RESUMO
Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids.
Assuntos
Ácidos Nucleicos Livres , Terminologia como Assunto , Animais , Ácidos Nucleicos Livres/sangue , HumanosRESUMO
We have designed a highly sensitive assay based on the Safe-SeqS technology to detect de novo mutations in the KIT gene and tested its performance. This assay was applied to plasma samples of GIST patients before and after treatment with regorafenib (GRID III trial) and mutations at known and novel sites of potential secondary resistance were identified.
Assuntos
Análise Mutacional de DNA/métodos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Antineoplásicos/uso terapêutico , Neoplasias Gastrointestinais/sangue , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/sangue , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Humanos , Mesilato de Imatinib/uso terapêutico , Indóis/uso terapêutico , Avaliação de Resultados em Cuidados de Saúde/métodos , Compostos de Fenilureia/uso terapêutico , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Reprodutibilidade dos Testes , SunitinibeRESUMO
Researchers working in industrial laboratories as well as in academic laboratories discussed topics related to the use of extracellular nucleic acids in different fields. These included areas like non-invasive prenatal diagnosis, the application of different methods for the analysis and characterization of patients with benign and malignant diseases and technical aspects associated with extracellular nucleic acids. In addition, the possibilities and chances for a cooperation of researchers working in different worlds, i.e. academia and industry, were discussed.
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Centros Médicos Acadêmicos/métodos , Indústrias/métodos , Pessoal de Laboratório , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/genética , Pesquisadores , Congressos como Assunto , Doença/genética , Espaço Extracelular/genética , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Ácidos Nucleicos/análise , Ácidos Nucleicos/sangueRESUMO
Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration-dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001-mediated anti-tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK-2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan-AKT inhibitor MK2206 in the CCA cell line TFK-1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK-2206 might be a new, effective strategy for the treatment of CCA.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Sinergismo Farmacológico , Everolimo , Citometria de Fluxo , Humanos , Imunoprecipitação , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.
RESUMO
We show that the times separating the birth of benign, invasive, and metastatic tumor cells can be determined by analysis of the mutations they have in common. When combined with prior clinical observations, these analyses suggest the following general conclusions about colorectal tumorigenesis: (i) It takes approximately 17 years for a large benign tumor to evolve into an advanced cancer but <2 years for cells within that cancer to acquire the ability to metastasize; (ii) it requires few, if any, selective events to transform a highly invasive cancer cell into one with the capacity to metastasize; (iii) the process of cell culture ex vivo does not introduce new clonal mutations into colorectal tumor cell populations; and (iv) the rates at which point mutations develop in advanced cancers are similar to those of normal cells. These results have important implications for understanding human tumor pathogenesis, particularly those associated with metastasis.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Adenoma/genética , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cinética , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação/genética , Metástase Neoplásica/genética , Estadiamento de Neoplasias , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND & AIMS: Somatic mutations provide uniquely specific markers for the early detection of neoplasia that can be detected in DNA purified from plasma or stool of patients with colorectal cancer. The primary purpose of the present investigation was to determine the parameters that were critical for detecting mutations using a quantitative assay. A secondary purpose was to compare the results of plasma and stool DNA testing using the same technology. METHODS: We examined DNA purified from the stool of 25 patients with colorectal cancers before surgery. In 16 of these cases, plasma samples also were available. Mutations in stool or plasma were assessed with an improved version of the BEAMing technology. RESULTS: Of the 25 stool DNA samples analyzed, 23 (92%) contained mutations that were present in the corresponding tumors from the same patients. In contrast, only 8 of the 16 (50%) plasma DNA samples analyzed had detectable levels of mutated DNA. We found that the DNA fragments containing mutations in both stool and plasma DNA typically were smaller than 150 bases in size. The sensitivity of the new method was superior to a widely used technique for detecting mutations, using single base extension and sequencing, when assessed on the same samples (92% vs 60%; P = .008, exact McNemar test). CONCLUSIONS: When assessed with sufficiently sensitive methods, mutant DNA fragments are detectable in the stool of more than 90% of colorectal cancer patients. DNA purified from stool provides a better template for mutation testing than plasma.
Assuntos
Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/análise , Fezes/química , Regulação Neoplásica da Expressão Gênica , Mutação , Idoso , Idoso de 80 Anos ou mais , DNA de Neoplasias/sangue , Emulsões , Feminino , Citometria de Fluxo , Humanos , Magnetismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
Internal human xenografts provide valuable animal models to study the microenvironments and metastatic processes occurring in human cancers. However, the use of such models is hampered by the logistical difficulties of reproducibly and simply assessing tumor burden. We developed a high-sensitivity assay for quantifying human DNA in small volumes of mouse plasma, enabling in-life monitoring of systemic tumor burden. Growth kinetics analyses of various xenograft models showed the utility of circulating human DNA as a biomarker. We found that human DNA concentration reproducibly increased with disease progression and decreased after successful therapeutic intervention. A marked, transient spike in circulating human tumor DNA occurred immediately after cytotoxic therapy or surgery. This simple assay may find broad utility in target validation studies and preclinical drug development programs.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias do Colo/sangue , DNA de Neoplasias/sangue , Animais , Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Primers do DNA , Modelos Animais de Doenças , Feminino , Células HCT116 , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL-labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL-lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro-translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.
Assuntos
Neoplasias Colorretais/genética , Ensaio Cometa/métodos , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Fezes/química , Espectrometria de Fluorescência/métodos , Análise Espectral/métodos , Coloração e Rotulagem/métodos , Compostos de Boro , DNA de Neoplasias/análise , Humanos , Biossíntese de Proteínas/genéticaRESUMO
For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.
Assuntos
DNA/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Candida albicans/genética , Linhagem Celular , DNA/genética , DNA/isolamento & purificação , Primers do DNA/genética , Primers do DNA/isolamento & purificação , Primers do DNA/metabolismo , Dessecação , Drosophila melanogaster/genética , Corantes Fluorescentes , Biblioteca Gênica , Vidro , Humanos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/economia , Fases de Leitura Aberta/genética , Polilisina , Reação em Cadeia da Polimerase , Pseudomonas putida/genética , Controle de Qualidade , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade , Silanos , Fatores de Tempo , Trypanosoma brucei brucei/genéticaRESUMO
OBJECTIVES: Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies. METHODS: Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS). RESULTS: Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes. CONCLUSION: Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Análise Mutacional de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Coleta de Amostras Sanguíneas/instrumentação , Análise Mutacional de DNA/métodos , DNA de Neoplasias/isolamento & purificação , Humanos , Mutação , TemperaturaRESUMO
Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 - 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71- 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 - 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 - 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with = 1% of KRAS mutant DNA (4.8 vs. 7.3 months, p=0.008). Similarly, 67 patients with >1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with = 1% of mutant cfDNA (5.5 vs. 9.8 months, p = 0.001), which was confirmed in multivariable analysis. [Corrected]
Assuntos
DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Feminino , Genes erbB-1/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias/mortalidade , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto JovemRESUMO
The Abstract is incorrect in PubMed. The corrected Abstract is provided here.
RESUMO
A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.
Assuntos
Genes de Protozoários , Análise de Sequência com Séries de Oligonucleotídeos , Trypanosoma brucei brucei/genética , Animais , Northern Blotting , Escherichia coli/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transcrição Gênica , Trypanosoma brucei brucei/isolamento & purificaçãoRESUMO
PURPOSE: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. EXPERIMENTAL DESIGN: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. RESULTS: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. CONCLUSIONS: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies.
Assuntos
Neoplasias da Mama/genética , DNA de Neoplasias/sangue , Fosfatidilinositol 3-Quinases/sangue , Fosfatidilinositol 3-Quinases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Classe I de Fosfatidilinositol 3-Quinases , Estudos de Coortes , Feminino , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Estudos RetrospectivosRESUMO
PURPOSE: Defining an adequate resection margin of colorectal cancer liver metastases is essential for optimizing surgical technique. We have attempted to evaluate the resection margin through a combination of histopathologic and genetic analyses. EXPERIMENTAL DESIGN: We evaluated 88 samples of tumor margins from 12 patients with metastatic colon cancer who each underwent partial hepatectomy of one to six liver metastases. Punch biopsies of surrounding liver tissue were obtained at 4, 8, 12, and 16 mm from the tumor border. DNA from these biopsies was analyzed by a sensitive PCR-based technique, called BEAMing, for mutations of KRAS, PIK3CA, APC, or TP53 identified in the corresponding tumor. RESULTS: Mutations were identified in each patient's resected tumor and used to analyze the 88 samples circumscribing the tumor-normal border. Tumor-specific mutant DNA was detectable in surrounding liver tissue in 5 of these 88 samples, all within 4 mm of the tumor border. Biopsies that were 8, 12, and 16 mm from the macroscopic visible margin were devoid of detectable mutant tumor DNA and of microscopically visible cancer cells. Tumors with a significant radiologic response to chemotherapy were not associated with any increase in mutant tumor DNA in beyond 4 mm of the main tumor. CONCLUSIONS: Mutant tumor-specific DNA can be detected beyond the visible tumor margin, but never beyond 4 mm, even in patients whose tumors were larger prior to chemotherapy. These data provide a rational basis for determining the extent of surgical excision required in patients undergoing resection of liver metastases.
Assuntos
Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Genes APC , Neoplasias Hepáticas/genética , Adulto , Idoso , Biópsia por Agulha , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , DNA de Neoplasias/análise , Feminino , Genes p53 , Hepatectomia , Humanos , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genéticaRESUMO
Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Rearranjo Gênico , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Medicina de Precisão , Análise de Sequência de DNA/métodos , Translocação Genética , Sequência de Bases , Biomarcadores Tumorais/sangue , DNA/sangue , DNA/genética , Humanos , Dados de Sequência Molecular , Medicina de Precisão/instrumentação , Medicina de Precisão/métodosRESUMO
Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in approximately 5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , DNA de Neoplasias/genética , Técnicas Genéticas , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/química , Fezes/química , Humanos , Sensibilidade e Especificidade , Vimentina/químicaRESUMO
The measurement of circulating nucleic acids has transformed the management of chronic viral infections such as HIV. The development of analogous markers for individuals with cancer could similarly enhance the management of their disease. DNA containing somatic mutations is highly tumor specific and thus, in theory, can provide optimum markers. However, the number of circulating mutant gene fragments is small compared to the number of normal circulating DNA fragments, making it difficult to detect and quantify them with the sensitivity required for meaningful clinical use. In this study, we applied a highly sensitive approach to quantify circulating tumor DNA (ctDNA) in 162 plasma samples from 18 subjects undergoing multimodality therapy for colorectal cancer. We found that ctDNA measurements could be used to reliably monitor tumor dynamics in subjects with cancer who were undergoing surgery or chemotherapy. We suggest that this personalized genetic approach could be generally applied to individuals with other types of cancer.
Assuntos
Biomarcadores/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/sangue , Técnicas de Diagnóstico Molecular , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/terapia , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Magnetismo , Técnicas de Amplificação de Ácido Nucleico , RecidivaRESUMO
Early detection of colorectal tumors through the identification of mutant DNA in serum or plasma could have a substantial impact on morbidity and mortality. Somatic mutations are specific biomarkers for neoplastic cells, but their detection requires sensitive assays, as the number of circulating mutant molecules is small compared to the number of normal DNA molecules. A newly developed method can provide this sensitivity and at the same time precisely quantify the fraction of mutant molecules present in the clinical sample. Using this technology, it has been found that more than half of patients with early stage disease contain mutant DNA in their circulation.