scholarly journals Detecting TP53 mutations in diagnostic and archival liquid-based Pap samples from ovarian cancer patients using an ultra-sensitive ddPCR method

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicolai Skovbjerg Arildsen ◽  
Laura Martin de la Fuente ◽  
Anna Måsbäck ◽  
Susanne Malander ◽  
Ola Forslund ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Diagnosis ◽  
Risk Groups ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Ovarian Cancer Screening ◽  
Assay Sensitivity ◽  
Diagnostic Samples ◽  
Very High ◽  
Generation Sequencing

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of epithelial ovarian cancer and early detection is challenging. TP53 mutations are a hallmark of HGSOC and detection of these mutations in liquid-based Pap samples could provide a method for early diagnosis. Here we evaluate the use of IBSAFE, an ultra-sensitive droplet digital PCR (ddPCR) method, for detecting TP53 mutations in liquid-based Pap samples collected from fifteen women at the time of diagnosis (diagnostic samples) and/or up to seven years prior to diagnosis (archival samples). We analysed tumours for somatic TP53 mutations with next generation sequencing and were able to detect the corresponding mutations in diagnostic samples from six of eight women, while one patient harboured a germline mutation. We further detected a mutation in an archival sample obtained 20 months prior to the ovarian cancer diagnosis. The custom designed IBSAFE assays detected minor allele frequencies (MAFs) with very high assay sensitivity (MAF = 0.0068%) and were successful despite low DNA abundance (0.17–206.14 ng, median: 17.27 ng). These results provide support for further evaluation of archival liquid-based Pap samples for diagnostic purposes and demonstrate that ultra-sensitive ddPCR should be evaluated for ovarian cancer screening in high-risk groups or in the recurrent setting.

scholarly journals A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Bangchuan Hu ◽  
Yue Tao ◽  
Ziqiang Shao ◽  
Yang Zheng ◽  
Run Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Culture ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Pathogen Detection ◽  
Bloodstream Infections ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Generation Sequencing

Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 blaKPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

scholarly journals Droplet Digital PCR for the Quantification of Alu Methylation Status in Hematological Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2612-2612
Author(s):  
Paola Orsini ◽  
Luciana Impera ◽  
Elisa Parciante ◽  
Cosimo Cumbo ◽  
Crescenzio Francesco Minervini ◽  
...  
Keyword(s):  
High Risk ◽  
Methylation Level ◽  
Hematological Malignancies ◽  
Methylation Status ◽  
Risk Groups ◽  
Digital Pcr ◽  
Differential Methylation ◽  
Droplet Digital Pcr ◽  
Alu Repeats ◽  
Alu Sequences

Abstract Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements. In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes. Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p<0.05 for all analyses. Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis. We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident. Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients. Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data. To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239819
Author(s):  
Matilda Holm ◽  
Emma Andersson ◽  
Emerik Osterlund ◽  
Ali Ovissi ◽  
Leena-Maija Soveri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Metastatic Colorectal Cancer ◽  
Disease Progression ◽  
Primary Tumor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Plasma Samples ◽  
Liquid Biopsies ◽  
Generation Sequencing

Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45–63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

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