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It is known that complete pathomorphological response (pCR) after neoadjuvant therapy (NAC) in patients with breast cancer (BC) correlates with higher rates of recurrence-free and overall survival. In turn, the widespread use of neoadjuvant therapy for the treatment of breast cancer defines the clinical need for prognostic markers of response to ongoing therapy. Currently, some clinicopathological prognostic factors are used to assess the potential benefit of neoadjuvant systemic therapy for female patients, but they have limited applicability. In the era of precision medicine and personalised treatment, a search for new prognostic markers is needed to better tailor patient-specific therapy. To date, novel factors have been proposed to predict response to preoperative treatment in breast cancer patients, but they are either not yet used in routine clinical practice or have limited application. Thus, this review summarises data on both established and proven biomarkers and the latest prognostic factors for response to neoadjuvant treatment in breast cancer patients. more...

Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs., Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified., Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer., Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance., Competing Interests: Authors DP and PK were employed by company Genomed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Larionova, Kiselev, Kazakova, Liu, Patysheva, Iamshchikov, Liu, Mossel, Riabov, Rakina, Sergushichev, Bezgodova, Vtorushin, Litviakov, Denisov, Koshkin, Pyankov, Tsyganov, Ibragimova, Cherdyntseva and Kzhyshkowska.) more...

Tumour stem cells (CSCs) are a self-renewing population that plays important roles in tumour initiation, recurrence, and metastasis. Although the medical literature is extensive, problems with CSC identification and cancer therapy remain. This review provides the main mechanisms of CSC action in breast cancer (BC): CSC markers and signalling pathways, heterogeneity, plasticity, and ecological behaviour. The dynamic heterogeneity of CSCs and the dynamic transitions of CSC - non-CSCs and their significance for metastasis are considered. more...

Aim: To study the peculiarities of ecological relationships of breast cancer (BC) cell lines MCF-7, BT-474 and MDA-MD-231 under co-culturing conditions., Materials and Methods: Three BC cell lines: luminal A - MCF-7, luminal B - BT-474 and triple-negative - MDA-MD-231 were co-cultured pairwise. Immunocytochemistry was used to differentiate the cell lines in the wells. The effect of the cell-free culture medium on the growth rate of the alternate cell line in the pair was also evaluated., Results: It was shown that when BT-474 cells were co-cultured with MCF-7 and BT-474 cells were co-cultured with MDA-MD-231, two types of ecological interactions could be observed: commensalism and amensalism, respectively. While the cells do not interact with each other in contact, the supernatants of single cultures of MCF-7 and MDA-MD-231 exert the same effect on BT-474 as co-cultivation of BT-474 with these cells., Conclusions: The paracrine mechanism of intercellular interaction between different human BC cell lines has been demonstrated. The models used in population ecology can be applicable to identify the types of interaction between cell lines. more...

The MYC and OCT4 genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal™ 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7 - EpCAM - CD44 +/- Myc +/- (Oct4, Snai2) +/- ) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7 + EpCam + CD44 + Snai2 - ). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7 + EpCam + CD44 - Myc + ) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7 + EpCam + CD44 - OCT4 + ). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326 + CD44 + CD24 - CD325 + ) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326 + CD44 + CD24 + Oct4 + ) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis. more...

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. more...

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-βR1 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. more...

One of the factors providing the diversity and heterogeneity of malignant tumors, particularly breast cancer, are genetic variations, due to gene polymorphism, and, especially, the phenomenon of loss of heterozygosity (LOH). It has been shown that LOH in some genes could be a good prognostic marker., Aim: To perform genome-wide study on LOH in association with metastasis-free survival in breast cancer., Materials and Methods: The study involved 68 patients with breast cancer. LOH status was detected by microarray analysis, using a high density DNA-chip CytoScanTM HD Array (Affymetrix, USA). The Chromosome Analysis Suite 3.1 (Affymetrix, USA) software was used for result processing., Results: 13,815 genes were examined, in order to detect LOH. The frequency of LOH varied from 0% to 63%. The association analysis identified four genes: EDA2R, PGK1, TAF9B and CYSLTR1 that demonstrated the presence of LOH associated with metastasis-free survival (log-rank test, p < 0.03)., Conclusions: The presence of LOH in EDA2R, TAF9B, and CYSLTR1 genes is associated with metastasis-free survival in breast cancer patients, indicating their potential value as prognostic markers. more...

Each patient has a unique history of cancer ecosystem development, resulting in intratumor heterogeneity. In order to effectively kill the tumor cells by chemotherapy, dynamic monitoring of driver molecular alterations is necessary to detect the markers for acquired drug resistance and find the new therapeutic targets. To perform the therapeutic monitoring, frequent tumor biopsy is needed, but it is not always possible due to small tumor size or its regression during the therapy or tumor inaccessibility in advanced cancer patients. Liquid biopsy appears to be a promising approach to overcome this problem, providing the testing of circulating tumor cells (CTC) and/or tumor-specific circulating nucleic acids. Their genomic characteristics make it possible to assess the clonal dynamics of tumors, comparing it with the clinical course and identification of driver mutation that confer resistance to therapy. The main attention in this review is paid to CTC. The biological behavior of the tumor is determined by specific cancer-promoting molecular and genetic alterations of tumor cells, and by the peculiarities of their interactions with the microenvironment that can result in the presence of wide spectrum of circulating tumor clones with various properties and potentialities to contribute to tumor progression and response to chemotherapy and prognostic value. Indeed, data on prognostic or predictive value of CTC are rather contradictory, because there is still no standard method of CTC identification, represented by different populations manifesting various biological behavior as well as different potency to metastasis. Circulating clasters of CTC appear to have essentially greater ability to metastasize in comparison with single CTC, as well as strong association with worse prognosis and chemoresistance in breast cancer patients. The Food and Drug Administration (USA) has approved the CTC-based prognostic test for clinical application in patients with advanced breast cancer. Prospective clinical trials have demonstrated that measuring changes in CTC numbers during treatment is useful for monitoring therapy response in breast cancer patients. Molecular and genetic analysis of CTC gives the opportunity to have timely information on emergence of resistant tumor clones and may shed light on the new targets for pathogenetic antitumor therapy. more...

Previously, we showed the association of neoadjuvant chemotherapy (NAC) response with changing the expression vector (increase or decrease) of multidrug resistance genes (MDR) in breast tumors during chemotherapy. The aim of the present study was to evaluate the relation between changes in the expression vector of MDR genes and distant metastasis-free survival. Patients (n = 120) with breast cancer (T1-4N0-3M0) treated by 2-4 cycles of NAC (CAX, FAC, and taxane regimes) and 4 cycles of adjuvant chemotherapy (FAC) were included. TaqMan-based quantitative reverse transcriptase PCR (qRT-PCR) was used to estimate the expression of the following MDR genes: ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2, GSTP1, and MVP--in biopsies before NAC and in tumor samples after chemotherapy. Comparing the corresponding expression levels allowed us to identify the vector of expression change during NAC. The results showed that 5-year distant metastasis-free survival was 73-78% in patients with a decrease in ABCB1, ABCC2, and ABCG1 expression. The up-regulation of these genes during NAC was related to a significant decrease (up to 50-55%) in metastasis-free survival (Kaplan-Meier analysis: log-rank p value = 0.006-0.03). The association of changing the expression vector of MDR genes with metastasis-free survival did not depend on tumor size, lymph node involvement, histological form, receptor status, molecular subtype, and others clinicopathological parameters of breast cancer. The obtained data suggest that changing the expression vector of MDR genes in breast tumors during NAC may be used as a new potential prognostic marker of breast cancer. An increase in tumor expression of ABCB1, ABCC2, and ABCG1 during chemotherapy is a factor of poor prognosis, whereas down-regulation of these genes--a favorable prognostic marker. more...

This study involved 525 breast cancer (BC) patients of T2-4N0-2M0 stages at the age of 35 years and older. Significant differences in clinical and pathological characteristics between premenopausal and postmenopausal BC patients were found. Mostly marked differences were shown for positive lymph node correlation with distant metastasis, multicentric growth and local recurrence depending on menopause status. The prevalence of various morphological structures in primary tumors was appeared to be associated with different forms of tumor progression in pre- and postmenopausal women. We have studied polymorphisms in 15 genes involved in major cancer related pathways (apoptosis, interleukins, folate metabolism enzymes genes). We found that variant genotypes of MTHFR and DHFR genes were associated with an increased BC risk among premenopausal women while polymorphism in IL-18, p53 genes were associated with BC among postmenopausal women. These results demonstrate novel biological information, which points the different mechanisms contributed to breast cancer progression in premenopausal and postmenopausal women. more...