Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations.
Ontology highlight
ABSTRACT:
SUBMITTER: Tsao SC
PROVIDER: S-EPMC4476039 | biostudies-other | 2015
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Circulating tumour DNA (ctDNA) can be used to identify gene alterations. The purpose of this study was to determine whether the detection of ctDNA, based on the identification of BRAF and NRAS mutations before systemic treatment initiation, was associated with the prognosis of metastatic melanoma. In total, 68 BRAF or NRAS-mutated stage IV or unresectable stage III metastatic cutaneous melanoma patients were included and tested for the presence of BRAF and NRAS mutations in circulating DNA before treatment initiation, using the Cobas BRAF/NRAS Mutation Test (Roche). The expected mutation was detected in the plasma of 34/68 patients (50% sensitivity). ctDNA detection was associated with AJCC stage, along with the number and nature of metastases. ctDNA was less frequently detected in NRAS-mutated than in BRAF-mutated melanoma (36% and 66%, respectively). At initiation of first-line treatment, ctDNA detection was associated with poor prognosis in Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis (log-rank: p = 0.002 and p < 0.0001, respectively). In multivariate analysis, ctDNA detection was an independent factor of poor prognosis in OS, after adjustment for AJCC stage, number and nature of metastases and gender (HR = 4.384; 95% CI: (1.308; 14.699); p = 0.017).
| S-EPMC7409003 | biostudies-literature
Project description:mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.
| S-EPMC9571659 | biostudies-literature
Project description:Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation - lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.
| S-EPMC6718424 | biostudies-literature
Project description:Legionella pneumophila (L. pneumophila) is a harmful pathogen often found in water systems. In hospitals, the absence of L. pneumophila in water systems is mandatory by law, therefore, frequent and effective monitoring of water is of fundamental importance. Molecular methods based on reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) have been proposed for the detection of L. pneumophila, however, the sensitivity and accuracy of these methods have not been validated yet. Therefore, it is important to evaluate other strategies able to overcome the limits of culture‑based and RT‑qPCR methods. On these bases, we compared the sensitivity and accuracy of droplet digital PCR (ddPCR) and RT‑qPCR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments. ddPCR showed a higher sensitivity rate and accuracy compared to RT‑qPCR in detecting low bacterial load. In addition, ddPCR is not affected by the presence of fragmented DNA and showed higher accuracy than RT‑qPCR in monitoring the efficacy of heat shock treatments. In conclusion, ddPCR represents an innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness, ddPCR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.
| S-EPMC7521553 | biostudies-literature
Project description:Primary mucosal melanomas represent a minority of melanomas, but have a significantly worse prognosis than cutaneous melanomas. A better characterization of the molecular pathogenesis of this melanoma subtype could help us understand the risk factors associated with the development of mucosal melanomas and highlight therapeutic targets. Because the Mitogen-Activated Protein Kinase (MAPK) pathway plays such a significant role in melanoma development, we explore v-raf murine sarcoma viral oncogene homolog B (BRAF) and neuroblastoma RAS viral oncogene homolog (NRAS) mutations in mucosal melanoma and compare them to the mutation profiles in cutaneous melanoma and other tumors with BRAF and NRAS mutations. We show that in addition to being less frequent, BRAF and NRAS mutations are different in mucosal melanoma compared to cutaneous melanomas. Strikingly, the BRAF and NRAS mutation profiles in mucosal melanoma are closer to those found in cancers such as lung cancer, suggesting that mutations in mucosal melanoma could be linked to some genotoxic agents that remain to be identified. We also show that the atypical BRAF and NRAS mutations found in mucosal melanomas have particular effects on protein activities, which could be essential for the transformation of mucosal melanocytes.
| S-EPMC6721527 | biostudies-literature
Project description:Repeated quantitative measurement of bacterial DNA on whole blood has been shown to be a promising method for monitoring bloodstream infection (BSI) with selected bacterial species. To enable broad use of this method, we developed a quantitative droplet digital PCR (ddPCR) method for 16S rDNA. It was validated with species-specific ddPCRs for Staphylococcus aureus (nuc), Streptococcus pneumoniae (lytA), and Escherichia coli (uidA) on spiked whole blood samples and on repeated whole blood samples (days 0, 1-2, 3-4, 6-8, and 13-15) from 83 patients with BSI with these pathogens. In these patients, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one time-point. The highest positivity rates were seen in S. aureus BSI, where 92% of the patients were 16S rDNA-positive and 85% nuc-positive. Quantitative 16S rDNA and species-specific DNA showed strong correlations in spiked samples (r = 0.98; p < 0.0001) and clinical samples (r = 0.84; p < 0.0001). Positivity for 16S rDNA was rapidly cleared in patients with S. pneumoniae and E. coli BSI, but more slowly and sometimes persisted, in those with S. aureus BSI. The initial 16S rDNA load was higher in BSI patients with sepsis (Sepsis-3 definition) than without sepsis (median 2.38 vs. 0 lg10 copies/mL; p = 0.031) and in non-survivors than in survivors (median 2.83 vs. 0 lg10 copies/mL; p = 0.006). 16S rDNA ddPCR appears to be a promising method for bacterial DNA monitoring during BSI. The clinical value of such monitoring should be further studied.
| S-EPMC6853374 | biostudies-literature
Project description:Droplet digital PCR is a particularly valuable tool for ratiometric assays because it provides simultaneous absolute quantification of two target sequences in a single assay. This manuscript addresses a challenge in establishing a new ratiometric droplet digital PCR assay for use in sputum, the rRNA synthesis ratio. In principle, the methods established to evaluate precision and determine the limit of quantification for a single measurand cannot be applied to a ratiometric assay. The precision of a ratio depends on precision in both the numerator and denominator. Here, we evaluated the MOVER approximated coefficient of variation as indicator of assay precision that does not require technical replicates. We estimated the MOVER approximated coefficient of variation in dilution series and routine assays and evaluated its agreement with the traditional coefficient of variation. We found that the MOVER approximated coefficient of variation was able to recapitulate the traditional coefficient of variation without the requirement for replicate assays. We also demonstrated that the MOVER approximated coefficient of variation threshold can be used to define the limit of quantification of the rRNA synthesis Ratio. In conclusion, the MOVER approximated coefficient of variation may be useful not only for the rRNA synthesis ratio but for other assays that measure ratios via droplet digital PCR.
| S-EPMC8923918 | biostudies-literature
Project description:BRAF and NRAS mutations arise early in melanoma development, but their associations with low-penetrance melanoma susceptibility loci remain unknown. In the Genes, Environment and Melanoma Study, 1,223 European-origin participants had their incident invasive primary melanomas screened for BRAF/NRAS mutations and germline DNA genotyped for 47 single-nucleotide polymorphisms identified as low-penetrant melanoma-risk variants. We used multinomial logistic regression to simultaneously examine each single-nucleotide polymorphism's relationship to BRAF V600E, BRAF V600K, BRAF other, and NRAS+ relative to BRAF-/NRAS- melanoma adjusted for study features. IRF4 rs12203592*T was associated with BRAF V600E (odds ratio [OR] = 0.59, 95% confidence interval [CI] = 0.43-0.79) and V600K (OR = 0.65, 95% CI = 0.41-1.03), but not BRAF other or NRAS+ melanoma. A global test of etiologic heterogeneity (Pglobal = 0.001) passed false discovery (Pglobal = 0.0026). PLA2G6 rs132985*T was associated with BRAF V600E (OR = 1.32, 95% CI = 1.05-1.67) and BRAF other (OR = 1.82, 95% CI = 1.11-2.98), but not BRAF V600K or NRAS+ melanoma. The test for etiologic heterogeneity (Pglobal) was 0.005. The IRF4 rs12203592 associations were slightly attenuated after adjustment for melanoma-risk phenotypes. The PLA2G6 rs132985 associations were independent of phenotypes. IRF4 and PLA2G6 inherited genotypes may influence melanoma BRAF/NRAS subtype development.
| S-EPMC6200630 | biostudies-literature
Project description:Droplet digital PCR was used to validate miR-1290 as circulating biomarker for pancreatic cancer (PC). The diagnostic performance of miR-1290 was evaluate in 167 PC patients and 267 healthy subjects at clinical risk of developing the disease (HS). MiR-1290 plasma levels were compared to CA 19-9 determinations, and the combination of the two biomarkers was also taken into account. Plasma levels of miR-1290 were higher in PC patients compared to HS (p = 2.55 × 10-16). A similar trend was observed for CA 19-9 determinations (p = 1.03 × 10-47). ROC curve analysis revealed that miR-1290 in combination with CA 19-9 was effective for discriminating between PC patients and HS (AUC = 0.956, 95% CI = 0.933-0.979) than the two biomarkers tested alone (miR-1290: AUC = 0.734, 0.678-0.789; CA 19-9: AUC = 0.914, 0.877-0.951). The discriminating ability was higher when only PC patients with low or slightly increased CA 19-9 levels were compared with HS. MiR-1290 concentrations were not able to differentiate between PC patients with single or multiple risk factors for developing PC. Our data suggest that the absolute quantification of circulating miR-1290 levels does not allow to select patients at clinical risk of PC for entry into a surveillance program, and underline the methodological challenges still existing in utilizing circulating miRNAs as new promising biomarkers for PC.
| S-EPMC6219528 | biostudies-literature
Project description:Quantification of tumour-specific molecular markers at the RNA and DNA level for treatment response monitoring is crucial for risk-adapted stratification and guidance of individualized therapy in leukaemia and other malignancies. Most pediatric leukaemias and solid tumours of mesenchymal origin are characterized by a relatively low mutation burden at the single nucleotide level and the presence of recurrent chromosomal translocations. The genomic fusion sites resulting from translocations are stable molecular tumour markers; however, repeat-rich DNA sequences flanking intronic breakpoints limit the design of high sensitivity PCR assays for minimal residual disease (MRD) monitoring. Here, we quantitatively evaluated the impact of repeat elements on assay selection and the feasibility of using extended amplicons (≤1330 bp) amplified by droplet digital PCR to monitor pediatric chronic myeloid leukaemia (CML). Molecular characterization of 178 genomic BCR-ABL1 fusion sites showed that 64% were located within sequence repeat elements, impeding optimal primer/probe design. Comparative quantification of DNA and RNA BCR-ABL1 copy numbers in 687 specimens from 55 pediatric patients revealed that their levels were highly correlated. The combination of droplet digital PCR, double quenched probes and extended amplicons represents a valuable tool for sensitive MRD assessment in CML and may be adapted to other translocation-positive tumours.
| S-EPMC6653534 | biostudies-literature