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Cosimo Cumbo, Luciana Impera, Crescenzio Francesco Minervini, Paola Orsini, Luisa Anelli, Antonella Zagaria, Nicoletta Coccaro, Giuseppina Tota, Angela Minervini, Paola Casieri, Claudia Brunetti, Antonella Russo Rossi, Elisa Parciante, Giorgina Specchia, Francesco Albano
For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology...
February 16, 2018: Oncotarget
Meenakshi Mehrotra, Rajesh R Singh, Sanam Loghavi, Dzifa Yawa Duose, Bedia A Barkoh, Carmen Behrens, Keyur P Patel, Mark J Routbort, Scott Kopetz, Russell R Broaddus, L Jeffrey Medeiros, Ignacio I Wistuba, Rajyalakshmi Luthra
A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton...
February 13, 2018: Oncotarget
Benedikt G Brink, Justin Meskas, Ryan R Brinkman
Motivation: Droplet digital PCR (ddPCR) is an emerging technology for quantifying DNA. By partitioning the target DNA into ∼20000 droplets, each serving as its own PCR reaction compartment, a very high sensitivity of DNA quantification can be achieved. However, manual analysis of the data is time consuming and algorithms for automated analysis of non-orthogonal, multiplexed ddPCR data are unavailable, presenting a major bottleneck for the advancement of ddPCR transitioning from low-throughput to high- throughput...
March 9, 2018: Bioinformatics
Liesbeth Van Wesenbeeck, Leen Janssens, Hanne Meeuws, Ole Lagatie, Lieven Stuyver
Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples...
March 12, 2018: Epigenetics: Official Journal of the DNA Methylation Society
Wei-Neng Feng, Wei-Quan Gu, Ning Zhao, Ying-Ming Pan, Wei Luo, Hua Zhang, Jian-Miao Liang, Jie Yang, Yan-Ming Deng
BACKGROUND: Liquid biopsy is emerging as an important approach for tumor genotyping in non-small cell lung cancer, ddPCR and SuperARMS are both methods with high sensitivity and specificity for detecting EGFR mutation in plasma. We aimed to compare ddPCR and SuperARMS to detect plasma EGFR status in a cohort of advanced NSCLC patients. METHOD: A total of 79 tumor tissues and paired plasma samples were collected. The EGFR mutation status in tissue was tested by ADx-ARMS, matched plasma was detected by ddPCR and SuperARMS, respectively...
March 8, 2018: Translational Oncology
S Robinson, M Follo, D Haenel, M Mauler, D Stallmann, M Tewari, D Duerschmied, K Peter, C Bode, I Ahrens, M Hortmann
BACKGROUND: micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. AIMS: We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). METHODS: A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR...
April 15, 2018: International Journal of Cardiology
Frida A Zink, Luke R Tembrock, Alicia E Timm, Todd M Gilligan
The silver Y moth [Autographa gamma (Linneaus) (Noctuidae: Plusiinae)] is a pervasive crop pest in its native range but has not been found in moth surveys in the United States. Specimens of A. gamma are often intercepted at U.S. ports of entry, so the risk of introduction of this invasive species is high. Currently, identification of Plusiinae adults captured in domestic surveys is done by morphlogical comparison; however, this method is time consuming and misidentifications have occurred in the past. A recent study outlined a real-time PCR assay capable of rapidly identifying individual A...
March 1, 2018: Journal of Economic Entomology
Anna Buder, Maximilian J Hochmair, Sophia Schwab, Tatjana Bundalo, Peter Schenk, Peter Errhalt, Romana E Mikes, Gudrun Absenger, Kurt Patocka, Bernhard Baumgartner, Ulrike Setinek, Otto C Burghuber, Helmut Prosch, Robert Pirker, Martin Filipits
INTRODUCTION: Osimertinib is standard treatment for patients with advanced EGFR T790M-mutated NSCLC who have been pre-treated with EGFR-TKIs. We studied whether cell-free plasma DNA for T790M detection can be used to select patients for osimertinib treatment in clinical routine. METHODS: From April 2015 to November 2016, we included 119 patients with advanced EGFR-mutated NSCLC who had progressed under treatment with an EGFR-TKI. The T790M mutation status was assessed in cell-free plasma DNA by droplet digital PCR (ddPCR) in all patients and by tissue analyses in selected patients...
March 2, 2018: Journal of Thoracic Oncology
Ling Wei, Li Xie, Xingwu Wang, Hongxin Ma, Liyan Lv, Lisheng Liu, Xianrang Song
Genotype-directed targeted therapy has become one of the standard treatment options for non-small cell lung cancer (NSCLC). There have been numerous limitations associated with mutation analysis of tissue samples. Consequently, mutational profile analysis of circulating cell-free DNA (cfDNA) by highly sensitive droplet digital PCR (ddPCR) assay has been developed. Possibly due to differences in cfDNA concentrations, previous studies have shown numerous discrepancies in mutation detection consistency between tissue and cfDNA...
March 1, 2018: Laboratory Investigation; a Journal of Technical Methods and Pathology
Fern Baedyananda, Arkom Chaiwongkot, Parvapan Bhattarakosol
OBJECTIVES: The primary replication protein, HPV E1, has been shown to play a role in mitigating host defence and disrupting normal cell cycle processes, leading to the development of cancer. This study investigated the expression profile of HPV16 E1 in various stages of cervical cancer development and the factors that control E1 expression. METHODS: One hundred and twenty-four HPV16-positive cervical samples ranging from normal to CIN 1, CIN 2/3, and SCC lesions were studied...
March 1, 2018: Intervirology
Sharna Tanzima Nuhat, Mamiko Sakata-Yanagimoto, Daisuke Komori, Keiichiro Hattori, Yasuhito Suehara, Kota Fukumoto, Manabu Fujisawa, Manabu Kusakabe, Kosei Matsue, Hirotake Wakamatsu, Mitsunobu Shimadzu, Shigeru Chiba
Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of nodal peripheral T-cell lymphomas. Somatic RHOA mutations, most frequently found at the hotspot site c.50G>T, p. Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next generation sequencing (NGS), droplet digital PCR (ddPCR) and PNA-LNA clamp method for detecting the G17V RHOA mutations...
March 1, 2018: Cancer Science
Ruimin Gao, Biruk A Feyissa, Mana Croft, Abdelali Hannoufa
The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa...
February 28, 2018: Planta
Steven A Yukl, Philipp Kaiser, Peggy Kim, Sushama Telwatte, Sunil K Joshi, Mai Vu, Harry Lampiris, Joseph K Wong
Latently infected CD4+ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4+ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription...
February 28, 2018: Science Translational Medicine
Heidi D Pharo, Kim Andresen, Kaja C G Berg, Ragnhild A Lothe, Marine Jeanmougin, Guro E Lind
Background: Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Methods: Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes...
2018: Clinical Epigenetics
Shetty Ravi Dyavar, Zhen Ye, Siddappa N Byrareddy, Kimberly K Scarsi, Lee C Winchester, Jonathan A Weinhold, Courtney V Fletcher, Anthony T Podany
Quantification of antiretroviral (ARV) drug concentrations in peripheral blood mononuclear cells (PBMCs) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMC) and rectum (RMC) is an important measure of bio-distribution. Normalization of drug concentrations is critical to represent tissue drug concentrations and to analyze both intra-individual and inter-individual variability in drug distribution. However, a molecular method to normalize intracellular drug concentrations in PBMCs and TIMCs methanol extracts is currently unavailable...
February 26, 2018: Scientific Reports
Sarah Talarico, Andrew S Korson, Christina K Leverich, Stephanie Park, Florencia G Jalikis, Melissa P Upton, Elizabeth Broussard, Nina R Salama
BACKGROUND: Treatment of Helicobacter pylori infection is often empiric; however, current guidelines for management of Helicobacter pylori infection advise against the use of standard triple therapy (clarithromycin, amoxicillin, and proton-pump inhibitor) when clarithromycin resistance exceeds 20%. We developed and tested a new culture-free assay to detect clarithromycin resistance-conferring mutations to determine the prevalence of H. pylori clarithromycin resistance in patients from the United States Pacific Northwest...
February 26, 2018: Helicobacter
Masaki Hirano, Fumiharu Ohka, Sachi Maeda, Lushun Chalise, Akane Yamamichi, Kosuke Aoki, Akira Kato, Kuniaki Tanahashi, Kazuya Motomura, Yusuke Nishimura, Masahito Hara, Keiko Shinjo, Yutaka Kondo, Toshihiko Wakabayashi, Atsushi Natsume
Detection of mutations in the isocitrate dehydrogenase 1 (IDH1) gene is useful for accurate diagnosis of lower grade gliomas, as described in the 2016 World Health Organization classification of tumors of the central nervous system. Conventional analysis tools, including Sanger DNA sequencing and immunohistochemistry, might fail to detect a small fraction of mutant IDH1 owing to their limited sensitivity. Considering that lower grade gliomas are infiltrative in nature, a highly sensitive detection assay for IDH1 mutation is required for their accurate diagnosis...
February 19, 2018: Brain Tumor Pathology
LaMont Cannon, Aditya Jagarapu, Cesar A Vargas-Garcia, Michael J Piovoso, Ryan Zurakowski
Time series measurements of circular viral episome (2-LTR) concentrations enable indirect quantification of persistent low-level Human Immunodeficiency Virus (HIV) replication in patients on Integrase-Inhibitor intensified Combined Antiretroviral Therapy (cART). In order to determine the magnitude of these low level infection events, blood has to be drawn from a patients at a frequency and volume that is strictly regulated by the Institutional Review Board (IRB). Once the blood is drawn, the 2-LTR concentration is determined by quantifying the amount of HIV DNA present in the sample via a PCR (Polymerase Chain Reaction) assay...
December 2017: Proceedings of the ... IEEE Conference on Decision & Control
Yazhen Zhu, Zhiwei Guo, Ying Liu, Xiyun Zheng, Guohua Yang, Guangjuan Zheng
Quantification of epidermal growth factor receptor (EGFR) mutations is important for the prediction of tyrosine kinase inhibitor (TKI) efficacy in patients with non-small cell lung cancer (NSCLC). However, clinicians lack a sensitive and convenient method to quantify EGFR mutant abundance. The present study introduces a novel method, namely amplification refractory mutation system (ARMS)-Plus, for the quantitative analysis of EGFR exon 19 deletion (19Del), L858R and T790M mutations. Formalin-fixed paraffin-embedded tumor samples were collected from 77 patients with lung adenocarcinoma...
March 2018: Oncology Letters
Ilaria J Russo, Yongwon Ju, Naheema S Gordon, Maurice P Zeegers, K K Cheng, Nicholas D James, Richard T Bryan, Douglas G Ward
Background: TERT promotor mutations are present in >75% of bladder tumours; these mutations are also detectable in urine. Previous studies have used urinary pellet DNA, and semi-quantitative methods unsuitable for detecting very low mutant allele frequencies. Objective: In this proof-of-principle study we use ddPCR to count the DNA molecules with wt and mutant TERT sequences in urinary cfDNA from patients whose bladder cancers harbour TERT mutations. Methods: Urinary cfDNA prepared from the urine from 104 bladder cancer patients was analysed...
January 20, 2018: Bladder Cancer
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