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1.
PLoS One ; 15(9): e0237802, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32976510

RESUMO

As availability of precision therapies expands, a well-validated circulating cell-free DNA (cfDNA)-based comprehensive genomic profiling assay has the potential to provide considerable value as a complement to tissue-based testing to ensure potentially life-extending therapies are administered to patients most likely to benefit. Additional data supporting the clinical validity of cfDNA-based testing is necessary to inform optimal use of these assays in the clinic. The FoundationOne®Liquid CDx assay is a pan-cancer cfDNA-based comprehensive genomic profiling assay that was recently approved by FDA. Validation studies included >7,500 tests and >30,000 unique variants across >300 genes and >30 cancer types. Clinical validity results across multiple tumor types are presented. Additionally, results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction for copy number amplifications, and 30.4% TF for copy number losses. The limit of detection for microsatellite instability and blood tumor mutational burden were also determined. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%. In comparison with an orthogonal method, an overall positive percent agreement of 96.3% and negative percent agreement of >99.9% was observed. These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction. Critically, clinical validity data is presented across multiple cancer types.


Assuntos
Bioensaio/métodos , Ácidos Nucleicos Livres/genética , Genômica , Neoplasias/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Receptores ErbB/genética , Éxons/genética , Humanos , Limite de Detecção , Mutação/genética , Intervalo Livre de Progressão , Reprodutibilidade dos Testes
2.
Ann Hematol ; 99(8): 1763-1769, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32577844

RESUMO

We aimed to detect the MYD88L265P and CXCR4S338X mutations in cell-free DNA (cfDNA) in patients with Waldenström macroglobulinemia (WM). We collected peripheral blood and paired bone marrow aspirates from 27 WM patients (including 16 patients with newly diagnosed WM, 3 patients with WM in relapse and 8 patients with WM during treatment). cfDNA was extracted from peripheral blood using a QIAamp Circulating Nucleic Acid Kit. The MYD88L265P and CXCR4S338X mutations were detected by real-time allele-specific PCR (AS-PCR) in cfDNA and genomic DNA (gDNA) extracted from bone marrow aspirates. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was determined using a serial dilution of 10%, 2%, 0.4% and 0.08% MYD88L265P cfDNA in wild-type cfDNA. Among the 27 patients, MYD88L265P was detected in 88.9% of them in gDNA and in 85.2% of them in cfDNA, with a concordance rate of 96.3%. The concordance rates were 93.8%, 100% and 100% in patients with newly diagnosed WM, patients with WM in relapse and patients with WM during treatment, respectively. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was 0.4%. CXCR4S338X was detected in 6.3% of the 16 newly diagnosed WM patients in both gDNA and cfDNA, with a concordance rate of 100.0%. It is feasible to apply cfDNA to detect MYD88L265P and CXCR4S338X in WM patients with a high concordance rate.


Assuntos
Ácidos Nucleicos Livres/genética , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Ácidos Nucleicos Livres/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/sangue , Proteínas de Neoplasias/sangue , Receptores CXCR4/sangue , Sensibilidade e Especificidade , Macroglobulinemia de Waldenstrom/sangue , Macroglobulinemia de Waldenstrom/terapia
3.
PLoS One ; 15(6): e0233782, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520974

RESUMO

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , Metilação de DNA , Neoplasias Pancreáticas/genética , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/normas , Ilhas de CpG , Detecção Precoce de Câncer/métodos , Detecção Precoce de Câncer/normas , Feminino , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas/diagnóstico , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Proteínas Proto-Oncogênicas p21(ras)/genética , Sensibilidade e Especificidade
4.
Nat Med ; 26(7): 1041-1043, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572266

RESUMO

Improving early cancer detection has the potential to substantially reduce cancer-related mortality. Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) is a highly sensitive assay capable of detecting early-stage tumors. We report accurate classification of patients across all stages of renal cell carcinoma (RCC) in plasma (area under the receiver operating characteristic (AUROC) curve of 0.99) and demonstrate the validity of this assay to identify patients with RCC using urine cell-free DNA (cfDNA; AUROC of 0.86).


Assuntos
Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Metilação de DNA/genética , Detecção Precoce de Câncer , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/urina , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/urina , Epigenoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
5.
Int J Clin Oncol ; 25(8): 1523-1532, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32394048

RESUMO

Oncogenic RAS mutations are negative biomarkers of response to epidermal growth factor receptor (EGFR) blockade. RAS mutations are usually detected in biopsies of primary colorectal tumors. However, the genomic profiles of primary tumors and metastases are not always concordant, and chemotherapeutic agents can alter the tumor molecular landscape. Cell-free DNA (cfDNA) is a novel tool to detect molecular heterogeneity. This study evaluated the clinical utility of cfDNA to predict primary or secondary resistance to EGFR blockade in patients with metastatic colorectal cancer. Thirty metastatic colorectal cancer patients without RAS and BRAF mutations were prospectively enrolled and treated with cytotoxic agents and EGFR blockade as first-line therapy. cfDNA was analyzed for the presence of RAS, BRAF, and EGFR (S492R) point mutations before initiating chemotherapy and every 2 months during chemotherapy. The analysis was performed in 223 plasma samples from all 30 patients. Of the 30 patients, five had RAS mutations in their cfDNA before starting chemotherapy and did not respond. Twenty-four of the remaining 25 patients without cfDNA RAS mutations had a response. Twenty of the 24 responders developed secondary resistance and cfDNA RAS mutations were found in 17 of the 20. cfDNA BRAF mutations were found in seven, and EGFR mutations were found in eight of the 20 patients. Emerging RAS, BRAF, and EGFR mutations occurred in patients with primary and secondary resistance to EGFR blockade. The detection of these mutations in cfDNA is a promising approach to predict treatment response and secondary resistance.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácidos Nucleicos Livres/genética , Cetuximab/administração & dosagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Fluoruracila/uso terapêutico , Humanos , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Compostos Organoplatínicos/uso terapêutico , Panitumumabe/administração & dosagem , Projetos Piloto , Resultado do Tratamento
6.
PLoS One ; 15(5): e0232754, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379795

RESUMO

Analyzing cell-free DNA (cfDNA) as a source of circulating tumor DNA is useful for diagnosing or monitoring patients with cancer. However, the concordance between cfDNA within liquid biopsy and genomic DNA (gDNA) within tumor tissue biopsy is still under debate. To evaluate the concordance in a clinical setting, we enrolled 54 patients with metastatic colorectal cancer and analyzed their plasma cfDNA, gDNA from peripheral blood mononuclear cells (PBMC), and gDNA from available matched tumor tissues using ultra-deep sequencing targeting 10 genes (38-kb size) recurrently mutated in colorectal cancer. We first established a highly reliable cut-off value using reference material. The sensitivity of detecting KRAS hotspot mutations in plasma was calculated as 100%, according to digital droplet PCR. We could selectively detect clinically important somatic alterations with a variant allele frequency as low as 0.18%. We next compared somatic mutations of the 10 genes between cfDNA and genomic DNA from tumor tissues and observed an overall 93% concordance rate between the two types of samples. Additionally, the concordance rate of patients with the time interval between liquid biopsy and tumor tissue biopsy within 6 months and no prior exposure to chemotherapy was much higher than those without. The patients with KRAS mutant fragments in plasma had poor prognosis than those without the mutant fragments (33 months vs. 63 months; p<0.05). Consequently, the profiling with our method could achieve highly concordant results and may facilitate the surveillance of the tumor status with liquid biopsy in CRC patients.


Assuntos
Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Adulto , Idoso , Neoplasias Colorretais/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
Gene ; 753: 144798, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32445925

RESUMO

Gastric carcinoma (GC) ranks fifth in terms of cancer morbidity and third in cancer-related death worldwide and imposes enormous health and economic burdens. The molecular mechanisms underlying GC formation and progression remain unclear. Our aim was to identify the involvement of circular RNA circFOXO3 in GC, and to determine the underlying mechanisms. In this study, we revealed a stimulatory role of circular RNA circFOXO3 in tumor growth in vivo. CircFOXO3 enhanced GC cell proliferation and migration in vitro and promoted tumor growth of GC cells in vivo. Bioinformatic analysis revealed that circFOXO3 might regulate USP44 expression by specifically binding to microRNA (miR)-143-3p. Existence of circFOXO3-miR-143-3p-USP44 axis in GC cells was confirmed by RNA-binding protein immunoprecipitation, luciferase reporter assay, and an RNA pull-down experiments. All the data indicate that circFOXO3 promotes GC cell proliferation and migration by upregulating USP44 expression via targeting of miR-143-3p.


Assuntos
Ácidos Nucleicos Livres/metabolismo , Proteína Forkhead Box O3/genética , MicroRNAs/metabolismo , Neoplasias Gástricas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ácidos Nucleicos Livres/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Proteína Forkhead Box O3/metabolismo , Células HEK293 , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ativação Transcricional , Ubiquitina Tiolesterase/genética , Regulação para Cima
9.
J Biotechnol ; 313: 48-56, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32165241

RESUMO

Circulating cell-free DNAs (cfDNAs) are DNA fragments which can be isolated from mammalian blood serum or plasma. In order to gain deeper insight into their origin(s), we have characterized the composition of human and cattle cfDNA via large-scale analyses of high-throughput sequencing data. We observed significant differences between the composition of cfDNA in serum/plasma and the corresponding DNA sequence composition of the human genome. Retrotransposable elements and non-telomeric satellite DNA were particularly overrepresented in the cfDNA population, while telomeric satellite DNA was underrepresented. This was consistently observed for human plasma, bovine serum and for the supernatant of human cancer cell cultures. Our results suggest that reverse transcription of retrotransposable elements and secondary-structure formation during the replication of satellite DNA are contributing to the composition of the cfDNA molecules in the mammalian blood stream. We believe that our work is an important step towards the understanding of the biogenesis of cfDNAs and thus may also facilitate the future exploitation of their diagnostic potential.


Assuntos
Ácidos Nucleicos Livres/genética , DNA Satélite/genética , Retroelementos/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Ácidos Nucleicos Livres/sangue , Exossomos/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Conformação de Ácido Nucleico , Análise de Sequência de DNA , Adulto Jovem
10.
Prostate ; 80(7): 547-558, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32153047

RESUMO

BACKGROUND: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. METHODS: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). RESULTS: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. CONCLUSION: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed.


Assuntos
Ácidos Nucleicos Livres/urina , Metilação de DNA , DNA/urina , Modelos Genéticos , Neoplasias da Próstata/genética , Neoplasias da Próstata/urina , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Ácidos Nucleicos Livres/genética , Estudos de Coortes , DNA/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Neoplasias da Próstata/patologia , Medição de Risco
11.
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
12.
Clin Chem ; 66(2): 352-362, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32040573

RESUMO

BACKGROUND: Identifying patients with high-grade serous ovarian cancer (HGSOC) who will respond to treatment remains a clinical challenge. We focused on miR-622, a miRNA involved in the homologous recombination repair (HRR) pathway, and we assessed its predictive value in serum prior to first-line chemotherapy and at relapse. METHODS: Serum miR-622 expression was assessed in serum prior to first-line platinum-based chemotherapy in a prospective multicenter study (miRNA Serum Analysis, miRSA, NCT01391351) and a retrospective cohort (Biological Resource Center, BRC), and was also studied at relapse. Progression-free survival (PFS) and overall survival (OS) were used as primary and secondary endpoints prior to first-line chemotherapy and OS as a primary endpoint at relapse. RESULTS: The group with high serum miR-622 expression was associated with a significantly lower PFS (15.4 versus 24.4 months; adjusted HR 2.11, 95% CI 1.2 3.8, P = 0.015) and OS (29.7 versus 40.6 months; adjusted HR 7.68, 95% CI 2.2-26.2, P = 0.0011) in the miRSA cohort. In the BRC cohort, a high expression of miR-622 was also associated with a significantly lower OS (22.8 versus 35.9 months; adjusted HR 1.98, 95% CI 1.1-3.6, P = 0.026). At relapse, high serum miR-622 was associated with a significantly lower OS (7.9 versus 20.6 months; adjusted HR 3.15, 95% CI 1.4-7.2, P = 0.0062). Serum miR-622 expression is a predictive independent biomarker of response to platinum-based chemotherapy for newly diagnosed and recurrent HGSOC. CONCLUSIONS: These results may open new perspectives for HGSOC patient stratification and monitoring of resistance to platinum-based and poly(ADP-ribose)-polymerase-inhibitor-maintenance therapies, facilitating better and personalized treatment decisions.


Assuntos
Ácidos Nucleicos Livres/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Antineoplásicos/uso terapêutico , Intervalo Livre de Doença , Feminino , Humanos , MicroRNAs/sangue , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/diagnóstico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Estudos Retrospectivos
13.
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
15.
J Cardiovasc Transl Res ; 13(2): 171-180, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31997261

RESUMO

Coronary collaterals can effectively improve myocardial blood supply to the area of CTO (chronic total coronary occlusion) and can, thus, reduce infarct size. LUNAR1(leukemia-induced noncoding activator RNA-1) is a specific LncRNA regulated by Notch signaling that not only can enhance the expression of IGFR-1 but also can promote angiogenesis and cell survival. Here, we investigated the relationship between LncRNA-LUNAR1 levels in peripheral plasma and the formation of coronary collaterals. In total, 172 patients with CTO were enrolled and followed up for 12 months. Coronary collaterals were scored according to the Rentrop scoring system. Preclinical tests of tube formation were used to address the mechanisms behind the association between LncRNA-LUNAR1 and development of collaterals. Clinical data and inflammatory factors, including comorbidity, CD14++CD16- monocytes, and CCL2 (chemokine motif ligand 2), were compared and analyzed. Real-time PCR was used to detect the expression of LncRNA-LUNAR1 in peripheral blood plasma. The Rentrop score was positively correlated with LncRNA-LUNAR1 levels in patients with CTO (R = 0.47, p < 0.001). Tube formation assay proved the direct association between LncRNA-LUNAR1 and development of collaterals (p = 0.011). The univariate Kaplan-Meier analysis revealed that patients with low LncRNA-LUNAR1 expression exhibited worse clinical outcomes than those with high LncRNA-LUNAR1 levels (p = 0.008). Receiver operating characteristic (ROC) curve and correlation analysis further confirmed that LncRNA-LUNAR1 expression was closely related to chronic inflammatory diseases, especially diabetes (area = 0.644, p = 0.001; 95% CI, 0.562-0.726). Furthermore, both CD14++CD16- monocytes (r = - 0.37; p < 0.001) and CCL2 levels (r = - 0.35; p < 0.001) negatively affected the expression of LncRNA-LUNAR1. LncRNA-LUNAR1 expression was positively correlated with coronary collaterals in patients with CTO. Inflammatory factors, including CD14++CD16- monocytes and CCL2, may be risk factors affecting LncRNA-LUNAR1 expression.


Assuntos
Ácidos Nucleicos Livres/sangue , Circulação Colateral , Circulação Coronária , Oclusão Coronária/sangue , Oclusão Coronária/fisiopatologia , RNA Longo não Codificante/sangue , Idoso , Ácidos Nucleicos Livres/genética , Células Cultivadas , Quimiocina CCL2/metabolismo , Doença Crônica , Oclusão Coronária/diagnóstico por imagem , Oclusão Coronária/genética , Células Progenitoras Endoteliais/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica , Prognóstico , RNA Longo não Codificante/genética , Receptores de IgG/metabolismo , Transdução de Sinais , Fatores de Tempo
16.
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
17.
Clin Exp Med ; 20(1): 97-108, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894424

RESUMO

Several meta-analyses have evaluated the value of biomarkers in diagnosing breast cancer, but which biomarker has the optimal diagnostic value remains unclear. This overview aimed to compare the accuracy of different biomarkers in diagnosing breast cancer. PubMed, Embase.com, the Cochrane Library of Systematic Reviews, and Web of Science were searched. The assessment of multiple systematic reviews-2 (AMSTAR-2) was used to assess the methodological quality and preferred reporting items for a systematic review and meta-analysis of diagnostic test accuracy (PRISMA-DTA) for reporting quality. Pairwise meta-analyses were performed to estimate the pooled results for each biomarker, and indirect comparisons were conducted to compare diagnostic accuracy between biomarkers. Eleven systematic reviews (SRs) involving 218 original studies were included. All SRs were of critically low methodological quality, 3 SRs had minimal reporting flaws and 8 SRs had minor flaws. The pooled sensitivity and specificity were 0.77 and 0.87 for miRNA, 0.70 and 0.87 for circulating cell-free DNA, 0.29 and 0.96 for APC gene promoter methylation, 0.69 and 0.99 for 14-3-3σ promoter methylation, 0.63 and 0.82 for CA153, 0.58 and 0.87 for CEA, and 0.73 and 0.56 for PSA. Compared with CA153 and PSA, miRNA had a higher sensitivity and specificity. The sensitivity of miRNA was higher than circulating cell-free DNA and CEA, although they had the same specificities. APC gene promoter methylation and 14-3-3σ promoter methylation were more specific than miRNA, but they had unacceptably low sensitivity. In conclusion, miRNA had better diagnostic accuracy than the other six biomarkers. But due to the low quality of included SRs, the results need to be interpreted with caution. Further study should investigate the diagnostic accuracy of different biomarkers in direct comparisons and focus on the value of combined biomarkers.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Proteínas 14-3-3/genética , Proteína da Polipose Adenomatosa do Colo/genética , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Antígeno Carcinoembrionário/metabolismo , Ácidos Nucleicos Livres/genética , Metilação de DNA , Exorribonucleases/genética , Feminino , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas , Sensibilidade e Especificidade
18.
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
19.
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
20.
Surgery ; 167(3): 646-652, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31898953

RESUMO

BACKGROUND: Although early survival from sepsis has improved with timely resuscitation and source control, survivors frequently experience persistent inflammation and develop chronic critical illness. We examined whether increased copy number of endogenous alarmins, mitochondrial DNA, and nuclear DNA are associated with the early "genomic storm" in blood leukocytes and the development of chronic critical illness in hospitalized patients with surgical sepsis. METHODS: A prospective, observational, cohort study of critically ill septic patients was performed at a United States tertiary health care center. Blood samples were obtained at multiple time points after the onset of sepsis. Droplet Digital polymerase chain reaction (Bio-Rad Laboratories, Hercules, CA) was performed to quantify RHO (nuclear DNA) and MT-CO2 (mitochondrial DNA) copies in plasma. Leukocyte transcriptomic expression of 63 genes was also measured in whole blood. RESULTS: We enrolled 112 patients with surgical sepsis. Two experienced early death, 69 recovered rapidly, and 41 developed chronic critical illness. Both mitochondrial DNA and nuclear DNA copy number were increased in all sepsis survivors, but early nuclear DNA, and not mitochondrial DNA, copy number was further increased in patients who developed chronic critical illness. Cell-free DNA copy number was associated with in-hospital but not long-term (180-day and 365-day) mortality and were only weakly correlated with leukocyte transcriptomics. CONCLUSION: Increased cell-free DNA copy number persists in survivors of sepsis but is not strongly associated with leukocyte transcriptomics. Nuclear DNA but not mitochondrial DNA copy number is associated with adverse, short-term, clinical trajectories and outcomes.


Assuntos
Alarminas/imunologia , Ácidos Nucleicos Livres/genética , Dosagem de Genes/imunologia , Sepse/imunologia , Sobreviventes , Idoso , Alarminas/genética , Ácidos Nucleicos Livres/sangue , Doença Crônica/mortalidade , Doença Crônica/terapia , Estado Terminal/mortalidade , Estado Terminal/terapia , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , DNA Mitocondrial/imunologia , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sepse/sangue , Sepse/mortalidade , Sepse/terapia , Centros de Atenção Terciária/estatística & dados numéricos , Fatores de Tempo , Resultado do Tratamento
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