We studied the association between the presence of 2 or more stemness gene amplifications as well as copy number aberrations (CNAs) of WNT signaling genes in residual breast tumor and metastasis. WNT pathway genes associated with metastasis were identified.Material and Methods. The study included 30 patients with breast cancer, who had 2 or more stemness gene amplifications in the residual tumor after neoadjuvant chemotherapy. Fifteen of the thirty patients developed hematogenous metastases; they constituted a group with metastases, the remaining 15 patients entered the second group without metastases. The tumor DNA was examined using a CytoScanHD Array microarray (Affymetrix, USA).Results. By subtracting amplification and deletion frequencies in 852 cytobands between groups with metastases and without metastases, 21 cytobands were identified with the largest difference in deletion and amplification frequencies. They contain 19/150 of WNT genes (12 activators: SKP1, WNT8A, MAPK9, CCND3, FZD9, WNT8B, CCND1, PLCB2, PRKCB, FZD2, WNT3, WNT9B and 7 negative regulators: GSK3B, APC, CSNK2B, SFRP5, BTRC, TCF7L2, CSNK2A2). A point system was developed: when amplifying WNT-signaling activators or deletion of negative regulators, one point was added to the total score, and vice versa when deleting WNT-signaling activators or amplification of negative regulators, one point was taken from the total amount. It was shown that 93% (14/15) of patients with metastases had a total score higher than 0, while 93% (14/15) of patients without metastases had a total score of zero or less than zero. The differences between the groups were statistically significant according to the two-sided Fisher test with a high level of confidence probability (p=0.000003) and the log-rank test (p=0.00004) when assessing non-metastatic survival by the Kaplan-Mayer method.Conclusion. Nineteen WNT signaling genes were identified. Copy number aberrations of these genes in combination with stemness gene amplifications in residual tumors were associated with metastasis. A new highly effective prognostic factor for breast cancer was identified.

Известно, что дефицит гомологичной рекомбинации в опухолевых клетках, обусловленный, в основном, дефектом генов BRCA1/2, связан с высокой эффективностью лечения и благоприятным прогнозом заболевания. Однако наличие других альтернативных путей репарации ДНК, таких как активация генов NF-κB или PARP1, может оказывать дополнительный негативный эффект. Таким образом, целью работы явилась оценка связи аберраций числа копий ДНК генов BRCA1, NF-κB, PARP1 в опухолевой ткани молочной железы с эффектом химиотерапии и прогнозом заболевания. Материалы и методы. В исследование было включено 85 больных раком молочной железы IIA-IIIB стадии. ДНК выделяли из биопсийных образцов опухолевой ткани с использованием набора QIAamp DNA mini Kit (Qiagen, Germany). Было проведено микроматричное исследование всех образцов опухоли на ДНК-чипах высокой плотности фирмы Affymetrix CytoScanTM HD Array. Для оценки аберраций числа копий ДНК использовали программу «Chromosome Analysis Suite 3.3». Результаты. Было установлено, что наибольшая частота делеций наблюдается в гене BRCA1 (28%, 24 случая из 85), и это статистически значимо сопряжено с объективным ответом на неоадъювантную химиотерапии (p=0,02). Частота амплификации гена PARP1 в исследуемой группе больных составляет 62%, что также определяет хороший ответ на неоадъювантную химиотерапию вне зависимости от наличия аберраций других исследуемых генов. При анализе прогностической значимости аберраций генов было показано, что амплификация гена PARP1 является неблагоприятным маркером безметастатической выживаемости (log-rank test, p=0,02). Выводы. На основании полученных данных можно полагать, что аберрантное состояние генов BRCA1 и PARP1 в опухоли молочной железы, может также являться перспективным маркером эффективности химиотерапии и прогноза заболевания, что подтверждает актуальность исследования, но и требует дальнейшего детального изучения. It is well known that the presence in the tumor cells of such a phenomenon as a deficiency of homologous recombination, caused mainly by a defect in the BRCA1/2 genes, is associated with a favorable treatment effect and prognosis of the disease. But it has been established that the presence of other alternative ways of DNA repair, such as activation of the NF-κB or PARP1 genes, may have an additional negative effect. Thus, the aim of the work was to assess the association of chromosomal aberrations of the BRCA1, NF-κB, PARP1 genes in tumor breast tissue with the effect of chemotherapy and the prognosis of the disease. Materials and methods. The study included 85 patients with stage IIA - IIIB breast cancer. DNA was isolated from biopsy specimens of tumor tissue using the QIAamp DNA mini Kit (Qiagen, Germany). A microarray was studied for all tumor samples on Affymetrix CytoScanTM HD Array high-density DNA chips. To estimate the aberrations of the number of DNA copies, the program Chromosome Analysis Suite 3.3 was used. Results. It was found that the highest frequency of deletions is observed in the BRCA1 gene (28%, 24 cases out of 85), and this is statistically significantly associated with an objective response to neoadjuvant chemotherapy (p=0.02). The frequency of amplification of PARP1 in the studied group of patients is 62%, which also determines the presence of a good response to neoadjuvant chemotherapy, regardless of the presence of chromosomal aberrations of other have study genes. When analyzing the prognostic significance of gene aberrations, it was shown that PARP1 amplification is an unfavorable marker of metastatic-free survival (log-rank test, p=0.02). Conclusion. Based on the data obtained, it can be assumed that the aberrant state of the BRCA1 and PARP1 genes in a breast cancer may also be a promising marker of the effectiveness of chemotherapy and disease prognosis, which confirms the undoubted relevance of the study, but also requires further detailed study.

Background: Hypothesis: Non-stem (differentiated and progenitor) cancer cells can form metastases by performing a reverse transition to cancer stem cells (CSC) in organs under the influence of cytokines (SST - Somatic-Stem Transition). The ability to SST is acquired in the course of evolution and is ensured by ectopic expression of several stemness genes (such as SOX2, OCT3/4, MYC, KLF4, NOTCH1, NANOG ...), due to amplifications in chromosomal regions of their localization (3q, 5p, 6p, 7q, 8q, 13q, 9p, 9q, 10p, 10q21.1, 16p, 18chr, 19p). If there are no amplifications in the tumor, it is not capable of SST and will not metastasize. Methods: Breast cancer cells of two patients were used to induce a SST. One patient had amplifications of 3q, 6q, 8q, 9q, 10q22.1 in the tumor, in which the SOX2, MYC, KLF4, NOTCH1, NODAL genes were localized. The other patient had no amplifications of stemness genes in the tumor. Magnetic separation was used to extract populations of EpCAM+CD44- tumor cells of both patients. SST was IL6-induced. Following this, the content of EpCAM+CD44+CD24- CSC was evaluated via flow cytometry, the increase in the number of cells after 3 passages, and the induction of mammospheres were measured. Results: Under the influence of IL6, CSC emerged in the population of EpCAM+CD44- tumor cells with amplifications, the number of cells after 3 passages increased by a factor of 33, and mammospheres of 7-15 cells were induced. While SST was not induced in the population of EpCAM+CD44- tumor cells taken from the patient with no amplifications, the number of cells after 3 passages was increased only by a factor of 5 and no mammospheres were formed. Prospective trials: in 11 breast cancer patients with stemness genes amplifications in the tumor, neoadjuvant chemotherapy (NAC) eliminated the clones with amplifications. All patients have a metastatic-free survival. 11 patients had no amplifications and they did not undergo NAC. All patients had100% survival. 9 patients had no amplifications of their stemness genes before the treatment, and NAC induced their occurrence, 90% of the patients developed metastases. Conclusions: We showed an SST in EpCam+CD44- tumor cells, the importance of amplifications of stemness genes loci for its induction, and the importance of amplifications of stemness genes for metastasis. Legal entity responsible for the study: Cancer Research Institute, Tomsk National Research Medical Center RAS, Russia. Funding: The Russian Science Foundation grant 17-15-01203. Disclosure: All authors have declared no conflicts of interest.

Background: Individual chemotherapy based on the determination of molecular biomarkers of chemosensitivity is a new way to treat patients with NSCLC. Promising markers for chemosensitivity are monoresistance genes such as BRCA1, RRM1, ERCC1, TOP1, TOP2α, TUBB3, TYMS, and ABCC5. Methods: We enrolled and analyzed 62 patients with stage III NSCLC. All the patients have received 2 courses of neoadjuvant chemotherapy vinorelbine/carboplatin and surgery. Then patients were randomly assigned (1:1 ratio) to either the personalized adjuvant chemotherapy arm (main group) or the adjuvant chemotherapy vinorelbine/carboplatin arm (control group). In the main group, carboplatin-containing doublets were assigned based on monoresistance gene expression levels ABCC5, RRM1, ERCC1, BRCA1, TOP1, TOP2α, TUBB3 and TYMS. RNA was extracted from tumor after neoadjuvant chemotherapy using “RNeasy Plus Mini Kit” (QIAGEN, Germany). The analysis of monoresistance genes expression was done by qRT-PCR method. A χ2 test was used to analyze gene expression in relation to clinicopathological parameters. The survival rates were calculated by the Kaplan-Meier method. Results: The follow-up period was 4 - 76 months. In the main group, the disease progression was observed in 6 patients (19.4%), in the control group - 15 patients (48.4%). Three-year disease-free survival in the main group was 80.7% (median DFS not achieved), in the control group - 51.6%, median DFS - 34 months (HR: 2.56, 95% CI: 1.09 - 6,03); differences are statistically significant: Log-Rank test χ2=4.196, p = 0.041. There was no difference in three-year overall survival (main group: 87.1%, control group: 67.7%, HR: 2.27, 95% CI: 0.79 - 6,47). Conclusions: Personalized postoperative chemotherapy based on the determination of monoresistance gene expression after neoadjuvant chemotherapy allows significant increase of patients 3-year disease-free survival by 29.1%. Legal entity responsible for the study: Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Purpose of work. To perform a genome-wide association study of loss of heterozygosity (LOH) with monoresistance genes expression during neoadjuvant chemotherapy (NAC) in breast cancer. Materials and methods. The study involved 68 patients with breast cancer. The tumour stages were IIAIIIB. RNA was extracted from tissue specimens (before and after NAC) using RNeasy Plus mini Kit (Qiagen, Germany). Expression profiling of the RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, BRCA1 genes was carried out using quantitative real-time PCR (qPCR). DNA was extracted from 68 biopsy specimens of tumour tissues using QIAamp DNA mini Kit (Qiagen, Germany). LOH status was detected using microarray analysis using high density DNA-chip manufactured by Affymetrix CytoScanTM HD Array company. Results. As a result of the study of loss of heterozygosity was evaluated in 13815 genes. The frequency of LOH varied from 0% to 63%. The highest incidence of heterozygosity loss events is characteristic for genes of 16, 17 and the X-chromosome. Our study established that the phenomenon of loss of heterozygosity in monoresistance genes (BRCA1, ERCC1, RRM1, TOP1, TOP2A, TUBB3 and TYMS), is not associated with their level of expression in the tumor. A statistical association has been found between LOH and level of expression of the studied genes in 54 genes. Among them it is necessary to note genes encoding miRNA and «zinc fingers» involved in the regulation of transcription of many genes, transmembrane drug transporters and ion channels, genes of the MAP kinase signaling pathway, and others. Conclusion. The results of this study allow for the more exact determination of the expression picture of monoresistance genes in the tumor and the indication of new candidate genes which are involved in the regulation of the expression of these genes. Evaluation of the loss of heterozygosity in tumor tissue can be used as an additional criterion for personalizing chemotherapy.