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Background: Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published. Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49). Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.
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Ensaios de Triagem em Larga Escala/métodos , Técnicas Analíticas Microfluídicas , Análise de Célula Única/métodos , Proliferação de Células , Sobrevivência Celular , Escherichia coli/isolamento & purificação , Células HL-60 , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica , Análise de Célula Única/instrumentaçãoRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive "real-time" biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). METHODS AND FINDINGS: We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01-3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30-3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66-6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations. CONCLUSIONS: In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.
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Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/genética , Neoplasias Pulmonares/genética , Mutação , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , DNA de Neoplasias/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos ProspectivosRESUMO
BACKGROUND: Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing. METHODS: We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer. RESULTS: Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90). CONCLUSIONS: Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.
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DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/genética , Mutação , Neoplasias Pancreáticas/genética , Análise de Sequência de DNA , Alelos , Humanos , Neoplasias Pulmonares/diagnóstico , Mutagênese Insercional , Neoplasias Pancreáticas/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Deleção de SequênciaRESUMO
In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.
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Receptores ErbB/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS: We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS: Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS: These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Biomarcadores Tumorais , Neoplasias Colorretais/genética , Metilação de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/química , Neuropeptídeo Y/genética , Proteínas Repressoras/genética , Idoso , Biomarcadores Tumorais/genética , Metilação de DNA/genética , DNA de Neoplasias/genética , Feminino , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis.
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DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Neoplasias/genética , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA de Neoplasias/análise , Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Neoplasias/sangue , Neoplasias/diagnóstico , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Espectrofotometria/instrumentação , Espectrofotometria/métodosRESUMO
Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.
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Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , Neoplasias/sangue , Reação em Cadeia da Polimerase/métodos , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , DNA de Neoplasias/isolamento & purificação , Detecção Precoce de Câncer , Feminino , Corantes Fluorescentes/análise , Amplificação de Genes , Genes erbB-1 , Genes erbB-2 , Genes ras , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Recidiva Local de Neoplasia/sangue , Neoplasias/genética , Plasma , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Soro , Processamento de Sinais Assistido por ComputadorRESUMO
The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.
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RNA Helicases DEAD-box/genética , MicroRNAs/genética , Processamento Alternativo , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/genética , Éxons , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células MCF-7 , Camundongos , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Mioblastos/enzimologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Transcrição GênicaRESUMO
Estrogen and androgen receptors (ER and AR) play key roles in breast and prostate cancers, respectively, where they regulate the transcription of large arrays of genes. The activities of ER and AR are controlled by large networks of protein kinases and transcriptional coregulators, including Ddx5 and its highly related paralog Ddx17. The Ddx5 and Ddx17 RNA helicases are also splicing regulators. Here, we report that Ddx5 and Ddx17 are master regulators of the estrogen- and androgen-signaling pathways by controlling transcription and splicing both upstream and downstream of the receptors. First, Ddx5 and Ddx17 are required downstream of ER and AR for the transcriptional and splicing regulation of a large number of steroid hormone target genes. Second, Ddx5 and Ddx17 act upstream of ER and AR by controlling the expression, at the splicing level, of several key regulators of ER and AR activities. Of particular interest, we demonstrate that Ddx5 and Ddx17 control alternative splicing of the GSK3ß kinase, which impacts on both ER and AR protein stability. We also provide a freely available online resource which gives information regarding splicing variants of genes involved in the estrogen- and androgen-signaling pathways.
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Processamento Alternativo , Androgênios/farmacologia , RNA Helicases DEAD-box/metabolismo , Estrogênios/farmacologia , Transdução de Sinais , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Células MCF-7 , Estabilidade Proteica , Receptores Androgênicos/metabolismoRESUMO
It is widely accepted that pre-mRNA maturation, including splicing, is tightly coupled to both transcription and mRNA export, but factors linking the three processes are less understood. By analysing the estrogen-regulated expression of the c-fos mRNA that is processed during transcription, we show that the ddx5 RNA helicase, is required throughout the major nuclear steps of the expression of the c-fos gene, from transcription to mRNA export. Indeed, ddx5, whose recruitment on the c-fos gene was increased upon estrogen treatment, was required for the full transcriptional activation of the c-fos gene. In addition, ddx5 was required for c-fos co-transcriptional RNA splicing. When splicing occurred post-transcriptionally in the absence of ddx5, the c-fos mRNA was poorly exported into the cytosol because of inefficient recruitment of the TAP mRNA export receptor. Finally, ddx5 was present in the c-fos messenger ribonucleoprotein together with mRNA export factors, which further supports that ddx5 is a key operator in the c-fos 'mRNA factory'.
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RNA Helicases DEAD-box/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Ativação Transcricional , Núcleo Celular/metabolismo , Estradiol/farmacologia , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismo , Splicing de RNA , Transporte de RNA , Ribonucleoproteínas/metabolismo , Transcrição GênicaRESUMO
Both epigenetic and splicing regulation contribute to tumor progression, but the potential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases Ddx17 and Ddx5 contribute to tumor-cell invasiveness by regulating alternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an opposite manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone variant isoforms that in turn drive a transcription program to switch tumor cells to an invasive phenotype.