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.

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.

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.

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.

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.