A Rapid Allele-Specific Assay for HLA-A*32:01 to Identify Patients at Risk for Vancomycin-Induced Drug Reaction with Eosinophilia and Systemic Symptoms
Human leukocyte antigen (HLA) alleles have been implicated as risk factors for immune-mediated adverse drug reactions. We recently reported a strong association between HLA-A*32:01 and vancomycin-induced drug reaction with eosinophilia and systemic symptoms (DRESS). Identification of individuals with the risk allele prior to or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality, improve drug safety and antibiotic treatment options. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 30, 2019 Category: Pathology Authors: Francois X. Rwandamuriye, Abha Chopra, Katherine C. Konvinse, Linda Choo, Jason A. Trubiano, Christian M. Shaffer, Mark Watson, Simon A. Mallal, Elizabeth J. Phillips Tags: Regular article Source Type: research

Recommendations for Clinical CYP2C9 Genotyping Allele Selection
The goals of the Association for Molecular Pathology Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee are to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for CYP2C9 testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 6, 2019 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special article Source Type: research

Detection of Tumor NTRK Gene Fusions to Identify Patients Who May Benefit from Tyrosine Kinase (TRK) Inhibitor Therapy
Chromosomal rearrangements involving the NTRK1, NTRK2, and NTRK3 genes (NTRK genes), which encode the high-affinity nerve growth factor receptor (TRKA), brain-derived neurotrophic factor/neurotrophin-3 (BDNF/NT-3) growth factor receptor (TRKB), and neurotrophin-3 (NT-3) growth factor receptor (TRKC) tyrosine kinases (TRK proteins), act as oncogenic drivers in a broad range of pediatric and adult tumor types. NTRK gene fusions have been shown to be actionable genomic events that are predictive of response to TRK kinase inhibitors, making their routine detection an evolving clinical priority. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 6, 2019 Category: Pathology Authors: Susan J. Hsiao, Ahmet Zehir, Anthony N. Sireci, Dara L. Aisner Tags: Review Source Type: research

Diagnostics Reform and Harmonization of Clinical Laboratory Testing
Developments in diagnostics reform legislation in the United States are occurring at a rapid pace. The framework for future regulatory oversight of clinical laboratory testing is currently under intensive debate among stakeholders that represent patients, providers, laboratories, diagnostic manufacturers, and regulators. The importance of clinical laboratory test standardization is a key component of any plan for regulatory reform. A laboratory-developed test is performed in a specific laboratory setting, often led by clinical laboratory professionals who possess specific expertise for developing and running the test to fi...
Source: Journal of Molecular Diagnostics - May 6, 2019 Category: Pathology Authors: Jeff Schreier, Robert Feeney, Peter Keeling Tags: Perspectives Source Type: research

Recommendations for Clinical CYP2C9 Genotyping Allele Selection: A Joint Recommendation of the Association for Molecular Pathology and College of American Pathologists
The goals of the Association for Molecular Pathology Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee are to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for CYP2C9 testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 6, 2019 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special Article Source Type: research

Detection of tumor NTRK gene fusions to identify patients who may benefit from TRK inhibitor therapy
Chromosomal rearrangements involving the NTRK1, NTRK2, and NTRK3 genes (NTRK gene fusions), which encode the TRKA, TRKB, and TRKC receptor tyrosine kinases, act as oncogenic drivers in a broad range of pediatric and adult tumor types. NTRK gene fusions have been shown to be actionable genomic events that are predictive of response to TRK kinase inhibitors, making their routine detection an evolving clinical priority. In certain exceedingly rare tumor types, NTRK gene fusions may be seen in the overwhelming majority of cases, whereas in a range of common cancers, reported incidences are in the range of 0.1% to 2%. (Source: ...
Source: Journal of Molecular Diagnostics - May 6, 2019 Category: Pathology Authors: Susan J. Hsiao, Ahmet Zehir, Anthony N. Sireci, Dara L. Aisner Tags: Review Source Type: research

Fixation Effects on Variant Calling in a Clinical Resequencing Panel
Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2019 Category: Pathology Authors: Jeremy D.K. Parker, Shyong Q. Yap, Elizabeth Starks, Jillian Slind, Lucas Swanson, T. Roderick Docking, Megan Fuller, Chen Zhou, Blair Walker, Douglas Filipenko, Wei Xiong, Ahmer A. Karimuddin, P. Terry Phang, Manoj Raval, Carl J. Brown, Aly Karsan Tags: Regular article Source Type: research

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele fractions (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2019 Category: Pathology Authors: Hua-Jun He, Erica V. Stein, Yves Konigshofer, Thomas Forbes, Farol L. Tomson, Russell Garlick, Emiko Yamada, Tony Godfrey, Toshiya Abe, Koji Tamura, Michael Borges, Michael Goggins, Sandra Elmore, Margaret L. Gulley, Jessica L. Larson, Lando Ringel, Brian Tags: Regular article Source Type: research

Fixation Effects on Variant-Calling in a Clinical Resequencing Panel
Formalin fixation is the standard method for preservation of tissue for diagnostic purposes, including pathological review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic tests. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade laboratory-developed targeted resequencing assay. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2019 Category: Pathology Authors: Jeremy D.K. Parker, Shyong Quin Yap, Elizabeth Starks, Jillian Slind, Lucas Swanson, T. Roderick Docking, Megan Fuller, Chen Zhou, Blair Walker, Douglas Filipenko, Wei Xiong, Ahmer A. Karimuddin, P. Terry Phang, Manoj Raval, Carl J. Brown, Aly Karsan Tags: Regular Article Source Type: research

Multi-Laboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements
We conducted a multi-laboratory assessment to determine the suitability of a new commercially-available reference material with 40 cancer variants in a background of wild type DNA at four different variant allele fractions (VAF): 2%, 0.5%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected both for their clinical importance, and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2019 Category: Pathology Authors: Hua-Jun He, Erica V. Stein, Yves Konigshofer, Thomas Forbes, Farol L. Tomson, Russell Garlick, Emiko Yamada, Tony Godfrey, Toshiya Abe, Koji Tamura, Michael Borges, Michael Goggins, Sandra Elmore, Margaret L. Gulley, Jessica L. Larson, Lando Ringel, Brian Tags: Regular Article Source Type: research

Promoter hypermethylation of genes encoding for RASSF/Hippo pathway members reveals specific alteration pattern in diffuse gliomas
RASSF/Hippo pathway alterations are poorly characterized in diffuse gliomas. We assayed promoter methylation of LATS1/2, MST1(STK4)/MST2(STK3), RASSF1, RASSF2, Nore1A/RASSF5, RASSF6, and RASSF10 genes in 133 diffuse Grade II-III-IV gliomas, using methylation-specific PCR or PCR coupled to Cobra. RASSF/Hippo pathway was highly silenced in gliomas, particularly RASSF1A (79.4%) and LATS2 (35.9%). Most gliomas (75.2%) exhibited at least hypermethylation for two promoters of the RASSF/Hippo member ’s genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2019 Category: Pathology Authors: Gu énaëlle Levallet, Christian Creveuil, Lien Bekaert, Elodie Péres, Gaëtane Planchard, Sylvie Lecot-Cotigny, Jean-Sébastien Guillamo, Evelyne Emery, Gérard Zalcman, Emmanuèle Lechapt-Zalcman Tags: Regular article Source Type: research

Abstracts of the 2nd Global Congress on Molecular Pathology
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 25, 2019 Category: Pathology Source Type: research

Author Index
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 25, 2019 Category: Pathology Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Source Type: research

Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell-Free Fetal DNA
During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free fetal DNA (cffDNA), constituting an accessible source for noninvasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component and the scarcity of the cffDNA itself make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study fetal aneuploidies. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Claudio Dello Russo, Anthony Cesta, Salvatore Longo, Maria A. Barone, Antonella Cima, Alvaro Mesoraca, Davide Sparacino, Antonella Viola, Claudio Giorlandino Tags: Technical advance Source Type: research

Adopting High-Resolution Allele Frequencies Substantially Expedites Variant Interpretation in Genetic Diagnostic Laboratories
A cohort of 1,242 individuals tested in a clinical diagnostic laboratory was used to test whether the “Filtering Allele Frequencies” (FAFs)-based framework, recently recommended for MHY7-associated cardiomyopathy, is extendable to 45 cardiomyopathy genes. Statistical analysis revealed a threshold of 0.00164% for the extreme outlier allele frequencies (AFs), based on the gnomAD (exome fraction) t otal AFs of 138 unique pathogenic and likely pathogenic variants; 135 of which (97.8%) had AFs (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Mahdi Ghani, Landry Nfonsam, Erinija Pranckeviciene, Hussein Daoud, Ryan Potter, Caitlin Chisholm, Patricia E. Harper, Audrey Schaffer, Leichelle Little, Elizabeth Sinclair-Bourque, Jean McGowan-Jordan, Amanda Smith, Lucas Bronicki, Olga Jarinova Tags: Regular article Source Type: research

Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell Free Fetal DNA
During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free foetal DNA (cffDNA), constituting an accessible source for non-invasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component, and the scarcity of the cffDNA itself, make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study foetal aneuploidies. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Claudio Dello Russo, Anthony Cesta, Salvatore Longo, Maria Antonietta Barone, Antonella Cima, Alvaro Mesoraca, Davide Sparacino, Antonella Viola, Claudio Giorlandino Tags: Technical Advance Source Type: research

Development of a Novel Next-Generation Sequencing Assay for Carrier Screening in Old Order Amish and Mennonite Populations of Pennsylvania
Genetically isolated populations such as the Old Order Amish and Old Order Mennonite communities have an increased incidence of specific autosomal recessive disorders due to the founder effect. In these populations, robust expanded carrier screening and diagnostic testing have the potential to reduce overall medical costs and improve patient outcomes. A novel next-generation sequencing assay was developed using anchored multiplex PCR technology (ArcherDX) for 162 different genetic syndromes caused by 202 pathogenic variants consisting of 150 single nucleotide changes, 43 small insertion/deletions, and nine large deletions ...
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Erin L. Crowgey, Michael C. Washburn, E. Anders Kolb, Erik G. Puffenberger Tags: Regular article Source Type: research

Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance
A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virological failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5 ’ and 3’ flanking sequences but having different centrally located nucleot...
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Dana S. Clutter, Gelareh Mazarei, Ruma Sinha, Justen Manasa, Janin Nouhin, Ellen LaPrade, Sara Bolouki, Philip L. Tzou, Jessica Hannita-Hui, Malaya K. Sahoo, Peter Kuimelis, Robert G. Kuimelis, Benjamin A. Pinsky, Gary K. Schoolnik, Arjang Hassibi, Robert Tags: Regular Article Source Type: research

An effective strategy to eliminate inherent cross-contamination in mtDNA next-generation sequencing of multiple samples
In this study, a novel sequencing strategy based on a unique double-barcode design was established. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Chun Yin, Yang Liu, Xu Guo, Deyang Li, Wan Fang, Jin Yang, Feng Zhou, Wancheng Niu, Yongfeng Jia, Hushan Yang, Jinliang Xing Tags: Regular Article Source Type: research

A reference system for BRCA mutation detection based on next-generation sequencing in the Chinese population
This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Shoufang Qu, Qiong Chen, Yuting Yi, Kang Shao, Wenxin Zhang, Yin Wang, Jian Bai, Xuchao Li, Zhiyuan Liu, Xiaowen Wang, Ruilin Jing, Yanfang Guan, Xin Yi, Miaoli Yan, Boyang Cao, Feng Chen, Shida Zhu, Xuexi Yang, Yingsong Wu, Jie Huang Tags: Regular article Source Type: research

Analytical validation of a highly sensitive, multiplexed chronic myeloid leukemia monitoring system targeting BCR-ABL1 RNA
This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Federal Drug Administration –cleared assay designed to monitor BCR-ABL1 fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex RT-qPCR assay amplifies both e13a2 and e14a2 major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided i n international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the WHO BCR-ABL1 Primary Reference Materials, without the necessity of dete...
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Justin T. Brown, Ion J. Beldorth, Walairat Laosinchai-Wolf, Marie E. Fahey, Keri L. Jefferson, Adam K. Ruskin, Jacquelyn J. Roth, Li Cai, Christopher D. Watt, Richard D. Press, Fei Yang, John B. Hedges, Bernard F. Andruss Tags: Regular Article Source Type: research

Clinical validation of a cell-free DNA gene panel
We describe herein the development and clinical validation of a 28 gene cell-free DNA panel that targets the most common genetic alterations in solid tumors. Bioinformatic and variant filtering solutions were developed to improve test sensitivity and specificity. The panel and these tools were used to analyze commercially available controls, allowing establishment of a limit of detection allele frequency cutoff of 0.25%, with 100% (95% CI: 81.5% to 100%) specificity and 89.8% (95% CI: 81.0% to 94.9%) sensitivity. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Ju Cheng, Yi Cao, Allison MacLeay, Jochen K. Lennerz, Aymen Baig, Ryan P. Frazier, Jesse Lee, Krista Hu, Maciej Pacula, Enrique Meneses, Hayley Robinson, Julie M. Batten, Priscilla K. Brastianos, Rebecca S. Heist, Aditya Bardia, Long P. Le, A. John Iafrat Tags: Regular Article Source Type: research

Tumor Heterogeneity Index to Detect Human Epidermal Growth Factor Receptor 2 Amplification by Next-Generation Sequencing
Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS) –based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journa...
Source: Journal of Molecular Diagnostics - April 21, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular article Source Type: research

Tumor Heterogeneity Index to Detect HER2 Amplification by Next-Generation Sequencing
Intratumoral heterogeneity of HER2 is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS) –based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 21, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular article Source Type: research

Tumor Heterogeneity Index to detect HER2 amplification by next-generation sequencing: A direct comparison study with immunohistochemistry
Intratumoral heterogeneity of HER2 is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS)-based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 21, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular Article Source Type: research

Leveraging Human Microbiome Features to Diagnose and Stratify Children with Irritable Bowel Syndrome
Accurate diagnosis and stratification of children with irritable bowel syndrome (IBS) remain challenging. Given the central role of recurrent abdominal pain in IBS, we evaluated the relationships of pediatric IBS and abdominal pain with intestinal microbes and fecal metabolites using a comprehensive clinical characterization and multiomics strategy. Using rigorous clinical phenotyping, we identified preadolescent children (aged 7 to 12 years) with Rome III IBS (n = 23) and healthy controls (n = 22) and characterized their fecal microbial communities using whole-genome shotgun metagenomics and global unbiased fecal metabolo...
Source: Journal of Molecular Diagnostics - April 16, 2019 Category: Pathology Authors: Emily B. Hollister, Numan Oezguen, Bruno P. Chumpitazi, Ruth Ann Luna, Erica M. Weidler, Michelle Rubio-Gonzales, Mahmoud Dahdouli, Julia L. Cope, Toni-Ann Mistretta, Sabeen Raza, Ginger A. Metcalf, Donna M. Muzny, Richard A. Gibbs, Joseph F. Petrosino, M Tags: Regular articles Source Type: research

Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples
Glioblastoma is a malignant brain tumor with dismal prognosis. Oncogenic mutations in glioblastoma frequently affect receptor tyrosine kinase pathway components that are challenging to quantify because of heterogeneous expression. EGFRvIII, a common oncogenic receptor tyrosine kinase mutant protein in glioblastoma, potentiates tumor malignancy and is an emerging tumor-specific immunotarget, underlining the need for its more accessible and quantitative detection. We used normalized next-generation sequencing data from 117 brain and 371 reference clinical tumor samples to detect focal gene amplifications across the commercia...
Source: Journal of Molecular Diagnostics - April 14, 2019 Category: Pathology Authors: Michael L. Miller, Jessica Tome-Garcia, Aneta Waluszko, Tatyana Sidorenko, Chitra Kumar, Fei Ye, Nadejda M. Tsankova Tags: Regular articles Source Type: research

Panel-Based Nuclear and Mitochondrial Next-Generation Sequencing Outcomes of an Ethnically Diverse Pediatric Patient Cohort with Mitochondrial Disease
We report on a molecular diagnostic study using mitochondrial DNA (mtDNA) and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African paediatric patients with clinical and biochemically defined MD. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 10, 2019 Category: Pathology Authors: Maryke Schoonen, Izelle Smuts, Roan Louw, Joanna L. Elson, Etresia van Dyk, Lindi-Maryn Jonck, Richard J.T. Rodenburg, Francois H. van der Westhuizen Tags: Regular article Source Type: research

A new fast phasing method based on haplotype subtraction
We developed a novel phasing approach, based solely on molecules and genotype frequency, and that does not rely on inference of new alleles. We initiated the project because of errors that were detected in the phased 1000 genomes data. The algorithm first combined identical genotypes into clusters and ranked them by descending frequency. Using alleles defined in homozygotes, it combined them to produce expected genotypes that were dismissed, and subtracted them from remaining genotypes to define additional new putative alleles. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 10, 2019 Category: Pathology Authors: Evelina Mocci, Marija Debeljak, Alison P. Klein, James R. Eshleman Tags: Regular Article Source Type: research

Validated Reference Panel from renewable source of genomic DNA available for standardization of blood group genotyping
Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic due to a disease state or recent transfusions. Additionally, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 10, 2019 Category: Pathology Authors: Evgeniya Volkova, Emilia Sippert, Meihong Liu, Teresita Mercado, Gregory A. Denomme, Orieji Illoh, Zhugong Liu, Maria Rios, Collaborative Study Group Tags: Regular Article Source Type: research

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics
The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 8, 2019 Category: Pathology Authors: Maristela L. Onozato, Clarence Yapp, Douglas Richardson, Tilak Sundaresan, Varun Chahal, Jesse Lee, James P. Sullivan, Marisa W. Madden, Hyo S. Shim, Mathew Liebers, Quan Ho, Shyamala Maheswaran, Daniel A. Haber, Zongli Zheng, Brian Clancy, Hunter L. Elli Tags: Technical advance Source Type: research

Highly Multiplexed FISH for in Situ Genomics
The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent “barcode” system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. (Source: Journa...
Source: Journal of Molecular Diagnostics - March 8, 2019 Category: Pathology Authors: Maristela L. Onozato, Clarence Yapp, Douglas Richardson, Tilak Sundaresan, Varun Chahal, Jesse Lee, James P. Sullivan, Marisa W. Madden, Hyo Sup Shim, Mathew Liebers, Quan Ho, Shyamala Maheswaran, Daniel A. Haber, Zongli Zheng, Brian Clancy, Hunter L. Ell Tags: Technical advance Source Type: research

Correction
In the article entitled, “The Development and Validation of Clinical Exome-Based Panels Using ExomeSlicer Considerations and Proof of Concept Using an Epilepsy Panel” (Volume 20, pages 643–652), which appeared in the September 2018 issue of The Journal of Molecular Diagnostics; https://doi.org/10.1016/j.jmoldx.2018.05 .003, there was an error during copyediting which updated the verb tense used in the first three paragraphs of the Results section. This language is meant to be in present tense to reflect general accepted practices, not past tense to reflect actions taken by the authors solely for this manu...
Source: Journal of Molecular Diagnostics - February 28, 2019 Category: Pathology Tags: Correction Source Type: research

Reviewer Acknowledgment
The Editors gratefully acknowledge the generous assistance of the following reviewers who served The Journal of Molecular Diagnostics between January 1 and December 31,  2018. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 28, 2019 Category: Pathology Tags: Reviewer acknowledgment Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 28, 2019 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 28, 2019 Category: Pathology Source Type: research

VarGrouper – a Bioinformatic Tool for Local Haplotyping of Deletion-Insertion Variants from Next-generation Sequencing Data Post Variant Calling
Accurate genetic variant representation through nomenclature and annotation is essential for understanding functional consequence and properly noting the presence of variants across time, assays, and laboratories. Current variant calling algorithms detect single deletion-insertion variants as multiple indel and/or substitution variants from next-generation sequencing data. Consequently, these variants are separately annotated in bioinformatics pipelines, leading to inaccurate variant representation. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 20, 2019 Category: Pathology Authors: Ryan J. Schmidt, Allison Macleay, Long Phi Le Tags: Technical Advance Source Type: research

A Model Information Management Plan for Molecular Pathology Sequence Data Using Standards: From Sequencer to Electronic Health Record
Incorporating genetic variant data into the electronic health record (EHR) in discrete computable fashion has vexed the informatics community for years. Genetic sequence test results are typically communicated by the molecular laboratory and stored in the EHR as textual documents. Although text documents are useful for human readability and initial use, they are not conducive for data retrieval and reuse. As a result, clinicians often struggle to find historical gene sequence results on a series of oncology patients within the EHR that might influence the care of the current patient. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 20, 2019 Category: Pathology Authors: Walter S. Campbell, Alexis B. Carter, Allison M. Cushman-Vokoun, Timothy C. Greiner, Rajesh C. Dash, Mark Routbort, Monica E. de Baca, James R. Campbell Tags: Regular article Source Type: research

Personalized chimerism test: selection of short tandem repeat or quantitative PCR depending on patient ’s chimerism status
Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplant for hematological malignancy. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%), but is less accurate than STR for patients in mixed chimerism. qPCR chimerism test allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR, based on patient chimerism status (recipient alleles (Source: Journal...
Source: Journal of Molecular Diagnostics - February 20, 2019 Category: Pathology Authors: Jennifer Tyler, Lorie Kumer, Carolyn Fisher, Heather Casey, Hiroko Shike Tags: Regular Article Source Type: research

Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
The emergence of highly sensitive molecular diagnostic approaches such as droplet digital PCR has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytical strategies that integrate molecular barcodes have rarely been applied to c...
Source: Journal of Molecular Diagnostics - February 20, 2019 Category: Pathology Authors: Saradhi Mallampati, Dzifa Y. Duose, Michael A. Harmon, Meenakshi Mehrotra, Rashmi Kanagal-Shamanna, Stephanie Zalles, Ignacio I. Wistuba, Xiaoping Sun, Rajyalakshmi Luthra Tags: Regular article Source Type: research

Analytical Validation of Variants to Aid in Genotype-Guided Therapy for Oncology
The Clinical Laboratory Improvement Amendments (CLIA) of 1988 requires that pharmacogenetic genotyping methods need to be established according to technical standards and laboratory practice guidelines before testing can be offered to patients. Testing methods for variants in ABCB1, CBR3, COMT, CYP3A7, C8ORF34, FCGR2A, FCGR3A, HAS3, NT5C2, NUDT15, SBF2, SEMA3C, SLC16A5, SLC28A3, SOD2, TLR4, and TPMT were validated in a CLIA-accredited laboratory. As no known reference materials were available, DNA samples that were from Coriell Cell Repositories (Camden, NJ) were used for the analytical validation studies. (Source: Journal...
Source: Journal of Molecular Diagnostics - February 20, 2019 Category: Pathology Authors: Marelize Swart, Wesley M. Stansberry, Victoria M. Pratt, Elizabeth B. Medeiros, Patrick J. Kiel, Fei Shen, Bryan P. Schneider, Todd C. Skaar Tags: Regular article Source Type: research

Personalized Chimerism Test that Uses Selection of Short Tandem Repeat or Quantitative PCR Depending on Patient's Chimerism Status
Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplantation for hematologic malignancies. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%) but is less accurate than STR for patients in mixed chimerism. qPCR chimerism allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR based on patient chimerism status (recipient alleles (Source: Journal o...
Source: Journal of Molecular Diagnostics - February 19, 2019 Category: Pathology Authors: Jennifer Tyler, Lorie Kumer, Carolyn Fisher, Heather Casey, Hiroko Shike Tags: Regular article Source Type: research

A Model Information Management Plan for Molecular Pathology Sequence Data Using Standards
Incorporating genetic variant data into the electronic health record (EHR) in discrete computable fashion has vexed the informatics community for years. Genetic sequence test results are typically communicated by the molecular laboratory and stored in the EHR as textual documents. Although text documents are useful for human readability and initial use, they are not conducive for data retrieval and reuse. As a result, clinicians often struggle to find historical gene sequence results on a series of oncology patients within the EHR that might influence the care of the current patient. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 19, 2019 Category: Pathology Authors: Walter S. Campbell, Alexis B. Carter, Allison M. Cushman-Vokoun, Timothy C. Greiner, Rajesh C. Dash, Mark Routbort, Monica E. de Baca, James R. Campbell Tags: Regular article Source Type: research

VarGrouper
Accurate genetic variant representation through nomenclature and annotation is essential for understanding functional consequence and properly noting the presence of variants across time, assays, and laboratories. Current variant calling algorithms detect single deletion –insertion variants as multiple indel and/or substitution variants from next-generation sequencing data. Consequently, these variants are separately annotated in bioinformatics pipelines, leading to inaccurate variant representation. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 19, 2019 Category: Pathology Authors: Ryan J. Schmidt, Allison Macleay, Long Phi Le Tags: Technical advance Source Type: research

A Novel Approach to Detect Programed Death Ligand 1 (PD-L1) Status and Multiple Tumor Mutations Using a Single Non –Small-Cell Lung Cancer (NSCLC) Bronchoscopy Specimen
Multiple biomarkers are under evaluation to guide the use of immune checkpoint inhibitors in non –small-cell lung cancer (NSCLC), including programed death ligand 1 (PD-L1) tumor cell staining. We have developed a new approach that accurately quantifies PD-L1 status and identifies multiple mutations by using a single bronchoscopy specimen. A novel molecular marker was identified to detect the presence of malignant cells in radial endobronchial ultrasound bronchial brushings from NSCLC (n = 15) and benign (n = 13) nodules by quantitative real-time RT-PCR (RT-qPCR). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 13, 2019 Category: Pathology Authors: Amanda Vannitamby, Shona Hendry, Tanvi Makadia, Janine Danks, John Slavin, Louis Irving, Daniel Steinfort, Steven Bozinovski Tags: Technical advance Source Type: research

Genetic Diagnostic Testing for Inherited Cardiomyopathies: Considerations for Offering Multi-Gene Tests in a Health Care Setting
Inherited cardiomyopathies (ICs) are a major cause of heart disease. Given their marked clinical and genetic heterogeneity, the content and clinical utility of IC multi-gene panels has been the topic of continuous debate. Our genetics diagnostic laboratory has been providing clinical diagnostic testing for ICs since 2012. We began by testing nine genes, and expanded our panel by 5-fold in 2015. Here, we describe the implementation of a cost-effective next-generation sequencing (NGS)-based assay for testing of IC genes, including a protocol that minimizes the amount of Sanger sequencing required to confirm variants identifi...
Source: Journal of Molecular Diagnostics - February 4, 2019 Category: Pathology Authors: Hussein Daoud, Mahdi Ghani, Landry Nfonsam, Ryan Potter, Shelley Ordorica, Virginia Haslett, Nathaniel Santos, Heather Derksen, Donelda Lahey, Martha McGill, Vanessa Trudel, Brittany Antoniuk, Nasim Vasli, Caitlin Chisholm, Gabrielle Mettler, Elizabeth Si Tags: Regular Article Source Type: research

Genomic Analysis of Circulating Tumor DNA Using a Melanoma-Specific UltraSEEK Oncogene Panel
The analysis of circulating tumor DNA (ctDNA) provides a minimally-invasive molecular interrogation that has the potential to guide treatment selection and disease monitoring. Here, we evaluated a custom UltraSEEK melanoma panel for the MassARRAY system, probing for 61 mutations over 13 genes. The analytical sensitivity and clinical accuracy of the UltraSEEK melanoma panel was compared to droplet digital PCR. The blinded analysis of 68 mutations detected in 48 plasma samples from stage IV melanoma patients revealed a concordance of 88% between the two platforms. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 4, 2019 Category: Pathology Authors: Elin S. Gray, Tom Witkowski, Michelle Pereira, Leslie Calapre, Karl Herron, Darryl Irwin, Brett Chapman, Muhammad A. Khattak, Jeanette Raleigh, Athena Hatzimihalis, Jonathan Cebon, Shahneen Sandhu, Grant A. McArthur, Michael Millward, Melanie Ziman, Alexa Tags: Regular article Source Type: research

Optimization of next-generation sequencing technologies for von Hippel Lindau (VHL) mosaic mutation detection and development of confirmation methods
Von Hippel Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients harboring a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotid...
Source: Journal of Molecular Diagnostics - February 4, 2019 Category: Pathology Authors: Lucie Coppin, Pascal Plouvier, Michel Cr épin, Anne-Sophie Jourdain, Emilie Ait Yahya, Stéphane Richard, Brigitte Bressac-de Paillerets, Catherine Cardot-Bauters, Sophie Lejeune, Julie Leclerc, Pascal Pigny Tags: Regular Article Source Type: research