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- Vaissière, Thomas, et al.
(författare)
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Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.
- 2009
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Ingår i: Epigenetics : official journal of the DNA Methylation Society. - 1559-2308. ; 4:4, s. 221-30
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Tidskriftsartikel (refereegranskat)abstract
- Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.
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- Zaidi, Syed H., et al.
(författare)
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Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival
- 2020
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR=0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR=1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features. Large scale sequencing study is of paramount importance to unravel the heterogeneity of colorectal cancer. Here, the authors sequenced 205 cancer genes in more than 2000 tumours and identified additional mutated driver genes, determined that mutational burden and specific mutations in TP53 are associated with survival odds.
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- Egyud, M., et al.
(författare)
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Detection of Circulating Tumor DNA in Plasma: A Potential Biomarker for Esophageal Adenocarcinoma
- 2019
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Ingår i: Annals of Thoracic Surgery. - : Elsevier BV. - 0003-4975. ; 108:2, s. 343-349
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Tidskriftsartikel (refereegranskat)abstract
- Background. Recent literature has demonstrated the potential of "liquid biopsy" and detection of circulating tumor (ct)DNA as a cancer biomarker. However, to date there is a lack of data specific to esophageal adenocarcinoma (EAC). This study was conducted to determine how detection and quantification of ctDNA changes with disease burden in patients with EAC and evaluate its potential as a biomarker in this population. Methods. Blood samples were obtained from patients with stage I to IV EAC. Longitudinal blood samples were collected from a subset of patients. Imaging studies and pathology reports were reviewed to determine disease course. Tumor samples were sequenced to identify mutations. Mutations in plasma DNA were detected using custom, barcoded, patient-specific sequencing libraries. Mutations in plasma were quantified, and associations with disease stage and response to therapy were explored. Results. Plasma samples from a final cohort of 38 patients were evaluated. Baseline plasma samples were ctDNA positive for 18 patients (47%) overall, with tumor allele frequencies ranging from 0.05% to 5.30%. Detection frequency of ctDNA and quantity of ctDNA increased with stage. Data from longitudinal samples indicate that ctDNA levels correlate with and precede evidence of response to therapy or recurrence. Conclusions. ctDNA can be detected in plasma of EAC patients and correlates with disease burden. Detection of ctDNA in early-stage EAC is challenging and may limit diagnostic applications. However, our data demonstrate the potential of ctDNA as a dynamic biomarker to monitor treatment response and disease recurrence in patients with EAC. (C) 2019 by The Society of Thoracic Surgeons
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- Ståhlberg, Anders, 1975, et al.
(författare)
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Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing
- 2017
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 12:4, s. 664-682
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Tidskriftsartikel (refereegranskat)abstract
- \Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next- generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (similar to 4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at < 0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.
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- Egyud, Matthew, et al.
(författare)
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Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer.
- 2019
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Ingår i: Head & neck. - : Wiley. - 1097-0347 .- 1043-3074. ; 41:5, s. 1351-1358
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Tidskriftsartikel (refereegranskat)abstract
- Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer.Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient-specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre-treatment blood samples and longitudinally during standard follow-up. Circulating tumor DNA status during follow-up was correlated to disease recurrence.Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence.Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.
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