Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects

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 cancerpromoting 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

Age-related function of tumor suppressor gene TP53:contribution to cancer risk and progression

Aim: To examine the influence of combined genotypes of TP53 (exon 4, intron 3, intron 6) and XRCC1 (codon 10) on lung cancer age of onset. Methods: TP53 polymorphisms in codon 72 of exon 4 (Arg72Pro), in intron 3 (16 bp duplication), in intron 6 (G/A transition) and XRCC1 polymorphism in codon 10 (Arg399Gln) were analyzed in blood cells of 177 lung cancer patients and 196 healthy donors with Restriction Fragment Lenth Polymorphism PCR. Results: We showed that combination of TP53 variant genotypes and XRCC1 variant genotype is associated with the increased lung cancer risk in younger, but not elderly, smokers. In contrast, wild allele combination increases lung cancer risk for individuals over the age of 60. Conclusion: Our data confirm antagonistic pleiotropy hypothesis indicating that p53 protects the organism against cancer early in life, but promoting aging phenotype, including late life cancer in older persons

The phenomenon of multi-drug resistance in the treatment of malignant tumors

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

Genome-wide association study of loss of heterozygosity and metastasis-free survival in breast cancer patients

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 metastasisfree 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

Smoking-related DNA adducts as potential diagnostic markers of lung cancer: new perspectives

In recent years, the new direction such as identification of informative circulating markers reflecting molecular genetic changes in the DNA of tumor cells was actively developed. Smoking-related DNA adducts are very promising research area, since they indicate high pathogenetic importance in the lung carcinogenesis and can be identified in biological samples with high accuracy and reliability using highly sensitive mass spectrometry methods (TOF/TOF, TOF/MS, MS/MS). The appearance of DNA adducts in blood or tissues is the result of the interaction of carcinogenic factors, such as tobacco constituents, and the body reaction which is determined by individual characteristics of metabolic and repair systems. So, DNA adducts may be considered as a cumulative mirror of heterogeneous response of different individuals to smoking carcinogens, which finally could determine the risk for lung cancer. This review is devoted to analysis of the role of DNA adducts in lung carcinogenesis in order to demonstrate their usefulness as cancer associated markers. Currently, there are some serious limitations impeding the widespread use of DNA adducts as cancer biomarkers, due to failure of standardization of mass spectrometry analysis in order to correctly measure the adduct level in each individual. However, it is known that all DNA adducts are immunogenic, their accumulation over some threshold concentration leads to the appearance of long-living autoantibodies. Thus, detection of an informative pattern of autoantibodies against DNA adducts using innovative multiplex ELISA immunoassay may be a promising approach to find lung cancer at an early stage in high-risk groups (smokers, manufacturing workers, urban dwellers). Key Words: lung cancer, DNA adducts, tobacco smoking

Different morphological structures of breast tumors demonstrate individual drug resistance gene expression profiles

Aim: To identify gene expression profiles involved in drug resistance of different morphological structures (tubular, alveolar, solid, trabecular, and discrete) presented in breast cancer. Material and Methods: Ten patients with luminal breast cancer have been included. A laser microdissection-assisted microarrays and qRT-PCR were used to perform whole-transcriptome profiling of different morphological structures, to select differentially expressed drug response genes, and to validate their expression. Results: We found 27 differentially expressed genes (p < 0.05) encoding drug uptake (SLC1A3, SLC23A2, etc.) and efflux (ABCC1, ABCG1, etc.) transporters, drug targets (TOP2A, TYMS, and Tubb3), and proteins that are involved in drug detoxification (NAT1 and ALDH1B1), cell cycle progression (CCND1, AKT1, etc.), apoptosis (CASP3, TXN2, etc.), and DNA repair (BRCA1 and USP11). Each type of structures showed an individual gene expression profile related to resistance and sensitivity to anticancer drugs. However, most of the genes (19/27; p < 0.05) were expressed in alveolar structures. Functional enrichment analysis showed that drug resistance is significantly associated with alveolar structures. Other structures demonstrated the similar number (10–13 out of 27) of expressed genes; however, the spectrum of resistance and sensitivity to different anticancer drugs varied. Conclusion: Different morphological structures of breast cancer show individual expression of drug resistance genes. Key Words: breast cancer, tumor heterogeneity, gene expression, chemotherapy, drug resistance

Different morphological structures of breast tumors demonstrate individual drug resistance gene expression profiles

Aim: To identify gene expression profiles involved in drug resistance of different morphological structures (tubular, alveolar, solid, trabecular, and discrete) presented in breast cancer. Material and Methods: Ten patients with luminal breast cancer have been included. A laser microdissection-assisted microarrays and qRT-PCR were used to perform whole-transcriptome profiling of different morphological structures, to select differentially expressed drug response genes, and to validate their expression. Results: We found 27 differentially expressed genes (p < 0.05) encoding drug uptake (SLC1A3, SLC23A2, etc.) and efflux (ABCC1, ABCG1, etc.) transporters, drug targets (TOP2A, TYMS, and Tubb3), and proteins that are involved in drug detoxification (NAT1 and ALDH1B1), cell cycle progression (CCND1, AKT1, etc.), apoptosis (CASP3, TXN2, etc.), and DNA repair (BRCA1 and USP11). Each type of structures showed an individual gene expression profile related to resistance and sensitivity to anticancer drugs. However, most of the genes (19/27; p < 0.05) were expressed in alveolar structures. Functional enrichment analysis showed that drug resistance is significantly associated with alveolar structures. Other structures demonstrated the similar number (10–13 out of 27) of expressed genes; however, the spectrum of resistance and sensitivity to different anticancer drugs varied. Conclusion: Different morphological structures of breast cancer show individual expression of drug resistance genes. Key Words: breast cancer, tumor heterogeneity, gene expression, chemotherapy, drug resistance

ЭТНИЧЕСКИЕ АСПЕКТЫ НАСЛЕДСТВЕННОГО РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ В РЕГИОНЕ СИБИРИ

Background: Ethnic diversity of the population in the region of Siberia suggests the existence of different germline mutations in the BRCA1/2 genes associated with breast and ovarian cancer in different ethnic populations, but spectrum of these mutations has not been studied. Objective: Our aim was to evaluate the frequency of the most common mutations BRCA1 / 2 (BRCA1 5382insC, BRCA1 185delAG, BRCA1 4153delAG, BRCA1 T300G, BRCA2 6174delT) in women diagnosed with breast cancer among indigenous people and newcomers living in Siberia. Methods: We tested 1281 genomic DNA samples for the presence of BRCA1 5382insC mutation in patients diagnosed with breast cancer considering no family history. 72 patients having hereditary cancer signs were tested for the mutations BRCA1 185delAG, BRCA1 4153delAG, BRCA1 T300G, BRCA2 6174delT. Results: Out of 765 patients of Slavic ethnic group, 27 women (3.5%) were carriers of allele BRCA1 5382insC. The frequencies of mutations in patients with signs of hereditary cancer were: 8.3% in group of young patients (under 40 years), 20.0% in patients with bilateral cancer and 5.7% in patients with family history of breast or ovarian cancers. We tested 516 BC patients residing on the territory of the Buryat-Aginsky district, Republics of Tyva and Altai. Out of them, there were 197 patients among the indigenous population (buryats, tuvinians, altaians), and 319 patients among newcomers (Slavic ethnics). Mutations BRCA1 5382insC were detected only in women from Slavic ethnic groups. The frequency of BRCA1 5382insC mutation was 6% in the group where family history was excluded and 14% in the group of patients with characteristics of family cancer. Allele BRCA1 5382insC was not found in indigenous breast cancer patients, although 59 patients had signs of hereditary cancer. In women from Slavic ethnic group, the BRCA1 185delAG, BRCA1 4153delAG and BRCA1 T300G mutations were detected in 9.1% of cases and were not found in patients among the indigenous population. Conclusion: studies of mutations in the BRCA1 gene in breast cancer patients from Siberia confirmed data on the high frequency of «founder mutation» BRCA1 5382insC in Slavic population and indicate the advisability of further studies to identify the genes responsible for the occurrence of hereditary breast cancer in the indigenous population. Обоснование: этническая разнородность населения Сибири предполагает наличие разных наследственных мутаций в генах BRCA1/2, ассоциированных с раком молочной железы (РМЖ) и раком яичников в различных популяциях, спектр которых не изучен. Цель исследования: оценить частоту встречаемости наиболее распространенных в РФ мутаций BRCA1/2 (BRCA1 5382insC, BRCA1 185delAG, BRCA1 4153delAG, BRCA1 T300G, BRCA2 6174delT) у представительниц коренного и пришлого населения Сибири. Методы: протестирован 1281 образец геномной ДНК на наличие распространенных мутаций у больных с диагнозом РМЖ. Результаты: из 765 больных РМЖ славянской принадлежности 27 человек (3,5%) были носителями аллеля BRCA1 5382insC. Частота мутации у пациенток с признаками наследственного рака составила: у молодых пациенток до 40 лет — 8,3%, у пациенток с билатеральным раком — 20,0%, при отягощенном семейном анамнезе — 5,7%. На наличие мутации BRCA15382insC протестировано 516 больных РМЖ из Агинского бурятского автономного округа, республик Тыва и Алтай, из них 319 женщин пришлого населения (славянки) и 197 представительниц коренного населения (бурятки, тувинки, алтайки). Мутации обнаружены только у славянок, частота без учета семейного анамнеза составила 6%, с учетом признаков семейного рака — около 14%. У больных РМЖ коренного населения мутация не обнаружена, хотя 59 пациенток имеют признаки наследственного рака. Мутации BRCA1 185delAG, BRCA1 4153delAG и BRCA1 T300G выявлены у славянок в 6,7% случаев и не выявлены у представительниц коренного населения. Заключение: получены данные о высокой частоте встречаемости «мутации-основателя» 5382insC у больных РМЖ славянской популяции и отсутствии «славянских» мутаций у больных РМЖ женщин монголоидного происхождения. Актуальны исследования по выявлению генов наследственного рака молочной железы у представительниц коренного населения.

DIFFERENT MORPHOLOGICAL STRUCTURES OF BREAST TUMORS DEMONSTRATE INDIVIDUAL DRUG RESISTANCE GENE EXPRESSION PROFILES

Aim: To identify gene expression profiles involved in drug resistance of different morphological structures (tubular, alveolar, solid, trabecular, and discrete) presented in breast cancer. Material and Methods: Ten patients with luminal breast cancer have been included. A laser microdissection-assisted microarrays and qRT-PCR were used to perform whole-transcriptome profiling of different morphological structures, to select differentially expressed drug response genes, and to validate their expression. Results: We found 27 differentially expressed genes (p < 0.05) encoding drug uptake (SLC1A3, SLC23A2, etc.) and efflux (ABCC1, ABCG1, etc.) transporters, drug targets (TOP2A, TYMS, and Tubb3), and proteins that are involved in drug detoxification (NAT1 and ALDH1B1), cell cycle progression (CCND1, AKT1, etc.), apoptosis (CASP3, TXN2, etc.), and DNA repair (BRCA1 and USP11). Each type of structures showed an individual gene expression profile related to resistance and sensitivity to anticancer drugs. However, most of the genes (19/27; p < 0.05) were expressed in alveolar structures. Functional enrichment analysis showed that drug resistance is significantly associated with alveolar structures. Other structures demonstrated the similar number (10–13 out of 27) of expressed genes; however, the spectrum of resistance and sensitivity to different anticancer drugs varied. Conclusion: Different morphological structures of breast cancer show individual expression of drug resistance genes. Key Words: breast cancer, tumor heterogeneity, gene expression, chemotherapy, drug resistance