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1.
Tumour Biol ; 42(4): 1010428320916314, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32338581

RESUMO

In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy of the method used for DNA purification, and is often a juncture of experimental confusion. Therefore, in this study, we compared six commercially available cell-free DNA isolation kits for the recovery of cell-free DNA from the cell culture supernatant of a human bone cancer cell line (143B), including two magnetic bead-based manual kits, one automated magnetic bead-based extraction method, and three manual spin-column kits. Based on cell-free DNA quantitation and sizing, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods showed significant variability concerning recovery, reproducibility, and size discrimination. These findings highlight the importance of selecting a cell-free DNA extraction method that is appropriate for the aims of a study. For example, mutational analysis of cell-free DNA may be enhanced by a method that favors a high yield or is biased toward the isolation of short cell-free DNA fragments. In contrast, quantitative analysis of cell-free DNA in a comparative setting (e.g. measuring the fluctuation of cell-free DNA levels over time) may require the selection of a cell-free DNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.


Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , Meios de Cultivo Condicionados/análise , Biópsia Líquida/métodos , Biópsia Líquida/normas , Biomarcadores Tumorais , Células Cultivadas , DNA de Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Reprodutibilidade dos Testes
2.
Zhonghua Fu Chan Ke Za Zhi ; 55(2): 100-105, 2020 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-32146738

RESUMO

Objective: To investigate the impact of maternal X chromosome aneuploidies on cell free DNA (cf-DNA) prenatal screening. Methods: After genetic counseling, invasive prenatal diagnosis was provided for the 124 cases with high risk of sex chromosome aneuploidie (SCA) indicated by cf-DNA prenatal screening. For cases with discordant results of fetal prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte was collected for copy number variation sequencing (CNV-seq) to detect whether the maternal X chromosome was carrying variations. Results: Totally, 124 cases with high risks of SCA indicated by cf-DNA prenatal screening, 9 cases refused to take invasive prenatal diagnosis, while the remaining 115 cases received. Among the 115 cases, 41 cases received accordant results with cf-DNA prenatal screening while 74 cases discordant. Among the 74 cases with discordant results, 19 cases were indicated with maternal X chromosome variations by maternal leukocyte CNV-seq, which accounting for 25.7% (19/74) of the SCA false positive cases, and 15.3% (19/124) of all SCA cases. Conclusions: Pregnant women with X chromosome variations may affect the results of cf-DNA prenatal screening, resulting in false positive or false negative outcomes, it should be emphasized that the cf-DNA results may be affected by maternal X chromosome variations. In cases with discordant results of prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte CNV-seq is recommended to find the reasons of false positive or negative results. And cf-DNA prenatal screening is not recommended for pregnant women who are already known with X chromosome variations.


Assuntos
Aneuploidia , Ácidos Nucleicos Livres/sangue , Cromossomos Humanos X/genética , Variações do Número de Cópias de DNA/genética , Testes para Triagem do Soro Materno/métodos , Diagnóstico Pré-Natal/métodos , Transtornos dos Cromossomos Sexuais/genética , Transtornos Cromossômicos , Feminino , Humanos , Gravidez
3.
Zhonghua Fu Chan Ke Za Zhi ; 55(2): 106-111, 2020 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-32146739

RESUMO

Objective: To evaluate the efficacy of cell free DNA (cf-DNA) screening in prenatal care by analyzing the follow-up information and pregnancy outcomes. Methods: All cf-DNA cases conducted in Women's Hospital of Nanjing Medical University from August 2011 to December 2017 were enrolled. The general information of the pregnancies, cf-DNA results, confirmatory testing results, and the follow-up results were collected. The pregnancy outcomes were analyzed in cases with low risk cf-DNA results as well as with high risk results for common trisomies, which were trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13). The sensitivity, specificity, positive predictive value and negative predictive value of cf-DNA screening were calculated. Results: (1) A total of 43 615 cf-DNA cases were involved, with 44 cases (0.10%, 44/43 615) test failure results, 314 cases (0.72%, 314/43 571) high risk results for common trisomies and 43 257 cases (99.27%, 43 257/43 571) low risk results. (2) Among 277 cases (88.21%, 277/314) high risk cases were successfully followed up, and 228 cases (82.31%, 228/277) underwent invasive confirmatory prenatal diagnosis. In the low risk results, 36 826 cases (85.13%, 36 826/43 257) were successfully followed up, and 572 (1.55%, 572/36 826) cases were found to have adverse pregnancy outcomes, among which 4 false negative cf-DNA results were confirmed. (3) In the 37 103 successfully followed up cf-DNA cases, the sensitivity for T21, T18, T13 were calculated as 97.96%, 96.67% and 100.00%, respectively; the specificity for T21, T18, T13 were calculated as 99.96%, 99.95% and 99.95%, respectively. The positive predictive value for T21, T18, T13 were calculated as 90.57%, 63.04% and 17.39%, respectively. The negative predictive value for T21, T18, T13 were calculated as 99.99%, 99.98% and 100.00%. Conclusions: Cf-DNA is effective in detecting common trisomies, with a high sensitivity and specificity. However, the follow-up information revealed several potential limitations in current clinical practice, such as a number of cases with high risk results rejected invasive confirmatory testing, as well as the genetic diagnostic results for most low risk cases with an adverse pregnancy outcome aren't obtained. Genetic counseling and the follow-up for all the cf-DNA cases should be emphasized in the future.


Assuntos
Ácidos Nucleicos Livres , Transtornos Cromossômicos/diagnóstico , Diagnóstico Pré-Natal/métodos , Trissomia/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 18/genética , Feminino , Seguimentos , Testes Genéticos/métodos , Humanos , Valor Preditivo dos Testes , Gravidez , Resultado da Gravidez , Sensibilidade e Especificidade , Trissomia/genética , Síndrome da Trissomia do Cromossomo 13 , Síndrome da Trissomía do Cromossomo 18 , Ultrassonografia Pré-Natal
4.
Magy Onkol ; 64(1): 70-72, 2020 Mar 17.
Artigo em Húngaro | MEDLINE | ID: mdl-32181765

RESUMO

During colorectal cancer (CRC) development, in addition to genetic alterations, several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumor cells. Cell-free DNA (cfDNA) in the circulatory system can originate also from tumor tissue; therefore the evaluation of methylated cfDNA in the plasma can be a promising method for early cancer screening. In my Ph.D., I have investigated the rate of cfDNA's release and stability using animal models. I aimed to compile an epigenetic marker panel, which contains genes with altered DNA methylation patterns in the healthy-colorectal adenoma-cancer sequence. I have found that the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters has already increased in adenoma stages in both tissue and plasma samples. Immunohistochemistry analyses indicated decreasing protein expression in parallel with elevated methylation. According to our results, cfDNA amount and the methylation have been influenced by DNA isolation and blood collection methods.


Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/química , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Metilação de DNA , DNA/química , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , DNA/sangue , DNA/genética , Humanos , Regiões Promotoras Genéticas/genética
5.
Am J Hum Genet ; 106(2): 202-214, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004449

RESUMO

Cell-free DNA (cf.DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf.DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFFB) with mice deficient in each of these nucleases. By analyzing the ends of cf.DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting preference that reveals the stepwise process of cf.DNA fragmentation. Essentially, we demonstrate that cf.DNA is generated first intracellularly with DFFB, intracellular DNASE1L3, and other nucleases. Then, cf.DNA fragmentation continues extracellularly with circulating DNASE1L3 and DNASE1. With the use of heparin to disrupt the nucleosomal structure, we also show that the 10 bp periodicity originates from the cutting of DNA within an intact nucleosomal structure. Altogether, this work establishes a model of cf.DNA fragmentation.


Assuntos
Ácidos Nucleicos Livres/metabolismo , Cromatina/metabolismo , Fragmentação do DNA , Desoxirribonuclease I/fisiologia , Desoxirribonucleases/fisiologia , Endodesoxirribonucleases/fisiologia , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Animais , Ácidos Nucleicos Livres/genética , Cromatina/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Nucleossomos/genética
6.
PLoS One ; 15(2): e0224001, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027658

RESUMO

The applications of liquid biopsy have attracted much attention in biomedical research in recent years. Circulating cell-free DNA (cfDNA) in the serum may serve as a unique tumor marker in various types of cancer. Circulating tumor DNA (ctDNA) is a type of serum cfDNA found in patients with cancer and contains abundant information regarding tumor characteristics, highlighting its potential diagnostic value in the clinical setting. However, the diagnostic value of cfDNA as a biomarker, especially circulating HPV DNA (HPV cDNA) in cervical cancer remains unclear. Here, we performed a meta-analysis to evaluate the applications of HPV cDNA as a biomarker in cervical cancer. A systematic literature search was performed using PubMed, Embase, and WANFANG MED ONLINE databases up to March 18, 2019. All literature was analyzed using Meta Disc 1.4 and STATA 14.0 software. Diagnostic measures of accuracy of HPV cDNA in cervical cancer were pooled and investigated. Fifteen studies comprising 684 patients with cervical cancer met our inclusion criteria and were subjected to analysis. The pooled sensitivity and specificity were 0.27 (95% confidence interval [CI], 0.24-0.30) and 0.94(95% CI, 0.92-0.96), respectively. The pooled positive likelihood ratio and negative likelihood ratio were 6.85 (95% CI, 3.09-15.21) and 0.60 (95% CI, 0.46-0.78), respectively. The diagnostic odds ratio was 15.25 (95% CI, 5.42-42.94), and the area under the summary receiver operating characteristic curve was 0.94 (95% CI, 0.89-0.99). There was no significant publication bias observed. In the included studies, HPV cDNA showed clear diagnostic value for diagnosing and monitoring cervical cancer. Our meta-analysis suggested that detection of HPV cDNA in patients with cervical cancer could be used as a noninvasive early dynamic biomarker of tumors, with high specificity and moderate sensitivity. Further large-scale prospective studies are required to validate the factors that may influence the accuracy of cervical cancer diagnosis and monitoring.


Assuntos
Ácidos Nucleicos Livres/sangue , Neoplasias do Colo do Útero/diagnóstico , Biomarcadores Tumorais/sangue , Feminino , Humanos , Infecções por Papillomavirus/genética , Curva ROC , Sensibilidade e Especificidade
7.
Crit Rev Oncol Hematol ; 146: 102863, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31935617

RESUMO

Lung cancer is the most frequent cancer for males and third most frequent cancer for females. Targeted therapy drugs based on molecular alterations, such as angiogenesis inhibitors, epidermal growth factor receptor (EGFR) inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors are important part of treatment of NSCLC. However, the quality of the available tumor biopsy and/or cytology material is sometimes not adequate to perform the necessary molecular testing, which has prompted the search for alternatives. This review examines the use of tumor-educated platelet (TEP) as a liquid biopsy in lung cancer patients. The development of sensitive and accurate techniques have made it possible to detect the specific genetic alterations for which targeted therapies are already available. Liquid biopsy offers opportunities to detect resistance mechanisms at an early stage. To conclude, tumor-educated platelet has the potential to be used as liquid biopsy for a variety of clinical and investigational applications.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácidos Nucleicos Livres/sangue , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Biópsia Líquida/métodos , Neoplasias Pulmonares/patologia , Biomarcadores Tumorais/genética , Plaquetas/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Ácidos Nucleicos Livres/genética , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Masculino , Mutação , Medicina de Precisão , Inibidores de Proteínas Quinases/uso terapêutico
8.
Nat Commun ; 11(1): 525, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988276

RESUMO

Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA (ctDNA) alterations through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative treatment in patients with operable gastric cancer. After filtering alterations from matched white blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer.


Assuntos
Ácidos Nucleicos Livres/química , DNA de Neoplasias/análise , Leucócitos/química , Recidiva Local de Neoplasia/diagnóstico , Análise de Sequência de DNA , Neoplasias Gástricas/diagnóstico , DNA de Neoplasias/química , Hematopoese , Humanos , Prognóstico , Estudo de Prova de Conceito , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias Gástricas/genética , Análise de Sobrevida
9.
BMC Med Genet ; 21(1): 3, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900123

RESUMO

BACKGROUND: Liquid biopsies of blood plasma cell free DNA can be used to monitor treatment response and potentially detect mutations that are present in resistant clones in metastatic cancer patients. CASE PRESENTATION: In our non-interventional liquid biopsy study, a male patient in his fifties diagnosed with stage IV colorectal cancer and polytope liver metastases rapidly progressed after completing chemotherapy and deceased 8 months after diagnosis. Retrospective cell free DNA testing showed that the APC/TP53/KRAS major clone responded quickly after 3 cycles of FOLFIRI + Bevacizumab. Retrospective exome sequencing of pre-chemotherapy and post-chemotherapy tissue samples including metastases confirmed that the APC/TP53/KRAS and other major clonal mutations (GPR50, SLC5A, ZIC3, SF3A1 and others) were present in all samples. After the last chemotherapy cycle, CT imaging, CEA and CA19-9 markers validated the cfDNA findings of treatment response. However, 5 weeks later, the tumour had rapidly progressed. CONCLUSION: As FOLFIRI+Bevacizumab has recently also been associated with sustained complete remission in a APC/TP53/KRAS triple-mutated patient, these driver genes should be tested and monitored in a more in-depth manner in future patients. Patients with metastatic disease should be monitored more closely during and after chemotherapy, ideally using cfDNA.


Assuntos
Proteína da Polipose Adenomatosa do Colo/sangue , Neoplasias Colorretais/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/sangue , Proteína Supressora de Tumor p53/sangue , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab/administração & dosagem , Bevacizumab/efeitos adversos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Proteínas Proto-Oncogênicas p21(ras)/genética , Indução de Remissão , Proteína Supressora de Tumor p53/genética
10.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R445-R452, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913687

RESUMO

Mitochondrial DNA (mtDNA) exposed to the extracellular space due to cell death has immunostimulatory properties. Case-control studies reported a positive association between odds of developing preeclampsia and circulating mtDNA. These findings are based on relative quantification protocols that do not allow determination of absolute concentrations of mtDNA and are highly sensitive to nuclear DNA contamination. Furthermore, circulating mtDNA concentrations in response to normal pregnancy, which is an inflammatory state characterized by continuous placental cell apoptosis, have not been established. The main objective of this study was to determine longitudinal changes in circulating mtDNA from preconception to first trimester, third trimester, and postpartum in healthy pregnant women. Absolute real-time PCR quantification of mtDNA and nuclear DNA (nDNA) was performed on whole genomic extracts from serum using TaqMan probes and chemistry. Serum cell-free mtDNA and nDNA concentrations were greater in late pregnancy as compared with early pregnancy and postpartum. Pregnant women carrying neonates at the upper quartile of birth length distribution had higher concentrations of mtDNA in late pregnancy compared with pregnancies carrying neonates at the lower quartile. The correlation between circulating mtDNA and nDNA concentrations varied by sex (i.e., pregnancies carrying female vs. male fetuses). This study is the first to establish temporal patterns of circulating cell-free mtDNA concentrations in normal human pregnancy using absolute DNA quantification techniques. Concentrations of circulating mtDNA in normal pregnancy may be used as reference values for the development of clinical prognostic or diagnostic tests in pregnant women with, or at risk of developing, gestational complications.


Assuntos
Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Adulto , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Feminino , Marcadores Genéticos , Voluntários Saudáveis , Humanos , Estudos Longitudinais , Período Pós-Parto/sangue , Gravidez , Trimestres da Gravidez/sangue , Estudos Prospectivos , Caracteres Sexuais , Processos de Determinação Sexual , Adulto Jovem
11.
PLoS One ; 15(1): e0227385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929557

RESUMO

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay's conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.


Assuntos
Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Transplantes/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Rejeição de Enxerto , Transplante de Coração , Humanos , Lactente , Masculino , Doadores de Tecidos , Adulto Jovem
12.
Nat Commun ; 11(1): 400, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964864

RESUMO

Circulating cell-free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, cf-mRNA composition and its potential clinical applications remain largely unexplored. Here we show, using Next Generation Sequencing-based profiling, that cf-mRNA is enriched in transcripts derived from the bone marrow compared to circulating cells. Further, longitudinal studies involving bone marrow ablation followed by hematopoietic stem cell transplantation in multiple myeloma and acute myeloid leukemia patients indicate that cf-mRNA levels reflect the transcriptional activity of bone marrow-resident hematopoietic lineages during bone marrow reconstitution. Mechanistically, stimulation of specific bone marrow cell populations in vivo using growth factor pharmacotherapy show that cf-mRNA reflects dynamic functional changes over time associated with cellular activity. Our results shed light on the biology of the circulating transcriptome and highlight the potential utility of cf-mRNA to non-invasively monitor bone marrow involved pathologies.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Medula Óssea/patologia , Ácidos Nucleicos Livres/isolamento & purificação , Leucemia Mieloide Aguda/diagnóstico , Mieloma Múltiplo/diagnóstico , RNA Mensageiro/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Medula Óssea/efeitos dos fármacos , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Estudos Longitudinais , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , RNA Mensageiro/sangue , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Resultado do Tratamento , Adulto Jovem
13.
Proc Natl Acad Sci U S A ; 117(3): 1658-1665, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31900366

RESUMO

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.


Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , DNA Circular/isolamento & purificação , Plasma/química , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/genética , DNA Circular/química , DNA Circular/genética , Feminino , Genoma Humano , Hong Kong , Humanos , Teste Pré-Natal não Invasivo , Gravidez
14.
Cell Mol Life Sci ; 77(3): 497-509, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31254045

RESUMO

Cell-free DNA (cfDNA) is described to mirror intratumoral heterogeneity and gives insight about clonal evolution for improved therapeutic decisions. We sequenced cfDNA of a hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) cohort with a high coverage to examine the prevalence and relevance of any detected variant. cfDNA of 44 MBC patients was isolated, followed by library construction using a customized targeted DNA panel with integrated unique molecular indices analyzing AKT1, AR, BRCA1, BRCA2, EGFR, ERCC4, ERBB2, ERBB3, ESR1, FGFR1, KRAS, MUC16, PIK3CA, PIK3R1, PTEN, PTGFR, and TGFB1. CfDNA was sequenced on the NextSeq® 550 platform (Illumina) and variants were analyzed with Ingenuity Variant Analysis (QIAGEN). We evaluated cfDNA variants in 40 of the 44 hormone receptor-positive and HER2-negative patients with a high mean coverage of 22,000×, resulting in MUC16, BRCA2, ERBB3, and AR variant calling in > 90% of the patients. 47% of all AR variants were pathogenic and at least one pathogenic or likely pathogenic variant was detected in each patient. A specific BRCA1 variant and > 3.5 pathogenic variants significantly associated with a reduced survival after diagnosis of metastasis. Longitudinal monitoring revealed an increase of pathogenic and likely pathogenic PIK3CA and ESR1 variant allele frequency under everolimus and exemestane, 8 months before proof of therapy failure by visual staging in one exemplary case. The identification of new variants with high prevalence, prognostic value, and dynamics under treatment by deep sequencing of cfDNA might empower sensitive monitoring and personalized therapeutic decisions.


Assuntos
Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , Variação Genética/genética , Receptores de Superfície Celular/genética , Alelos , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Receptor alfa de Estrogênio/genética , Feminino , Humanos
15.
Int J Cancer ; 146(1): 103-114, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199508

RESUMO

Next-generation sequencing of cell-free circulating DNA (cfDNA) has emerged as promising technique for identifying minimally invasive genomic profiling of tumor cells recently. However, it remains relatively unknown in LAM disease. In our study, paired cfDNA and genomic DNA (gDNA) in blood samples were obtained from 23 LAM patients and seven healthy controls to explore mutations profiles of targeted 70 cancer-related genes. As results, log2-based allele frequencies of mutations in cfDNA were significantly different from those of gDNA. By comparing the mutual mutations identified both in cfDNA and gDNA, a significant correlation was also observed. After removing mutations in gDNA, distinct somatic mutation profiles of cfDNA were observed in LAM patients. Forty of 70 targeted genes had recurrent mutations, of which ATM, BRCA2 and APC showed the highest frequency. Based on the mutation, correlation network constructed of 40 mutated genes, 11 hub genes bearing intensive interactions were highlighted, including BRCA1, BRCA2, RAD50, RB1, NF1, APC, MLH3, ATM, PDGFRA, PALB2 and BLM. Expression of the hub genes showed significant clusters between LAM patients and controls and that RAD50 and BRCA2 had the strongest associations with subject phenotypes. Myogenesis and estrogen response were confirmed to be positively regulated in LAM patients. Collectively, our study provided a landscape of genomic alterations in LAM and discovered several potential driver genes, that is, BRCA2 and RAD50, which shed a substantial light on the clinical application of key molecular markers and potential therapy targets for precision diagnosis and treatment in the future.


Assuntos
Ácidos Nucleicos Livres/genética , Neoplasias Pulmonares/genética , Linfangioleiomiomatose/genética , Mutação/genética , Adulto , Proteína BRCA2/genética , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Feminino , Frequência do Gene/genética , Genoma/genética , Genômica/métodos , Humanos , Masculino , Pessoa de Meia-Idade
16.
Cancer Treat Rev ; 83: 101951, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874446

RESUMO

Molecular profiling of tumor derived cell free DNA (cfDNA) is gaining ground as a prognostic and predictive biomarker. However to what extent cfDNA reflects the full metastatic landscape as currently determined by tumor tissue analysis remains controversial. Though technically challenging, whole exome sequencing (WES) of cfDNA enables thorough evaluation of somatic alterations. Here, we review the feasibility of WES of cfDNA and determine the sensitivity of WES-detected single nucleotide variants (SNVs) in cfDNA on individual patient data level using paired tumor tissue as reference (sharedSNVsAlltissueSNVs×100%). The pooled sensitivity was 50% (95% credible interval (CI): 29-72%). The tissue mutant allele frequency (MAF) of variants exclusively identified in tissue was significantly lower (12.5%, range: 0.5-18%) than the tissue MAF of variants identified in both tissue and cfDNA (23.9%, range: 17-38%), p = 0.004. The overall agreement (sharedSNVsAllSNVs×100%)between SNVs in cfDNA and tumor tissue was 31% (95% CI: 15-49%). The number of detected SNVs was positively correlated with circulating tumor DNA (ctDNA) fraction (p = 0.016). A sub analysis of samples with ctDNA fractions ≥ 25% improved the sensitivity to 69% (95% CI: 46-89%) and agreement to 46% (95% CI: 36-59%), suggesting that WES is mainly feasible for patients with high ctDNA fractions. Pre- and post-analytical procedures were highly variable between studies rendering comparisons problematic. In conclusion, various aspects of WES of cfDNA are largely in its investigative phase, standardization of methodologies is highly needed to bring this promising technique to its clinical potential.


Assuntos
Teorema de Bayes , Biomarcadores Tumorais/análise , Ácidos Nucleicos Livres/análise , DNA Tumoral Circulante/análise , DNA de Neoplasias/análise , Neoplasias/patologia , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Neoplasias/genética , Neoplasias/terapia , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma
17.
Am J Clin Pathol ; 153(1): 126-130, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585003

RESUMO

OBJECTIVES: Tuberculosis meningitis (TBM) is one of the most severe forms of tuberculosis. However, TBM diagnosis is quite challenging due to nonspecific clinical presentation and the paucity of the pathogen in cerebrospinal fluid (CSF) samples. In this study, we report a new method for detecting cell-free Mycobacterium tuberculosis DNA (cf-TB) in CSF and evaluate its clinical value for TBM diagnosis. METHODS: Of 68 patients prospectively recruited, 46 were diagnosed as having TBM and 22 as non-TBM. We compared the cf-TB method with CSF smear microscopy, mycobacterial culture, and the Xpert MTB/RIF assay (Xpert) using the consensus case definition for TBM proposed in 2009 as a reference standard. RESULTS: The sensitivity of the cf-TB test was 56.5% (26/46) in patients with TBM, and it was significantly higher than other methods: microscopy (2.2%, 1/46; P < .001), mycobacterial culture (13.0%, 6/46; P < .001), and Xpert (23.9%, 11/46; P = .001). For specificity, none of the four methods reported false-positive results in the non-TBM group. CONCLUSIONS: The new method detecting cell-free M tuberculosis DNA in CSF is rapid and accurate for diagnosis of TBM and easily incorporated into regular laboratory tests.


Assuntos
Ácidos Nucleicos Livres/líquido cefalorraquidiano , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Meníngea/diagnóstico , Adolescente , Adulto , DNA Bacteriano/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/microbiologia , Tuberculose Meníngea/patologia , Adulto Jovem
18.
Anticancer Res ; 39(12): 6595-6602, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810925

RESUMO

BACKGROUND/AIM: Non-invasive biomarker detection using DNA from cell-free circulating DNA (cfDNA) and circulating tumor cells (ctcDNA) are emerging as they can be used for early diagnosis, prognosis and therapeutic target selection for cancer. However, cfDNA and ctcDNA from the same patient have not yet been compared extensively on how different the genetic characteristics of the two are in terms of the overlap between them. MATERIALS AND METHODS: The performance of a customized NGS panel was used to compare the variants found in the 20 pairs of cfDNA and ctcDNA from gynecological cancer patients. RESULTS: A genetic variant analysis revealed that there were only nine common overlapping variants out of 63 between the cfDNA and ctcDNA pairs, while 31 and 22 were unique to cfDNA and ctcDNA, respectively. CONCLUSION: A combinatory analysis of both cfDNA and CTCs from cancer patients can improve the sensitivity of liquid biopsies. These results are expected to provide better genetic target information for guiding clinical strategies for cancer.


Assuntos
Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Variação Genética , Neoplasias dos Genitais Femininos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Feminino , Neoplasias dos Genitais Femininos/tratamento farmacológico , Humanos , Pessoa de Meia-Idade , Análise de Sequência de DNA/métodos
19.
Sheng Wu Gong Cheng Xue Bao ; 35(12): 2284-2294, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31880136

RESUMO

With the development of liquid biopsy technology, plasma cell-free DNA (cfDNA) becomes one of the research hotspots. Whole-genome bisulfite sequencing of plasma cell-free DNA has shown great potential medical applications such as cancer detection. However, the practical stability evaluation is still lacking. In this study, plasma cell-free DNA samples from two volunteers at different time were collected and prepared for sequencing in multiple laboratories. The library preparation strategies include pre-bisulfite, post-bisulfite and regular whole-genome sequencing. We established a set of quality control references for plasma cell-free DNA sequencing data and evaluated practical stability of blood collection, DNA extraction, and library preparation and sequencing depth. This work provided a technical practice guide for the application of plasma cfDNA methylation sequencing for clinical applications.


Assuntos
Sequenciamento Completo do Genoma , Ácidos Nucleicos Livres , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , Sulfitos
20.
Int J Mol Sci ; 21(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861832

RESUMO

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Ácidos Nucleicos Livres/genética , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Projetos Piloto
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