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Journal of Molecular Diagnostics: JMD

Aurélie Dupuy, François Lemonnier, Virginie Fataccioli, Nadine Martin-Garcia, Cyrielle Robe, Romain Pelletier, Elsa Poullot, Anissa Moktefi, Karima Mokhtari, Marie Christine Rousselet, Alexandra Traverse-Glehen, Richard Delarue, Olivier Tournilhac, Marie Hélène Delfau-Larue, Corinne Haioun, Nicolas Ortonne, Christiane Copie-Bergman, Laurence de Leval, Anaïs Pujals, Philippe Gaulard
Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma associated with chemoresistance and a poor prognosis. Various non-synonymous mutations in the R172 residue of IDH2 are present in 20% to 30% of AITL patients. In addition to their diagnostic value, these mutations are potentially targetable, especially by IDH2 inhibitor, and therefore their identification in a routine setting is clinically relevant. However, in AITL, the neoplastic cells may be scarce making the identification of molecular anomalies difficult...
July 5, 2018: Journal of Molecular Diagnostics: JMD
Agnes Balla, Ken J Hampel, Mukesh K Sharma, Catherine E Cottrell, Nikoletta Sidiropoulos
Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived from fine needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods as compared to surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, fine needle aspiration-derived specimens most commonly accepted for molecular analysis are cell blocks (CB) as they are processed most similarly to surgical biopsy tissue...
July 5, 2018: Journal of Molecular Diagnostics: JMD
Maria P Bettinotti, Deborah Ferriola, Jamie L Duke, Timothy L Mosbruger, Nikolaos Tairis, Lawrence Jennings, Lisa V Kalman, Dimitri Monos
The highly polymorphic human leukocyte antigen (HLA) genes, located in the human major histocompatibility complex, encode the class I and II antigen-presenting molecules which are centrally involved in the immune response. HLA typing is used for several clinical applications such as transplantation, pharmacogenetics, and diagnosis of autoimmune disease. HLA typing is highly complex due to the homology of HLA genes and pseudogenes and the extensive polymorphism in the population. The Centers for Disease Control and Prevention (CDC) established the Genetic Testing Reference Materials Coordination Program (GeT-RM) in partnership with the genetics community to improve the availability of genomic DNA reference materials necessary to assure the quality of genetic laboratory testing...
June 26, 2018: Journal of Molecular Diagnostics: JMD
Megan H Cleveland, Justin M Zook, Marc Salit, Peter M Vallone
The National Institute of Standards and Technology has developed reference materials for five human genomes. DNA aliquots are available for purchase and the data, analyses, and high-confidence small variant and homozygous reference calls are freely available on the web. These reference materials are useful for evaluating whole-genome sequencing methods and can also be used to benchmark targeted sequencing panels, which are commonly used in clinical settings. This paper describes how to use the Genome in a Bottle samples to obtain performance metrics on any germline-targeted sequencing panel of interest, as well as the limitations of the reference materials...
June 26, 2018: Journal of Molecular Diagnostics: JMD
François M J Lamoury, Behzad Hajarizadeh, Angelica Soker, Danica Martinez, Camelia Quek, Philip Cunningham, Beth Catlett, Gavin Cloherty, Pip Marks, Janaki Amin, Jason Grebely, Gregory J Dore, Tanya L Applegate
Simplified, affordable tools to diagnose active hepatitis C virus (HCV) infection are needed to scale up treatment. This study evaluated the analytical performance of HCV core antigen (HCVcAg) detection in plasma and dried blood spot (DBS) samples. Paired plasma and venous DBS samples were prepared from remnant diagnostic samples. Plasma HCV RNA was quantified by AmpliPrep/COBAS Taqman (Roche) and HCVcAg measured by ARCHITECT HCV Ag (Abbott Diagnostics). Sensitivity and specificity for HCVcAg (>3 fmol/L) at two HCV RNA thresholds (≥15 IU/mL and ≥3,000 IU/mL) were calculated...
June 26, 2018: Journal of Molecular Diagnostics: JMD
Iván Prieto-Potin, Clara Montagut, Beatriz Bellosillo, Matthew Evans, Matthew Smith, Linea Melchior, Werner Reiltin, Michael Bennett, Veronica Pennati, Francesca Castiglione, Karl-Friedrich Bürrig, Ulrike Cooper, Barbara Dockhorn-Dworniczak, Christiana Rossenbach, Claudia Maribel Luna-Aguirre, Hugo Alberto Barrera-Saldaña, José Carlos Machado, José Luis Costa, Rinat Yacobi, Hilla Tabibian-Keissar, Simonetta Buglioni, Livia Ronchetti, Lotte Douglas-Berger, Hendrikus Jan Dubbink, Mohammed Alorini, Jean-Christophe Sabourin, Federico Rojo
Treatment of colorectal cancer (CRC) with monoclonal antibodies against epidermal growth factor receptor requires the assessment of the mutational status of exons 2, 3, and 4 of the NRAS and KRAS oncogenes. Moreover, the mutational status of exon 15 of the BRAF oncogene is a marker of poor prognosis in CRC. The Idylla™ NRAS-BRAF Mutation Test is a very reliable, simple (<2 minutes hands-on time), and quick (<2 hours turnaround time) sample-to-result solution, enabling the detection of clinically relevant mutations in NRAS (18 mutations) and BRAF (five mutations)...
June 26, 2018: Journal of Molecular Diagnostics: JMD
Ryoji Fujiki, Makoto Ikeda, Akiko Yoshida, Maeda Akiko, Yue Yao, Motio Nishimura, Kazuyuki Matsushita, Tomohiko Ichikawa, Tomoaki Tanaka, Hiroko Morisaki, Takayuki Morisaki, Osamu Ohara
There is significant debate within the diagnostics community regarding the accuracy of variant identification by next-generation sequencing and the necessity of confirmatory testing of detected variants. Since the quality threshold to discriminate false-positives depends on the nature of the workflow, no regulatory standard regarding this matter has yet been published. The goal of this study was to empirically determine the threshold to perform additional Sanger sequencing and to reduce the experimental cost to a practical level...
June 25, 2018: Journal of Molecular Diagnostics: JMD
Rojeen Niazi, Michael A Gonzalez, Jorune Balciuniene, Perry Evans, Mahdi Sarmady, Ahmad N Abou Tayoun
Exome-based panels are becoming the preferred diagnostic strategy in clinical laboratories. This approach enables dynamic gene content update and, if needed, cost-effective reflex to whole exome sequencing. There are currently no guidelines or appropriate resources to support the clinical implementation of exome-based panels. Here, we highlight principles and important considerations for the clinical development and validation of exome-based panels. In addition, we developed ExomeSlicer, a novel, web-based resource, which uses empirical exon-level next-generation sequencing quality metrics to predict and visualize technically challenging exome-wide regions in any gene(s) of interest...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Travis A Clark, Jon H Chung, Mark Kennedy, Jason D Hughes, Niru Chennagiri, Daniel S Lieber, Bernard Fendler, Lauren Young, Mandy Zhao, Michael Coyne, Virginia Breese, Geneva Young, Amy Donahue, Dean Pavlick, Alyssa Tsiros, Timothy Brennan, Shan Zhong, Tariq Mughal, Mark Bailey, Jie He, Steven Roels, Garrett M Frampton, Jill M Spoerke, Steven Gendreau, Mark Lackner, Erica Schleifman, Eric Peters, Jeffrey S Ross, Siraj M Ali, Vincent A Miller, Jeffrey P Gregg, Philip J Stephens, Allison Welsh, Geoff A Otto, Doron Lipson
Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a non-invasive method for the detection of genomic biomarkers to guide clinical decision-making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT®). High sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including base substitutions, short insertions/deletions, and genomic rearrangements at low allele frequencies (AF), and copy number amplifications...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Keith M Callenberg, Lucas Santana-Santos, Liang Chen, Wayne L Ernst, Michelle Barbi De Moura, Yuri E Nikiforov, Marina N Nikiforova, Somak Roy
Human Genome Variation Society (HGVS) nomenclature is a de facto clinical standard for reporting DNA sequence variants. With increasing use of high-throughput sequencing, manual generation of HGVS nomenclatures for all variants is impractical and error-prone. It is therefore beneficial to include one or more HGVS generator tools in next-generation sequencing (NGS) bioinformatics pipelines to enable automated, consistent, and accurate generation of HGVS nomenclature after appropriate validation. We implemented an HGVS nomenclature tool, hgvs package, by integrating it into our custom-developed NGS variant management and reporting software...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Marthe M de Jonge, Dina Ruano, Ronald van Eijk, Nienke van der Stoep, Maartje Nielsen, Juul T Wijnen, Natalja T Ter Haar, Astrid Baalbergen, Monique E M M Bos, Marjolein J Kagie, Maaike P G Vreeswijk, Katja N Gaarenstroom, Judith R Kroep, Vincent T H B M Smit, Tjalling Bosse, Tom van Wezel, Christi J van Asperen
BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort (Clinical implementation Of BRCA1/2 screening in ovarian tumor tissue: COBRA-cohort) in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Anthony Y Y Hsieh, Matthew Budd, David Deng, Izabella Gadawska, Hélène C F Côté
Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, Human immunodeficiency virus, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to rise as research uncovers its biological relevance. Here, we present a novel mtDNA content assay developed on monochrome, multiplex qPCR (MMqPCR) principles. This assay offers a >2-fold improvement on time- and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Reuben J Pengelly, Babatunde Rowaiye, Karen Pickard, Brendan Moran, Sanjeev Dayal, William Tapper, Alex Mirnezami, Tom Cecil, Faheez Mohamed, Norman Carr, Sarah Ennis
Pseudomyxoma peritonei is a clinical syndrome characterized by gross mucinous ascites originating from a disseminated intraperitoneal neoplasm. Although typically confined to the abdomen, mortality is high if untreated. Biomarkers, including genetic mutation profiles, may aid treatment selection and decision making. We applied whole-exome sequencing to five patients diagnosed with low grade appendiceal mucinous neoplasms, utilizing paired tumor and germline samples identify biomarkers. Multiple bioinformatic approaches were applied to these data to assess both somatic mutation profiles and loss of heterozygosity events...
June 21, 2018: Journal of Molecular Diagnostics: JMD
David A Armstrong, John A Dessaint, Carol S Ringelberg, Haley F Hazlett, Louisa Howard, Moemen A K Abdalla, Roxanna L Barnaby, Bruce A Stanton, Mark A Cervinski, Alix Ashare
There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for microRNA profiling studies. MicroRNA is an attractive candidate as a potential biomarker due to high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. Here, we present a comparison of small RNA recovery/stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of microRNA from cell-free urine and urine extracellular vesicles (EVs)...
June 21, 2018: Journal of Molecular Diagnostics: JMD
Cleo Keppens, John F Palma, Partha M Das, Sidney Scudder, Wei Wen, Nicola Normanno, J Han Van Krieken, Alessandra Sacco, Francesca Fenizia, David Gonzalez de Castro, Selma Hönigschnabl, Izidor Kern, Fernando Lopez-Rios, Maria D Lozano, Antonio Marchetti, Philippe Halfon, Ed Schuuring, Ulrike Setinek, Boe Sorensen, Phillipe Taniere, Markus Tiemann, Hana Vosmikova, Elisabeth M C Dequeker
Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter...
April 26, 2018: Journal of Molecular Diagnostics: JMD
Wenjun Kang, Sabah Kadri, Rutika Puranik, Michelle N Wurst, Sushant A Patil, Ibro Mujacic, Sonia Benhamed, Nifang Niu, Chao Jie Zhen, Bekim Ameti, Bradley C Long, Filipo Galbo, David Montes, Crystal Iracheta, Venessa L Gamboa, Daisy Lopez, Michael Yourshaw, Carolyn A Lawrence, Dara L Aisner, Carrie Fitzpatrick, Megan E McNerney, Y Lynn Wang, Jorge Andrade, Samuel L Volchenboum, Larissa V Furtado, Lauren L Ritterhouse, Jeremy P Segal
Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive...
April 24, 2018: Journal of Molecular Diagnostics: JMD
Diana Y Vargas, Salvatore A E Marras, Sanjay Tyagi, Fred R Kramer
In PCR assays designed to detect rare somatic mutations, SuperSelective primers, by virtue of their short 3'-foot sequences, selectively initiate synthesis on mutant DNA target fragments, while suppressing the synthesis of related wild-type fragments, and the resulting threshold cycle reflects the quantity of mutant targets present. However, when there are ≤10 mutant target fragments in a sample, the threshold cycle that is observed occurs so late that it can be confused with the threshold cycle that arises from samples that contain only abundant related wild-type fragments...
April 24, 2018: Journal of Molecular Diagnostics: JMD
Kevin Yauy, David Baux, Henri Pegeot, Charles Van Goethem, Charly Mathieu, Thomas Guignard, Raul J Morales, Delphine Lacourt, Martin Krahn, Vilma-Lotta Lehtokari, Gisele Bonne, Sylvie Tuffery-Giraud, Michel Koenig, Mireille Cossée
Interpretation of next-generation sequencing constitutes the main limitation of molecular diagnostics. In diagnosing myopathies and muscular dystrophies, another issue is efficiency in predicting the pathogenicity of variants identified in large genes, especially TTN; current in silico prediction tools show limitations in predicting and ranking the numerous variants of such genes. We propose a variant-prioritization tool, the MoBiDiCprioritization algorithm (MPA). MPA is based on curated interpretation of data on previously reported variants, biological assumptions, and splice and missense predictors, and is used to prioritize all types of single-nucleotide variants...
April 22, 2018: Journal of Molecular Diagnostics: JMD
Adrian Selim, Andrew S Moore
The ability to sensitively monitor minimal residual disease (MRD) has played a key role in improving the management and outcomes for a number of leukemias, particularly acute promyelocytic leukemia and childhood acute lymphoblastic leukemia. By contrast, MRD monitoring in acute myeloid leukemia (AML) has been limited by variable assay methodologies and a relative paucity of patient-specific MRD markers. Inter- and intratumor genetic heterogeneity poses significant challenges for the identification of molecular markers suitable for MRD monitoring in AML, particularly for those cases without structural chromosomal rearrangements associated with fusion genes...
April 22, 2018: Journal of Molecular Diagnostics: JMD
Junichi Hashiguchi, Masahiro Onozawa, Satoshi Oguri, Shinichi Fujisawa, Masahisa Tsuji, Kohei Okada, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Takeshi Kondo, Chikara Shimizu, Takanori Teshima
Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3; red) and a bacterial artificial chromosomes clone probe for detecting the 3' flanking region (Spectrum Green)...
July 2018: Journal of Molecular Diagnostics: JMD
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