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Journal of Molecular Diagnostics: JMD

Victoria M Pratt, Andria L Del Tredici, Houda Hachad, Yuan Ji, Lisa V Kalman, Stuart A Scott, Karen E Weck
This document was developed by the Pharmacogenetics (PGx) Working Group of the Association for Molecular Pathology (AMP) Clinical Practice Committee, whose aim is to recommend variants for inclusion in clinical pharmacogenetic testing panels. The goals of the AMP PGx Working Group are to define the key attributes of PGx alleles recommended for clinical testing, and to define a minimum set of variants that should be included in clinical PGx genotyping assays. These recommendations include a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing PGx assays...
February 20, 2018: Journal of Molecular Diagnostics: JMD
Anu Aggarwal, Manu Jamwal, Ganesh Kumar V, Prashant Sharma, Man Updesh Singh Sachdeva, Deepak Bansal, Pankaj Malhotra, Reena Das
Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and due to several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected from published literature...
February 20, 2018: Journal of Molecular Diagnostics: JMD
Susanne Kricke, Lana Mhaldien, Rozendo Fernandes, Charizel Villanueva, Alistair Shaw, Paul Veys, Stuart Adams
Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA extraction step and performing PCR and fragment analysis directly on bone marrow, whole-blood, and individual cell fractions. For chimerism analysis, this is possible with the use of a robust commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pre-treatment...
February 20, 2018: Journal of Molecular Diagnostics: JMD
Anieta M Sieuwerts, Bianca Mostert, Michelle van der Vlugt-Daane, Jaco Kraan, Corine Beaufort, Mai Van, Wendy J C Prager, Bram De Laere, Nick Beije, Paul Hamberg, Hans M Westgeest, Metin Tascilar, Luc Y Dirix, Wendy Onstenk, Ronald de Wit, Martijn P Lolkema, Ron H J Mathijssen, John W M Martens, Stefan Sleijfer
Recent reports have emphasized the clinical relevance of detecting the androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by RT-qPCR in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7 positive/negative score in a clinically acceptable time range. CellSearch-enirched CTCs from metastatic castration-resistant prostate cancer patients were characterized by RT-qPCR. After optimization it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study)...
February 20, 2018: Journal of Molecular Diagnostics: JMD
Yang Wang, Cindy L Vnencak-Jones, Justin M Cates, Chanjuan Shi
Elevated microsatellite alterations at selected tetranucleotide repeats are common in colorectal cancers (CRCs). Its association with classic mono/dinucleotide microsatellite instability (MSI) is unknown. We assessed the stability of 13 tetranucleotide and three pentanucleotide repeat markers on both tumor and normal tissue from a cohort of 22 MSI-high (MSI-H) and 107 microsatellite stable (MSS) CRCs. When present, instability was observed at tetra/pentanucleotide repeats and defined as elevated microsatellite alterations at selected tetra/pentanucleotide repeats-high (EMASTP-H) (≥ 30% instability), EMASTP-low (<30% instability), and EMASTP-stable...
February 20, 2018: Journal of Molecular Diagnostics: JMD
Andreas Bräuninger, Wolfgang Blau, Kristin Kunze, Ann-Kathrin Desch, Alexander Brobeil, Mehmet Kemal Tur, Benjamin Etschmann, Ulrich Günther, Dieter Körholz, Georg Schliesser, Andreas Käbisch, Michael Kiehl, Mathias Rummel, Stefan Gattenlöhner
Myelodysplastic syndromes are hematological neoplasias in which immunohistological examination of bone-marrow trephines is important for a definite diagnosis. Unequivocal distinction from reactive bone-marrow changes is, however, sometimes difficult. As neoplastic clones in myelodysplastic syndrome carry mutations in recurrent genes, mutation detection by targeted next-generation sequencing may be a useful support for differential diagnosis. To elucidate the accuracy of this approach in the clinical diagnostic setting, we analyzed single and consecutive bone-marrow trephines processed for immunohistological examination from 145 patients by targeted next-generation sequencing of 12 genes recurrently mutated in myelodysplastic syndromes...
February 19, 2018: Journal of Molecular Diagnostics: JMD
Shelly R Gunn, Shailin Govender, Cynthe L Sims, Aditi Khurana, Samuel Koo, Jayne Scoggin, Mathew W Moore, Philip D Cotter
Cancer genome copy number alterations (CNAs) assist clinicians in selecting targeted therapeutics. Solid tumor CNAs are most commonly evaluated in formalin-fixed, paraffin-embedded (FFPE) tissue by fluorescence in situ hybridization. Although fluorescence in situ hybridization is a sensitive and specific assay for interrogating pre-selected genomic regions, it provides no information about co-existing clinically significant copy number changes. Chromosomal microarray analysis is an alternative DNA-based method for interrogating genome-wide CNAs in solid tumors...
February 19, 2018: Journal of Molecular Diagnostics: JMD
Thomas Schneider, Geoffrey H Smith, Michael R Rossi, Charles E Hill, Linsheng Zhang
Bioinformatic analysis is an integral and critical part of clinical next-generation sequencing. It is especially challenging for some pipelines to consistently identify insertions and deletions. We present the validation of an open source tumor amplicon pipeline (OTA-pipeline) for clinical next-generation sequencing targeting solid tumor-associated variants. Raw data generated from 557 TruSight Tumor 26 samples as well as in silico data were analyzed by the OTA-pipeline and legacy pipeline and compared. Discrepant results were confirmed by orthogonal methods...
February 19, 2018: Journal of Molecular Diagnostics: JMD
Julie Denis, Faezeh Forouzanfar, Raoul Herbrecht, Elise Toussaint, Romain Kessler, Marcela Sabou, Ermanno Candolfi, Valérie Letsher-Bru
Invasive pulmonary aspergillosis (IPA) is a common complication of immunosuppression. Rapid diagnosis using molecular techniques is essential to improve patient survival. PCR techniques are promising in enhancing Aspergillus detection in blood and respiratory samples. Here, we evaluate for the first time the performances of two commercial Real-Time PCR kits, the A. fumigatus® Bio-Evolution and the MycoGENIE® A. fumigatus for the detection of A. fumigatus DNA in broncho-alveolar lavage (BAL) from patients with and without IPA...
February 19, 2018: Journal of Molecular Diagnostics: JMD
Claudia Cagnoli, Alessandro Brussino, Cecilia Mancini, Marina Ferrone, Laura Orsi, Paola Salmin, Patrizia Pappi, Elisa Giorgio, Elisa Pozzi, Simona Cavalieri, Eleonora Di Gregorio, Marta Ferrero, Alessandro Filla, Giuseppe De Michele, Cinzia Gellera, Caterina Mariotti, Suran Nethisinghe, Paola Giunti, Giovanni Stevanin, Alfredo Brusco
Spinocerebellar ataxias (SCA) type 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson's disease and amyotrophic lateral sclerosis. Their diagnosis is presently based on a PCR to identify small expanded alleles, followed by a second-level test whenever the suspect of false normal homozygous, or a CAT interruption in SCA1 needs to be verified...
February 17, 2018: Journal of Molecular Diagnostics: JMD
Michael Erlichster, Jason A Tye-Din, Michael D Varney, Efstratios Skafidas, Patrick Kwan
Human leukocyte antigen (HLA) genotyping has become a useful investigation in the diagnostic work-up of coeliac disease (CD), with utility in risk stratification and screening. However, broad application of this technology has been hindered by the cost and time burden of conventional laboratory-based assays. We have developed and validated CD-loop-mediated isothermal amplification (CD-LAMP), a LAMP assay, which enables rapid identification of the signature CD risk genotypes, HLA-DQ2.5, HLA-DQ8, HLA-DQ2.2, and HLA-DQA1*05...
February 16, 2018: Journal of Molecular Diagnostics: JMD
Stacy S Hung, Barbara Meissner, Elizabeth A Chavez, Susana Ben-Neriah, Daisuke Ennishi, Martin R Jones, Hennady P Shulha, Fong Chun Chan, Merrill Boyle, Robert Kridel, Randy D Gascoyne, Andrew J Mungall, Marco A Marra, David W Scott, Joseph M Connors, Christian Steidl
Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients...
February 7, 2018: Journal of Molecular Diagnostics: JMD
Neal I Lindeman, Philip T Cagle, Dara L Aisner, Maria E Arcila, Mary Beth Beasley, Eric Bernicker, Carol Colasacco, Sanja Dacic, Fred R Hirsch, Keith Kerr, David J Kwiatkowski, Marc Ladanyi, Jan A Nowak, Lynette Sholl, Robyn Temple-Smolkin, Benjamin Solomon, Lesley H Souter, Erik Thunnissen, Ming S Tsao, Christina B Ventura, Murry W Wynes, Yasushi Yatabe
CONTEXT: In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing. OBJECTIVE: To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update...
January 23, 2018: Journal of Molecular Diagnostics: JMD
Barbara Zehnbauer
This editorial comments on the updated molecular testing guideline for lung cancer patients.
January 19, 2018: Journal of Molecular Diagnostics: JMD
Robert S Ohgami, Andreas Rosenwald, Adam Bagg
This commentary highlights the article by Hung et al that details the design and implementation of a 32-gene next-generation sequencing (NGS) panel for lymphomas, and compares hybrid-capture versus amplicon-based NGS approaches.
January 18, 2018: Journal of Molecular Diagnostics: JMD
Eline Eberhardt, Magali Van den Kerkhof, Dimitri Bulté, Dorien Mabille, Lieselotte Van Bockstal, Séverine Monnerat, Fabiana Alves, Jane Mbui, Peter Delputte, Paul Cos, Sarah Hendrickx, Louis Maes, Guy Caljon
Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA qPCR assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0...
January 17, 2018: Journal of Molecular Diagnostics: JMD
Ashleigh C McEvoy, Benjamin A Wood, Nima M Ardakani, Michelle Pereira, Robert Pearce, Lester Cowell, Cleo Robinson, Fabienne Grieu-Iacopetta, Alexander J Spicer, Benhur Amanuel, Melanie Ziman, Elin S Gray
Identification of somatic mutations is crucial to guide therapeutic decisions for personalized melanoma treatment. However, genetic analysis of the tumor is usually performed on limited and often low-quality DNA, from tumors with low tumor cellularity and high tumor heterogeneity. Different mutation detection platforms exist with varying analytical sensitivities. Here we evaluated the detection of common mutations in BRAF, NRAS, and TERT-promoter in 40 melanoma formalin-fixed, paraffin-embedded tissues using droplet digital PCR (ddPCR), and compared the results to the detection rate obtained by Sanger sequencing and pyrosequencing...
January 2, 2018: Journal of Molecular Diagnostics: JMD
Job van Riet, Niels M G Krol, Peggy N Atmodimedjo, Erwin Brosens, Wilfred F J van IJcken, Maurice P H M Jansen, John W M Martens, Leendert H Looijenga, Guido Jenster, Hendrikus J Dubbink, Winand N M Dinjens, Harmen J G van de Werken
Exploration and visualization of next-generation sequencing data is crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions-of-interest coupled with dynamic heuristic filtering of genetic aberrations is however lacking. Therefore, we developed the web-application SNPitty that allows interactive visualization and interrogation of variant call format (VCF) files by utilizing B-allele frequencies of single nucleotide polymorphisms and single nucleotide variants, coverage metrics and copy-numbers analysis results...
January 2, 2018: Journal of Molecular Diagnostics: JMD
Victor Bobée, Philippe Ruminy, Vinciane Marchand, Pierre-Julien Viailly, Ahmad Abdel Sater, Liana Veresezan, Fanny Drieux, Caroline Bérard, Elodie Bohers, Sylvain Mareschal, Sydney Dubois, Jean-Philippe Jais, Karen Leroy, Martin Figeac, Jean-Michel Picquenot, Thierry J Molina, Gilles Salles, Corinne Haioun, Hervé Tilly, Fabrice Jardin
Authors' Reply to the Letter to the Editor by Y. Lynn Wang (MYD88 mutations and sensitivity to ibrutinib therapy).
March 2018: Journal of Molecular Diagnostics: JMD
Y Lynn Wang
This Correspondence relates to the article by Bobee et al (Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier: A CALYM Study. J Mol Diagn 2017,19:892-904).
March 2018: Journal of Molecular Diagnostics: JMD
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