Use of germline genetic variability for prediction of chemoresistance and prognosis of breast cancer patients

The aim of our study was to set up a panel for targeted sequencing of chemoresistance genes and the main transcription factors driving their expression and to evaluate their predictive and prognostic value in breast cancer patients. Coding and regulatory regions of 509 genes, selected from PharmGKB and Phenopedia, were sequenced using massive parallel sequencing in blood DNA from 105 breast cancer patients in the testing phase. In total, 18,245 variants were identified of which 2565 were novel variants (without rs number in dbSNP build 150) in the testing phase. Variants with major allele frequency over 0.05 were further prioritized for validation phase based on a newly developed decision tree. Using emerging in silico tools and pharmacogenomic databases for functional predictions and associations with response to cytotoxic therapy or disease-free survival of patients, 55 putative variants were identified and used for validation in 805 patients with clinical follow up using KASP™ technology. In conclusion, associations of rs2227291, rs2293194, and rs4376673 (located in ATP7A, KCNAB1, and DFFB genes, respectively) with response to neoadjuvant cytotoxic therapy and rs1801160 in DPYD with disease-free survival of patients treated with cytotoxic drugs were validated and should be further functionally characterized. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.Czech Medical Council [15-25618A]; Charles University [GAUK 1776218

Role of genetic variation in ABC transporters in breast cancer prognosis and therapy response

Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 43 prioritized variants associating with response or survival in the above testing phase were then analyzed by allelic discrimination in the large validation set (n = 802). Variants in ABCA4, ABCA9, ABCA12, ABCB5, ABCC5, ABCC8, ABCC11, and ABCD4 associated with response and variants in ABCA7, ABCA13, ABCC4, and ABCG8 with survival of the patients. No association passed a false discovery rate test, however, the rs17822931 (Gly180Arg) in ABCC11, associating with response, and the synonymous rs17548783 in ABCA13 (survival) have a strong support in the literature and are, thus, interesting for further research. Although replicated associations have not reached robust statistical significance, the role of ABC transporters in breast cancer should not be ruled out. Future research and careful validation of findings will be essential for assessment of genetic variation which was not in the focus of this study, e.g., non-coding sequences, copy numbers, and structural variations together with somatic mutations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Czech Medical Council [17-28470A]; Czech Ministry of Education, Youth and SportsMinistry of Education, Youth & Sports - Czech Republic [CZ.02.1.01/0.0/0.0/16_013/0001634]; Grant Agency of Charles University [UNCE/MED/006]; Grant Agency of the Czech RepublicGrant Agency of the Czech Republic [19-03063S

Role of genetic variation in cytochromes P450 in breast cancer prognosis and therapy response

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer.Czech Medical Council [NV19-08-00113]; Czech Ministry of Education, Youth and Sports, INTER-EXCELLENCE LTA-USA grant [19032]; Charles University Research Fund [Q39]NV19-08-00113; Univerzita Karlova v Praze, UK; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT: 1903

The role of cytochromes P450 and aldo-keto reductases in prognosis of breast carcinoma patients

Metabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and survival of breast carcinoma patients.Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting.AKR1A1 was significantly overexpressed and AKR1C1-4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro.Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in breast carcinoma patients and models.13-25222J, GACR, Czech Science FoundationCzech Science Foundation [13-25222J]; Internal Grant Agency of the Czech Ministry of Health [NT/14055-3

Methods for DNA extraction from historic sceletal material

Department of Anthropology and Human GeneticsKatedra antropologie a genetiky člověkaPřírodovědecká fakultaFaculty of Scienc

STR genotyping of Czech medieval population: archeologocal site in Mlekojedy (Litoměřice)

The aim of this diploma thesis was the initial genetic analysis of early mediaeval burial site from Mlekojedy polycultural locality (Litoměřice District, Czech Republic). Autosomal STR markers were chosen because of the following reasons. The high degree of polymorphism of these markers and the high extent of heterozygosity favor the use of STRs instead of mitochondrial DNA for the structural analysis of small populations. Usefulness of STR typing for validation purposes was demonstrated many times before. We used primers for miniSTRs to obtain the fullest results. Nuclear DNA was extracted from 35 % of bone samples and 91 % of teeth. We detected lower PCR amplification success rate of fragments longer than 150 bp and very high rate of allele drop-out which is sign of degraded DNA. Twelve reliable genotypes were determined for TH01 marker. Observed allele frequency and genetic diversity values were discussed in comparison with recent populations and other aDNA studies of burial sites. Keywords: ancient DNA, STR markers, miniSTR, early medieval burial site, Czech populatio

Role of genetic factors in the prognosis and prediction of efficacy of chemotherapy in breast carcinoma patients

Changes in the regulation of apoptosis and cell cycle are involved in tumor development, progression, and resistance to antitumor therapy. The aim of this work was to evaluate the importance of apoptotic caspases and regulators of cytokineses as possible prognostic and predictive markers in breast carcinoma patients. In addition to determining the transcript levels of selected genes in tumor and control tissues obtained from breast carcinoma patients, we have also focused on the importance of alternative splice variants of caspases and their potential genetially determined regulation. We analysed the obtained data in relation to the clinical-pathological characteristics of the tumors, the progression-free survival of patients and to the response of the patients to the neoadjuvant chemotherapeutic treatment. Part of the work was determination of protein expression levels and verification of the importance of selected candidates for the effect of chemotherapy by functional study. The transcript levels of caspase 2, 3, 7, 8, 9, 10, the specifically detected splice variants caspase 2S, 2L, 3A and B, 3S, 9A, 9B, 8L, and the transcript levels of KIF14 and CIT in breast carcinomas were unrelated to the progression-free survival of patients, or to the response of patients to neoadjuvant treatment. The..

Role of Genetic Variation in ABC Transporters in Breast Cancer Prognosis and Therapy Response

Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 43 prioritized variants associating with response or survival in the above testing phase were then analyzed by allelic discrimination in the large validation set (n = 802). Variants in ABCA4, ABCA9, ABCA12, ABCB5, ABCC5, ABCC8, ABCC11, and ABCD4 associated with response and variants in ABCA7, ABCA13, ABCC4, and ABCG8 with survival of the patients. No association passed a false discovery rate test, however, the rs17822931 (Gly180Arg) in ABCC11, associating with response, and the synonymous rs17548783 in ABCA13 (survival) have a strong support in the literature and are, thus, interesting for further research. Although replicated associations have not reached robust statistical significance, the role of ABC transporters in breast cancer should not be ruled out. Future research and careful validation of findings will be essential for assessment of genetic variation which was not in the focus of this study, e.g., non-coding sequences, copy numbers, and structural variations together with somatic mutations

Expression of oxysterol pathway genes in oestrogen-positive breast carcinomas

Objective: This study investigated whether gene expression levels of key modulators of the oxysterol signalling pathway modify the prognosis of patients with oestrogen receptor-positive (ER+) breast carcinomas via interaction with endocrine therapy. Context: The prognosis of patients with ER+ breast carcinoma depends on several factors. Previous studies have suggested that some oxygenated forms of cholesterol (oxysterols) bind to oestrogen receptor and anti-oestrogen binding site which may deregulate cholesterol homoeostasis and influence effect of therapy. Design: The expression levels of 70 oxysterol pathway genes were evaluated in a test set of breast carcinomas differing in ER expression. The genes differentially expressed in ER+ tumours were assessed in a comprehensive set of ER+ tumours to evaluate their clinical significance. Patients: A total of 193 primary patients with breast carcinoma were included. Measurements: The transcript levels were determined by quantitative real-time polymerase chain reaction. Results: The expression levels of 23 genes were found to be specifically dysregulated in ER+ tumours compared to ER− tumours of the test set. The expression levels of ABCG2, CYP7B1, CYP24A1, CYP39A1 and CH25H genes were found to be strongly associated with disease stage; however, none of the gene expression levels were associated with disease-free survival in patients treated with endocrine therapy. Conclusions: The expression of a number of oxysterol pathway genes is significantly modulated by ER expression and associated with the clinical stage of patients. However, the expression of oxysterol pathway genes was not found to modify the prognosis of ER+ patients with breast carcinoma treated with endocrine therapy. © 2017 John Wiley & Sons Ltd15-25618A, MZCR, Ministerstvo Zdravotnictví Ceské RepublikyGrantova Agentura Ceske Republiky [13-25222J]; Ministry of Health of the Czech Republic [15-25618A]; National Sustainability Program I [LO1503

Role of Genetic Variation in Cytochromes P450 in Breast Cancer Prognosis and Therapy Response

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer