Publikácie

@article{Rendek2024b,

title = {Current Challenges of Methylation-Based Liquid Biopsies in Cancer Diagnostics},

author = {T. Rendek and O. Pos and T. Duranova and R. Saade and J. Budis and V. Repiska and T. Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85195678349&doi=10.3390%2fcancers16112001&partnerID=40&md5=a00b727d255d45e6c52a69ea1697e97a},

doi = {10.3390/cancers16112001},

issn = {20726694},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {Cancers},

volume = {16},

number = {11},

publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},

abstract = {In current clinical practice, effective cancer testing and screening paradigms are limited to specific types of cancer, exhibiting varying efficiency, acceptance, and adherence. Cell-free DNA (cfDNA) methylation profiling holds promise in providing information about the presence of malignity regardless of its type and location while leveraging blood-based liquid biopsies as a method to obtain analytical samples. However, technical difficulties, costs and challenges resulting from biological variations, tumor heterogeneity, and exogenous factors persist. This method exploits the mechanisms behind cfDNA release but faces issues like fragmentation, low concentrations, and high background noise. This review explores cfDNA methylation’s origins, means of detection, and profiling for cancer diagnostics. The critical evaluation of currently available multi-cancer early detection methods (MCEDs) as well as tests targeting single genes, emphasizing their potential and limits to refine strategies for early cancer detection, are explained. The current methodology limitations, workflows, comparisons of clinically approved liquid biopsy-based methylation tests for cancer, their utilization in companion diagnostics as well as the biological limitations of the epigenetics approach are discussed, aiming to help healthcare providers as well as researchers to orient themselves in this increasingly complex and evolving field of diagnostics. © 2024 by the authors.},

keywords = {Cell-free nucleic acids, Liquid biopsy, Oncology, Review},

pubstate = {published},

tppubtype = {article}

}

@article{Lukacova2024,

title = {Novel liquid biopsy CNV biomarkers in malignant melanoma},

author = {E. Lukacova and Z. Hanzlikova and P. Podlesnyi and T. Sedlackova and T. Szemes and M. Grendar and M. Samec and T. Hurtova and B. Malicherova and K. Leskova and J. Budis and T. Burjanivova},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85197798133&doi=10.1038%2fs41598-024-65928-y&partnerID=40&md5=5640df31219fd97cf224ab24649c395c},

doi = {10.1038/s41598-024-65928-y},

issn = {20452322},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {Scientific Reports},

volume = {14},

number = {1},

publisher = {Nature Research},

abstract = {Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis. © The Author(s) 2024.},

keywords = {Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Styk2023,

title = {Microsatellite instability assessment is instrumental for Predictive, Preventive and Personalised Medicine: status quo and outlook},

author = {J. Styk and Z. Pös and O. Pös and J. Radvanszky and E. H. Turnova and G. Buglyó and D. Klimova and J. Budis and V. Repiska and B. Nagy and T. Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85146857319&doi=10.1007%2fs13167-023-00312-w&partnerID=40&md5=3b63e993459e80e985772551f60bbc4e},

doi = {10.1007/s13167-023-00312-w},

issn = {18785077},

year  = {2023},

date = {2023-01-01},

urldate = {2023-01-01},

journal = {EPMA Journal},

publisher = {Springer Science and Business Media Deutschland GmbH},

abstract = {A form of genomic alteration called microsatellite instability (MSI) occurs in a class of tandem repeats (TRs) called microsatellites (MSs) or short tandem repeats (STRs) due to the failure of a post-replicative DNA mismatch repair (MMR) system. Traditionally, the strategies for determining MSI events have been low-throughput procedures that typically require assessment of tumours as well as healthy samples. On the other hand, recent large-scale pan-tumour studies have consistently highlighted the potential of massively parallel sequencing (MPS) on the MSI scale. As a result of recent innovations, minimally invasive methods show a high potential to be integrated into the clinical routine and delivery of adapted medical care to all patients. Along with advances in sequencing technologies and their ever-increasing cost-effectiveness, they may bring about a new era of Predictive, Preventive and Personalised Medicine (3PM). In this paper, we offered a comprehensive analysis of high-throughput strategies and computational tools for the calling and assessment of MSI events, including whole-genome, whole-exome and targeted sequencing approaches. We also discussed in detail the detection of MSI status by current MPS blood-based methods and we hypothesised how they may contribute to the shift from conventional medicine to predictive diagnosis, targeted prevention and personalised medical services. Increasing the efficacy of patient stratification based on MSI status is crucial for tailored decision-making. Contextually, this paper highlights drawbacks both at the technical level and those embedded deeper in cellular/molecular processes and future applications in routine clinical testing. © 2023, The Author(s).},

keywords = {Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Holesova2023,

title = {Telomere Length Changes in Cancer: Insights on Carcinogenesis and Potential for Non-Invasive Diagnostic Strategies},

author = {Z. Holesova and L. Krasnicanova and R. Saade and O. Pös and J. Budis and J. Gazdarica and V. Repiska and T. Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85151112952&doi=10.3390%2fgenes14030715&partnerID=40&md5=d89df2608717b306d353873f39018ecb},

doi = {10.3390/genes14030715},

issn = {20734425},

year  = {2023},

date = {2023-01-01},

urldate = {2023-01-01},

journal = {Genes},

volume = {14},

number = {3},

publisher = {MDPI},

abstract = {Telomere dynamics play a crucial role in the maintenance of chromosome integrity; changes in telomere length may thus contribute to the development of various diseases including cancer. Understanding the role of telomeric DNA in carcinogenesis and detecting the presence of cell-free telomeric DNA (cf-telDNA) in body fluids offer a potential biomarker for novel cancer screening and diagnostic strategies. Liquid biopsy is becoming increasingly popular due to its undeniable benefits over conventional invasive methods. However, the organization and function of cf-telDNA in the extracellular milieu are understudied. This paper provides a review based on 3,398,017 cancer patients, patients with other conditions, and control individuals with the aim to shed more light on the inconsistent nature of telomere lengthening/shortening in oncological contexts. To gain a better understanding of biological factors (e.g., telomerase activation, alternative lengthening of telomeres) affecting telomere homeostasis across different types of cancer, we summarize mechanisms responsible for telomere length maintenance. In conclusion, we compare tissue- and liquid biopsy-based approaches in cancer assessment and provide a brief outlook on the methodology used for telomere length evaluation, highlighting the advances of state-of-the-art approaches in the field. © 2023 by the authors.},

keywords = {Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Soltész2022,

title = {The role of exosomes in cancer progression},

author = {B. Soltész and G. Buglyó and N. Németh and M. Szilágyi and O. Pös and T. Szemes and I. Balogh and B. Nagy},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85121366077&doi=10.3390%2fijms23010008&partnerID=40&md5=a6c0cdd2a0624326aa4378c4af43611b},

doi = {10.3390/ijms23010008},

issn = {16616596},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {International Journal of Molecular Sciences},

volume = {23},

number = {1},

publisher = {MDPI},

abstract = {Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non‐invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell‐to‐cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next‐generation treatment of cancer patients. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Cell-free nucleic acids, Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Buglyó2022,

title = {Liquid Biopsy as a Source of Nucleic Acid Biomarkers in the Diagnosis and Management of Lynch Syndrome},

author = {G. Buglyó and J. Styk and O. Pös and Á. Csók and V. Repiska and B. Soltész and T. Szemes and B. Nagy},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85128130388&doi=10.3390%2fijms23084284&partnerID=40&md5=b115dc9efc5482690694163145c4f23e},

doi = {10.3390/ijms23084284},

issn = {16616596},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {International Journal of Molecular Sciences},

volume = {23},

number = {8},

publisher = {MDPI},

abstract = {Lynch syndrome (LS) is an autosomal dominant inherited cancer predisposition disorder, which may manifest as colorectal cancer (CRC), endometrial cancer (EC) or other malignancies of the gastrointestinal and genitourinary tract as well as the skin and brain. Its genetic cause is a defect in one of the four key DNA mismatch repair (MMR) loci. Testing of patients at risk is currently based on the absence of MMR protein staining and detection of mutations in cancer tissue and the germline, microsatellite instability (MSI) and the hypermethylated state of the MLH1 promoter. If LS is shown to have caused CRC, lifetime follow-up with regular screening (most importantly, colonoscopy) is required. In recent years, DNA and RNA markers extracted from liquid biopsies have found some use in the clinical diagnosis of LS. They have the potential to greatly enhance the efficiency of the follow-up process by making it minimally invasive, reproducible, and time effective. Here, we review markers reported in the literature and their current clinical applications, and we comment on possible future directions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Styk2022,

title = {Extracellular Nucleic Acids in the Diagnosis and Progression of Colorectal Cancer},

author = {J. Styk and G. Buglyó and O. Pös and Á. Csók and B. Soltész and P. Lukasz and V. Repiská and B. Nagy and T. Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85136586949&doi=10.3390%2fcancers14153712&partnerID=40&md5=c1693e930ca57ea69fb9f5147e1de219},

doi = {10.3390/cancers14153712},

issn = {20726694},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Cancers},

volume = {14},

number = {15},

publisher = {MDPI},

abstract = {Colorectal cancer (CRC) is the 3rd most common malignant neoplasm worldwide, with more than two million new cases diagnosed yearly. Despite increasing efforts in screening, many cases are still diagnosed at a late stage, when mortality is high. This paper briefly reviews known genetic causes of CRC (distinguishing between sporadic and familial forms) and discusses potential and confirmed nucleic acid biomarkers obtainable from liquid biopsies, classified by their molecular features, focusing on clinical relevance. We comment on advantageous aspects such as better patient compliance due to blood sampling being minimally invasive, the possibility to monitor mutation characteristics of sporadic and hereditary CRC in a disease showing genetic heterogeneity, and using up- or down-regulated circulating RNA markers to reveal metastasis or disease recurrence. Current difficulties and thoughts on some possible future directions are also discussed. We explore current evidence in the field pointing towards the introduction of personalized CRC management. © 2022 by the authors.},

keywords = {Cell-free nucleic acids, Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Kucharík2021,

title = {Copy number variant detection with low-coverage whole-genome sequencing represents a viable alternative to the conventional array-cgh},

author = {M Kucharík and J Budiš and M Hýblová and G Minárik and T Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85109087040&doi=10.3390%2fdiagnostics11040708&partnerID=40&md5=6fdfa35027032bf889399d967bb1cce9},

doi = {10.3390/diagnostics11040708},

issn = {20754418},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {Diagnostics},

volume = {11},

number = {4},

publisher = {MDPI AG},

abstract = {Copy number variations (CNVs) represent a type of structural variant involving alterations in the number of copies of specific regions of DNA that can either be deleted or duplicated. CNVs contribute substantially to normal population variability, however, abnormal CNVs cause numerous genetic disorders. At present, several methods for CNV detection are applied, ranging from the conventional cytogenetic analysis, through microarray-based methods (aCGH), to next-generation sequencing (NGS). In this paper, we present GenomeScreen, an NGS-based CNV detection method for low-coverage, whole-genome sequencing. We determined the theoretical limits of its accuracy and obtained confirmation in an extensive in silico study and in real patient samples with known genotypes. In theory, at least 6 M uniquely mapped reads are required to detect a CNV with the length of 100 kilobases (kb) or more with high confidence (Z-score > 7). In practice, the in silico analysis required at least 8 M to obtain >99% accuracy (for 100 kb deviations). We compared GenomeScreen with one of the currently used aCGH methods in diagnostic laboratories, which has mean resolution of 200 kb. GenomeScreen and aCGH both detected 59 deviations, while GenomeScreen furthermore detected 134 other (usually) smaller variations. When compared to aCGH, overall performance of the proposed GenemoScreen tool is comparable or superior in terms of accuracy, turn-around time, and cost-effectiveness, thus providing reasonable benefits, particularly in a prenatal diagnosis setting. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Cell-free nucleic acids, Computational method, Copy number variation, Liquid biopsy, Non-invasive prenatal testing},

pubstate = {published},

tppubtype = {article}

}

@article{Pös20201,

title = {Technical and methodological aspects of cell‐free nucleic acids analyzes},

author = {Z Pös and O Pös and J Styk and A Mocova and L Strieskova and J Budis and L Kadasi and J Radvanszky and T Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85096148893&doi=10.3390%2fijms21228634&partnerID=40&md5=826434c64ac9dbe5f81fc0ee5b62eaa3},

doi = {10.3390/ijms21228634},

issn = {16616596},

year  = {2020},

date = {2020-01-01},

journal = {International Journal of Molecular Sciences},

volume = {21},

number = {22},

pages = {1-43},

publisher = {MDPI AG},

abstract = {Analyzes of cell‐free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Body fluids, Cell-free nucleic acids, Liquid biopsy, Non-invasive prenatal testing, Review},

pubstate = {published},

tppubtype = {article}

}

@article{Soltysova20201,

title = {Monosomy 3 influences epithelial-mesenchymal transition gene expression in uveal melanoma patients; consequences for liquid biopsy},

author = {A Soltysova and T Sedlackova and D Dvorska and K Jasek and P C Baradaran and V H Kajabova and L Demkova and V Buocikova and T Kurucova and D Lyskova and A Furdova and G Minarik and P Babal and Z Dankova and B Smolkova},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85097833518&doi=10.3390%2fijms21249651&partnerID=40&md5=133b8d5d56ebac3bf7a09024f007273e},

doi = {10.3390/ijms21249651},

issn = {16616596},

year  = {2020},

date = {2020-01-01},

journal = {International Journal of Molecular Sciences},

volume = {21},

number = {24},

pages = {1-24},

publisher = {MDPI AG},

abstract = {Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCβ4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Aneuploidy, Body fluids, Circulating tumor cells, Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Smolkova20201,

title = {Increased stromal infiltrating lymphocytes are associated with the risk of disease progression in mesenchymal circulating tumor cell-positive primary breast cancer patients},

author = {B Smolkova and Z Cierna and K Kalavska and S Miklikova and J Plava and G Minarik and T Sedlackova and D Cholujova and P Gronesova and M Cihova and K Majerova and M Karaba and J Benca and D Pindak and J Mardiak and M Mego},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85097678410&doi=10.3390%2fijms21249460&partnerID=40&md5=8dd0aa352be241f64188de23774ac3c4},

doi = {10.3390/ijms21249460},

issn = {16616596},

year  = {2020},

date = {2020-01-01},

journal = {International Journal of Molecular Sciences},

volume = {21},

number = {24},

pages = {1-16},

publisher = {MDPI AG},

abstract = {Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30–10.37, p < 0.001 for CD3high; HR = 6.17, 95% CI 2.75–13.80, p < 0.001 for CD8high; HR = 6.93, 95% CI 2.86–16.81, p < 0.001 for CD45ROhigh). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048–11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.},

keywords = {Body fluids, Circulating tumor cells, Liquid biopsy, Oncology},

pubstate = {published},

tppubtype = {article}

}

@article{Sedlackova20141543,

title = {Selection of an optimal method for co-isolation of circulating DNA and miRNA from the plasma of pregnant women},

author = {T Sedlackova and G Repiska and G Minarik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84908102134&doi=10.1515%2fcclm-2014-0021&partnerID=40&md5=ff635286396a625ebe2934f00e7b7772},

doi = {10.1515/cclm-2014-0021},

issn = {14346621},

year  = {2014},

date = {2014-01-01},

journal = {Clinical Chemistry and Laboratory Medicine},

volume = {52},

number = {11},

pages = {1543-1548},

publisher = {Walter de Gruyter GmbH},

abstract = {Background: Circulating nucleic acids acquired non-invasively have been confirmed as useful biomarkers in cancer and prenatal medicine. The most important molecules in the field of circulating nucleic acids research are circulating DNA and miRNA. In this study, the possibility of co-isolation of total circulating DNA, cell-free fetal DNA and miRNA from the plasma of pregnant women was tested, and the yields of co-isolated circulating nucleic acids using two commercial kits and three protocols were compared. 

Methods: Cell-free fetal DNA and miRNA from the plasma of pregnant women carrying male fetuses were co-isolated with the miRCURY™ RNA Isolation Kit according to the original protocol and the QIAamp Circulating Nucleic Acid Kit (CNA kit) according to the manufacturer's protocol for DNA isolation and miRNA isolation. For comparison of DNA isolation, the AR and DYS14 gene-based assays were used for the detection and quantification of total circulating and cell-free fetal DNA. For miRNA detection and quantification, the miR-16 and miR-451 assays were used. Results: Two different protocols for isolation using the CNA kit did not significantly differ in the yields of isolated tcDNA and cffDNA; however, the amount of isolated cffDNA using the miRCURY™ RNA Isolation Kit was significantly less (p<0.05},

keywords = {Cell-free nucleic acids, Liquid biopsy, Non-invasive prenatal testing},

pubstate = {published},

tppubtype = {article}

}

@article{Repiská20131185,

title = {Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma},

author = {G Repiská and T Sedláčková and T Szemes and P Celec and G Minárik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84882355382&doi=10.1515%2fcclm-2012-0418&partnerID=40&md5=f476653212c66d4f5b9ec534582f5921},

doi = {10.1515/cclm-2012-0418},

issn = {14346621},

year  = {2013},

date = {2013-01-01},

journal = {Clinical Chemistry and Laboratory Medicine},

volume = {51},

number = {6},

pages = {1185-1189},

abstract = {Background: The cell free fetal DNA (cffDNA) present in plasma of pregnant women represents an important alternative source of DNA for non-invasive prenatal diagnosis. Due to the low quantity and increased fragmentation of cffDNA, the choice of DNA extraction method is a crucial step for downstream analyses. Methods: In our study, the three spin column-based kits for isolation of cffDNA [DNA Blood Mini Kit (DBM), DSP Virus Kit (DSP) and Circulating Nucleic Acid (CNA) Kit] were compared. Original and optimized protocol were used in comparison and applied in the two phases of the study. Results: A statistically significant difference in performance of the kits was determined based on the comparison of genomic equivalents per mL (GEq/mL) values (p < 0.0001). The GEq/mL of isolated DNA was significantly higher using CNA and DSP Kits than DBM Kit. The CNA Kit and DSP Kit did not significantly differ in the GEq/mL values, although all tested samples isolated with CNA Kit showed higher values. Conclusions: According to our results the commonly used DBM Kit could be successfully replaced with CNA or DSP Kits. The replacement could be beneficial in qualitative as well quantitative tests (e.g., gender determination, aneu ploidy detection) when the isolation yield limits subsequent analyses. However, there is an important decision to be made when switching DBM Kit for DSP or CNA Kits. The price of DBM Kit is two and six times lower than DSP and CNA Kits, respectively.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Sedlackova2013,

title = {Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods},

author = {T Sedlackova and G Repiska and P Celec and T Szemes and G Minarik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84874073176&doi=10.1186%2f1480-9222-15-5&partnerID=40&md5=723b0e9568bb2d83456bbc497a945237},

doi = {10.1186/1480-9222-15-5},

issn = {14809222},

year  = {2013},

date = {2013-01-01},

journal = {Biological Procedures Online},

volume = {15},

number = {1},

abstract = {Background: Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results: In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions: Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA. © 2013 Sedlackova et al.; licensee BioMed Central Ltd.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Vlková2010b,

title = {Advances in the research of fetal DNA in maternal plasma for noninvasive prenatal diagnostics},

author = {B Vlková and T Szemes and G Minárik and J Turňa and P Celec},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-77954303943&partnerID=40&md5=f8502e85159556021100dec5321b7865},

issn = {12341010},

year  = {2010},

date = {2010-01-01},

journal = {Medical Science Monitor},

volume = {16},

number = {4},

pages = {RA85-RA91},

abstract = {Molecular analysis of fetal DNA present in the maternal circulation allows noninvasive, early, and precise determination of fetal genetic status in prenatal diagnostics. The most common clinical applications, i.e. prenatal gender determination and fetal RhD genotyping, are possible already in the first trimester using specialized protocols for DNA isolation from plasma and subsequent realtime PCR detection. Recent advances in molecular techniques enable other applications of fetal DNA purified from maternal plasma samples. Chromosomal abnormalities (e.g. trisomy 21) can be diagnosed by digital PCR, which offers higher accuracy in quantifying DNA sequences than standard real-time PCR. Digital PCR, but also MALDI-TOF, are suitable for detecting point mutations, widening the spectrum of applications to monogenic diseases. The ongoing lowering of costs for massively parallel sequencing might lead to replacement of most of the other currently used approaches. Adopting specialized protocols for the purification of fragmented circulating fetal DNA and improving the bioinformatic analysis of raw data can bring us closer to sequencing the fetal genome as the ultimate goal of prenatal DNA diagnostics, with wide-ranging medical applications. The discussion and solution of ethical issues beyond early fetal gender or paternity determination is hanging just behind the rapid technical progress of noninvasive prenatal DNA diagnostics. © Med Sci Monit, 2010.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Review, Validation},

pubstate = {published},

tppubtype = {article}

}