance in breast and prostate cancers; however, there are currently no data on molecular markers associated with resistance. The conflicting data were reported regarding the AKT/m-TOR signaling pathway components as markers predicting resistance. The AKT/m-TOR signaling pathway is involved in the development of many human cancers; its activation is related to cell proliferation, angiogenesis, apoptosis, as well as to therapy resistance. Molecular alterations in the AKT/m-TOR signaling pathway provide a platform to identify universal markers associated with the development of resistance to cancer therapy.

Predictive value of vascular endothelial growth factor receptor type 2 in triple-negative breast cancer patients treated with neoadjuvant chemotherapy.

The identification of informative biomarkers that could predict the treatment response is particularly important in the triple-negative (TN) breast cancer, which is characterized by biological diversity. The aim of this study was to investigate the impact of vascular endothelial growth factor receptor (VEGFR2) expression and its gene polymorphisms on pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) in Russian patients with TN breast cancer. We performed a retrospective analysis of 70 women with operable TN breast cancer, who underwent NCT with 5-fluorouracil, adriamycin, and cyclophosphamide (FAC) or cyclophosphamide, adriamycin, and capecitabine (CAX) between 2007 and 2013. VEGFR2 expression was evaluated before NCT by immunohistochemistry. TaqMan SNP assays were used for genotyping KDR - 604T>C (rs2071559) and KDR 1192G>A (rs2305948) polymorphisms. The pCR was used as an end-point in the treatment efficacy analysis. In the univariate analysis, the pCR rate was strongly associated with young age (P = 0.004), high Ki67 expression (P = 0.012), lymph node negativity (P = 0.023) as well as with positive VEGFR2 expression (P = 0.019) and the CAX regimen (P = 0.005). In the multivariate analysis, only patient's age (P = 0.005) and pre-NCT VEGFR2 expression (P = 0.048) remained significant predictors of pCR. The pCR rate was higher in the CAX-treated patients than that in the FAC-treated patients (P = 0.005). Our results revealed that - 604TT genotype of rs2071559 and age < 50 years were correlated with a pCR in the CAX-treated patients. VEGFR2 expression in pre-NCT tumors and KDR gene polymorphism can be considered as additional predictive molecular markers of pCR in Russian TN breast cancer patients treated with NCT.

Effect of Targeted Therapy With Pazopanib on Expression Levels of Transcription, Growth Factors and Components of AKT/m-TOR Signaling Pathway in Patients with Renal Cell Carcinoma

Background: The effect of the targeted therapy on cancer molecular markers remains currently unknown. The aim of the study was to investigate the expression and content of transcription, growth factors and components of the AKT/m-TOR signaling pathway in kidney cancer patients before and after targeted therapy with pazopanib. Methods: A total of 157 patients with renal cell carcinoma were enrolled into the study. The level of mRNA expression was investigated by real-time PCR, and the contents of transcription and growth factors, as well as the levels of AKT/m- TOR signaling pathway components were determined by ELISA and Western blotting. Results: Targeted therapy with pazopanib resulted in a 3.1-fold decrease in HIF-2α expression that was accompanied by a reduction in the levels of NF-κB p65 and p50, HIF-1α and CAIX. The levels of GSK-3ß and AKT mRNA were increased; however, the levels of corresponding proteins remained low. The targeted therapy with pazopanib did not influence the level of PTEN phosphatase. A 1.9-fold increase in the level of p70 S6 (S371) was observed after therapy. Conclusion: The efficacy of tyrosine kinase inhibitors is associated with the changes in the angiogenic factors. Molecular characteristics of cancer could determine markers of disease progression as well as potential targets for anticancer therapies

Genetic variability in the regulation of the expression cluster of MDR genes in patients with breast cancer.

PURPOSE: We aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC). MATERIALS AND METHODS: The MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates. RESULTS: The SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs. CONCLUSIONS: Four MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

The distribution pattern of ERα expression, ESR1 genetic variation and expression of growth factor receptors: association with breast cancer prognosis in Russian patients treated with adjuvant tamoxifen.

Identification of additional biomarkers associated with ER genomic and nongenomic pathways could be very useful to distinguish patients who will benefit from tamoxifen treatment. The aim of this study was to analyze the prognostic significance of the distribution pattern of ERα expression, ESR1 gene single-nucleotide polymorphisms and expression levels of growth factor receptors in Russian hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen. Formalin-fixed paraffin-embedded tumor tissue samples from 97 patients were examined for the distribution pattern of ERα expression, as well as for EGFR and TGF-ßR1 expression by immunohistochemistry. Genotypes for ESR1 +30T>C (rs2077647) and ESR1 2014G>A (rs2228480) were analyzed using a TaqMan assay. Progression-free survival (PFS) was used as an endpoint for the survival analyses. We found that patients with the heterogeneous distribution of ERα expression had poor prognosis on tamoxifen treatment (P = 0.021). We identified a high EGFR expression in patients who developed distant metastasis or recurrence during tamoxifen treatment (a tamoxifen-resistant group-TR) in contrast to the distant metastasis-free patients (a tamoxifen-sensitive group-TS) (80.0 vs. 41.9 %, respectively, P = 0.009). Carriers of the ESR12014A mutant allele were more prevalent among the TR patients compared to the TS patients (26.3 vs. 8.0 %, respectively, P = 0.009). EGFR expression and the distribution pattern of ERα expression were associated with the response to tamoxifen by both univariate and multivariate logistic regression analyses. The presence of these markers either alone or in combination was correlated with the worse PFS for all patients. Analysis of the distribution pattern of ERα expression and the EGFR status in tumor tissue may be valuable for patient selection for tamoxifen adjuvant therapy.