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Objective To research a simple and sensitive K-ras gene mutations detection method in order to be suitable for the routine mutation detection.Methods The corresponding detection locus oligonucleotide probe was designed.By the connection,amplification,labeling and ELISA reaction in probe,the mutation locus genotype was determined by the ELISA reaction detection value.With the six point mutations of G12S,G12R,G12C,G12D,G12A and G12V in 12 codons of K-ras gene as the detection objects,the plasma circulation DNA sample in 72 cases of lung cancer was detected,then the results were compared with those obtained by the direct sequencing.Results Three samples were identified as the G12S,G12R and G12A mutatins by the established method.But no K-ras mutations were detected in the samples by using the direct sequencing,indicating that the direct sequencing had lower sensitivity and was not suitable for the mutation detection of heterogeneous samples such as circulating DNA.Conclusion The simple and sensitive K-ras gene mutation detection method is established and can conduct the routine mutation detection for the heterogeneous samples.
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Circulating DNA is defined as a kind of extracellular DNA that exists in plasma,cerebrospinal fluid and synovial fluid.The concentration of circulating DNA of cancer patients is significantly higher than that in healthy people.The genetic and epigenetic alterations of circulating cell-free nucleic acids are relevant to cancer development and progression,for example,gene mutation,DNA methylation and microsatellite instability and so on.The quantitative and qualitative detection of circulating DNA shows promising potential value in cancer screening,diagnosis,disease monitoring treatment and prognosis.
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Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNAdamage- repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications for ageing and a multitude of human pathologies including initiation of cancer.
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Objective Plasma circulating DNA can be em-ployed in place of bone marrow examination for the auxiliary diagnosis of leukemia.This study aimed to explore the clinical application of the plasma DNA level in evaluating the effect of chemotherapy on chronic leukemia. Methods We collected blood samples from 52 patients with chronic myelogenous leukemia (CML) (33 in the chronic phase, 7 in the acceleration phase, and 12 in the blast phase) , 85 with chron-ic lymphocytic leukemia (CLL) (28 with complete remission, 27 with partial remission, and 30 with no remission), 4 patients with hairy cell leukemia (HCL), and 80 healthy subjects.We simultaneously obtained plasma DNA and recombinant plasmid DNA using the BI-LATEST DNA Kit and examined the human β-actin gene and the level of plasmid DNA by real-time quantitative PCR. Results Before chemotherapy, the median value of plasma DNA was 149.46(30.63-496.91)ng/ml in the CML and 101.54(69.10-258.14) ng/ml in the CLL patients, both significantly higher than in the healthy controls (19.05[12.67-25.92]ng/ml) (P<0.01).After chemotherapy, the plasma DNA level of the CML patients was remarkably decreased, but still higher than that of the controls ( P<0.01).The CML patients in the chronic phase showed a markedly higher level of plasma DNA (302.89[93.33-541.52]ng/ml) than those in the blast phase (43.19[23.54-70.03]ng/ml) and acceleration phase (28.11[16.21-92.07]ng/ml) (P<0.05).The CLL patients with CR exhibited a significantly lower level of plasma DNA (24.29[14.64-30.74]ng/ml) than those with PR (106.88 [96.23-143.25]ng/ml) and NR (460.73[284.57-653.38〗ng/ml) (P<0.01), but all dramatically higher than that of the healthy controls (P<0.01) Conclusion The quantification of plasma DNA has a clinical application value in evaluating the effect of chemo-therapy on chronic leukemia.
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Mutations detection of circulating tumor DNA can be divided into quantitative and qualitative classifications:the forumer mainly detects the total amount of circulating DNA (serum or plasma),whereas the latter mainly detects the specific genetic variations in serum or plasma DNA,such as gene mutations,methylations of tumor suppressor genes,and microsatellite alterations,etc.Both of them may reflect the tumor presence and disease severity.In this paper,mutations detection and its clinical significance of circulating tumor DNA in patients with hepatocellular carcinoma are reviewed.
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O tratamento padrão para pacientes com câncer de reto localmente avançado consiste no uso de quimioradioterapia neoadjuvante (QRTn), seguida por cirurgia. Uma fração significativa dos pacientes responde completamente ao tratamento e no momento da reavaliação não apresenta evidência clínica nem radiológica de doença. Uma abordagem alternativa, Watch and Wait, propõe não operar imediatamente esses pacientes e submetê-los a um protocolo de observação frequente, a fim de evitar as morbidades associadas à cirurgia. No entanto, a avaliação da resposta ao tratamento ainda é um desafio, devido à subjetividade da avaliação clínica e a ausência de exames radiológicos suficientemente sensíveis e específicos para garantir a ausência de células tumorais residuais ou capazes de detectar a recorrência precoce da doença. DNA circulante contendo alterações genéticas específicas do tumor (ctDNA) pode ser encontrado na fração livre de células do sangue e tem sido utilizado para monitorar a dinâmica tumoral em tumores sólidos. Avanços recentes das tecnologias de sequenciamento permitem a identificação eficiente e rápida e a um custo relativamente baixo de alterações genéticas em tumores individuais, superando o problema imposto pela ausência de alterações genéticas recorrentes nesses tumores. Essas alterações podem ser utilizadas como biomarcadores personalizados para monitorar a resposta ao tratamento, detectar doença residual e a recidiva precoce do tumor. O objetivo deste trabalho foi identificar e estudar biomarcadores personalizados em pacientes com câncer de reto localmente avançado tratados com QRTn e avaliar a capacidade desses biomarcadores para monitorar a dinâmica tumoral, e auxiliar na definição da conduta cirúrgica e na detecção da recidiva precoce da doença. Biópsias de seis pacientes com adenocarcinoma de reto distal (cT2- 3N0-1M0), foram coletadas prospectivamente pré-tratamento. O DNA genômico extraído a partir das biópsias foi usado para construir bibliotecas tipo mate-pair para o sequenciamento do genoma completo, utilizando a plataforma SOLiD. Rearranjos inter e intracromossômicos foram identificados utilizando programas computacionais desenvolvidos pelo nosso grupo de pesquisa e em seguida foram validados utilizando PCR e sequenciamento Sanger. Foram validadas, pelo menos, três variações estruturais para cada paciente. Amostras de plasma foram coletadas no momento do diagnóstico, depois da QRTn e durante o seguimento. DNA circulante total foi extraído a partir das amostras de plasma e ensaios personalizados foram desenvolvidos para monitorar a presença de variações estruturais através de PCR Digital. ctDNA foi detectado em todas amostras de plasma pré-tratamento de pacientes com tumores T3. A detecção desses biomarcadores apresentou boa correlação com a resposta ao tratamento, no entanto, esta abordagem não foi sensível o suficiente para detectar doença residual. Para dois pacientes que desenvolveram doença metastática foi verificado um aumento nos níveis de ctDNA com pelo menos 36 semanas antes do diagnóstico clínico de doença metastática, sendo possível correlacionar os níveis de ctDNA detectados em coletas subsequentes com a resposta ao tratamento sistêmico de segunda linha. Este estudo, embora de caráter exploratório, gerou dados relevantes e suficientes para justificar a realização de estudos adicionais para avaliar a aplicação dos biomarcadores personalizados na definição da conduta cirúrgica e no acompanhamento de pacientes com câncer de reto tratados com QRTn
The standard treatment for patients with locally advanced rectal cancer comprises in neoadjuvant chemo radiotherapy (nCRT), followed by surgery. A significant fraction of these patients show complete response to the treatment and at the time of reassessment, there are no clinical and nor radiological evidence of residual tumor. An alternative approach, Watch and Wait, proposes not to immediately operate these patients, but to submit them to a protocol of frequent observation in order to avoid the morbidities associated with radical surgery. However, assessment of treatment response remains a significant challenge due to the subjectivity of the clinical examination and to the lack of sufficiently sensitive tools to ensure the absence of tumor cells or to detect early disease recurrence. Circulating DNA carrying tumor-specific genetic alterations (circulating tumor DNA - ctDNA) can be found in the cell-free fraction of the blood and has been successfully used to monitor the tumor dynamics in solid tumors. Recent advances in sequencing technologies have enabled the rapid and cost effective identification of genetic alterations in individual tumors, overcoming the problem imposed by the absence of recurrent genetic alterations in these tumors. These alterations can be used as personalized biomarkers to monitor treatment response, detect residual disease and early tumor recurrence. The purpose of this work was to identify and validate the use of personalized biomarkers for patients with locally advanced rectal cancer treated with nCRT and to evaluate the ability of these biomarkers to monitor the tumor dynamics, to define surgical approach and to detect early recurrence of the disease. Pre-treatment biopsies from 6 patients with cT2-3N0-1M0 distal rectal adenocarcinoma were prospectively collected. Genomic DNA extracted from the biopsies was used to construct mate-pair libraries for whole genome sequencing using SOLiD platform. Inter and intrachromosomal rearrangements were identified using an in-house bioinformatics pipeline and validated using PCR amplification and Sanger sequencing. At least three structural variations were validated for each patient. Plasma samples were collect at diagnosis, after nCRT and follow-up. Circulating DNA was obtained from the plasma samples and personalized assays were designed to monitor the presence of structural variations using Droplet Digital PCR. ctDNA was detected in all pre-treatment plasma samples for patients with T3 tumors. The detection of these biomarkers showed a good correlation with the treatment response, nonetheless, the approach was not sensitive enough to detect residual disease. In two patients who developed metastatic disease, an increase in ctDNA levels was observed at least 36 weeks before clinical detection of metastatic disease, and it was possible to correlate the level of ctDNA in subsequent plasma samples with response to the second-line treatment. This study, although exploratory, generated relevant and sufficient data to support additional studies to evaluate the use of personalized biomarkers in the surgical management and follow-up of rectal cancer patients treated with nCRT
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Masculino , Femenino , Biomarcadores de Tumor/análisis , ADN/genética , Terapia Neoadyuvante/clasificación , Neoplasias del Recto/patología , Biblioteca de Genes , Células Neoplásicas Circulantes/metabolismo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Background and purpose:Aberrant DNA methylation that leads to the inactivation of tumor suppressor genes plays important roles in development and progression of breast cancer. Clinically, related gene methylation is considered to be a promising biomarker for tumor diagnosis and prognosis. This study aimed to investigate the methylation status ofSox17 gene in breast cancer tissue and its corresponding plasma circulating DNA, as well as to investigate its value in breast cancer early diagnosis and prognosis.Methods:TheSox17 gene promoter methylation status was detected by MSP in 86 cases of breast cancer, 36 normal breast tissues and its paired plasma DNA, the results were analyzed with corresponding clinical and pathological features.Results:The frequency ofSox17 gene methylation rate among 86 breast cancer tissues was 77.9%(67/86), and was 61.6%(53/86)in plasma circulating DNA, however, noSox17 gene methylation was found in normal breast tissues.Sox17 gene promoter methylation in plasma circulating DNA was signiifcantly associated with the methylation status in tumor tissues (r=0.502,P=0.000). In breast cancer tissue specimens,Sox17 methylation status was significantly correlated with tumor stage (χ2=6.18,P=0.041) and lymph node metastasis (χ2=13.54,P=0.001);Sox17 gene methylation rate was signiifcantly correlated with tumor stage (χ2=27.06,P=0.000), tumor size (χ2=9.65,P=0.007) and lymph node metastasis (χ2=20.80,P=0.000) in plasma samples, and there was no signiifcant difference ofSox17 gene methylation between patient age, histological grade and ER, PR, HER-2/neu status.Conclusion:Sox17 gene promoter methylation plays an important role in the carcinogenesis and development of breast cancer, and may be associated with the prognosis of breast cancer. Furthermore, methylatedSox17 gene may be a useful tumor biomarker in plasma circulating DNA for breast cancer detection and disease monitoring.
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Traditional immunological strategy which largely based on antigen-antibody techniques such as CA153,cytokeratin CK8/18/19,is still the main diagnostic biomarkers for breast cancer.Molecular biology markers such as BRCA1/2 and HER2 are becoming popular and hot.This article summarizes recent progress of breast cancer diagnosis based on nucleic acid related strategies such as circulating DNA,miRNAs,aptamers and circulating tumor cells.
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Objective: To quantitatively detect the serum circulating DNA in patients with breast cancer and evaluate its potential applications. Methods: The copy number of GAPDH gene in serum circulating DNA was measured by real-time quantitative PCR from 100 healthy female volunteers, 100 patients with benign breast lesions, and 200 patients with breast cancer. The copy number of GAPDH gene was used as an indicator for circulating DNA content. Results: With a cut-off value of ≥1×103 copies, the results of serum circulating DNA detection in 93% of healthy females and 95% of patients with benign breast lesions were negative, and 84.5% of patients with breast cancer were positive. The positive rate of serum circulating DNA in patients with I-II stage breast cancer was 84%. The DNA content was significantly different among healthy females, patients with benign breast lesions and the patients with breast cancer (P<0.005). Conclusion: There is a certain amount of tumor-derived circulating DNA in patients with early stage breast cancer. These genetic materials may serve as a type of specific biomarkers. Copyright© 2011 by TUMOR.
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Circulating DNA is cell-free DNA existing in plasma or serum. It has already been verified that circulating DNA of cancer patients is derived from tumor cells. Therefore, it is of great value to detect the changes in the quantity and quality of the circulating DNA in cancer patients for early diagnosis and prognosis. The advantages of the detection of circulating DNA such as micro-trauma, convenient access to samples, possibility of continuous and dynamic monitoring, make it a promising tumor mark. This review recapitulates the application of circulating DNA analysis in hepatocellular carcinoma patients for diagnosis and prognosis.
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Objective: To detect somatic mutations in epidermial growth factor receptor (EGFR) in the serum circulating DNA, screen the EGFR-mutated lung cancer patients, and evaluate the clinical effects of the genetically targeted drug gefitinib. Methods: DNA was extracted from the serum specimens of patients and amplified by PCR. The EGFR mutations in PCR product were detected by direct sequence analysis. Results: Somatic mutations of EGFR were identified in serum from 46 of the 116 cases (39.7%), including 30 cases (65.2%) in exon 19 and 16 cases (34.8%) in exon 21. Female patients and those patients with adenocarcinoma (including adenosquamous carcinoma) had an increased frequency of mutations. Further analysis on 20 lung cancer patients demonstrated that the EGFR mutation in serum was consistant with that detected in tumor tissues, suggesting that the EGFR mutations in serum originated from the primary tumor. Based on the screening assay, 19 patients with EGFR mutations who failed prior chemotherapy were treated with gefitinib. The response rate was 52.6% and the disease-controlling rate was 89.5%. The median progression-free survival time was 8 months, the median overall survival time was 12 months, and 2-year survival rate was 33.3%. Conclusions: The EGFR mutations in serum circulating DNA are consistant with the mutation in lung cancer tissue. Gefitinib treatment has significant effects on the advanced lung cancer based on the screening results of EGFR mutation.
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Background and purpose:It has been confirmed that homozygous deletion of p16/p15 gene and its co-deletion of p16/p15 genes were related to the occurrence, progress and prognosis of epithelial ovarian cancer. However, the mono-deletion and co-deletion of the genes has been detected with tissue but not in serum DNA of the epithelial ovarian cancer. In this article, we studied the relationship between homozygous deletion of p16/p15 gene and its co-deletion of p16/p15 genes in serum DNA of the epithelial ovarian cancer.Methods:Primers were used to amplify exon 2 of p16 and exon 2 of p15 gene by polymerase chain reaction. Homozygous deletions of the p16, p15 and co-deletion of p16/p15 genes were studied in either serum DNA of 165 patients with epithelial ovarian cancer, their counterpart lymphocytes DNA, serum DNA of 25 benign ovarian cyst or of 15 health donors.Results:The homozygous deletion rates of either p15 or p16 gene were 27.9%(46/165)and 27.3%(45/165)serum DNA in the patients with epithelial ovarian cancer respectively, while the co-deletion rate of p16/p15 genes was 24.2% (40/165). However, the deletions of p15/p16 genes and its co-deletion were not found in serum DNA of the counterpart lymphocytes,25 benign ovarian cyst and 15 health donors (The P values were 0.000、0.000 and 0.000 respectively). The deletions of either p15 or p16 gene for the patients with stage Ⅰ~Ⅱ were 14.3%(5/35) and 11.4%(4/35), 33.3%(25/75) and 32.0%(24/75) for the patients with stage Ⅲ, 29.1%(16/55) and 30.9% (17/55) for stage Ⅳ, respectively. Although there was no significant differences among the groups, the deletion of p15 and p16 genes in the patients with advanced stage were higher than that with early stage. The deletion was not found to be associated with histopathology of epithelial ovarian cancer.Conclusions:Homozygous deletions of the p16, p15 genes and its co-deletion of p15/p16 genes were commonly found in the serum DNA of epithelial ovarian cancer and might be associated with clinical stage of the disease. It was suggested that detection with serum DNA may be used as a micro-invasive approach and the deletion of genes might served as biological markers for the development and prognosis of the patients with epithelial ovarian cancer.
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DNA methylation is one of the most common epigenetic events in eukaryotic cell.Aberrant DNA methylation is tightly related to tumorigenesis.Recent evidence indicates that DNA methylation is a promosing biomarker for cancer.The cancer-associated DNA methylation events could be detected from serum(plasma),urine,stool and bronchoalveolar lavage,providing a new choice for noninvasive cancer diagnosis.In this review,we summarized the advances on DNA methylation for noninvasive cancer diagnosis.