A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens
miRNAs represent an emerging class of promising biomarkers for cancer diagnostics. To perform reliable miRNA expression analysis using quantitative PCR, adequate data normalization is essential to remove nonbiological, technical variations. Ideal reference genes should be biologically stable and reduce technical variability of miRNA expression analysis. Herein is a new strategy for the identification and evaluation of reference genes that can be applied for miRNA-based diagnostic tests without entailing excessive additional experiments. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Authors: Iris Babion, Barbara C. Snoek, Mark A. van de Wiel, Saskia M. Wilting, Renske D.M. Steenbergen Tags: Technical advance Source Type: research

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening
Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Authors: Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S. Abraham, Devin Oglesbee Tags: Regular article Source Type: research

High-Throughput, Multiplex Genotyping Directly from Blood or Dried Blood Spot without DNA Extraction for the Screening of Multiple G6PD Gene Variants at Risk for Drug-Induced Hemolysis
Clinical or epidemiologic screening of single-nucleotide polymorphism markers requires large-scale multiplexed genotyping. Available genotyping tools require DNA extraction and multiplex PCR, which may limit throughput and suffer amplification bias. Herein, a novel genotyping approach has been developed, multiplex extension and ligation-based probe amplification (MELPA), which eliminates DNA extraction and achieves uniform PCR amplification. MELPA lyses blood or dried blood spot and directly captures specific target DNA to 96-well plates using tailed probes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Authors: Xiaoyi Tian, Jun Zhou, Ye Zhao, Zhibin Cheng, Wenqi Song, Yu Sun, Xiaodong Sun, Zhi Zheng Tags: Technical advance Source Type: research

Calling Chromosome Alterations, DNA Methylation Statuses, and Mutations in Tumors by Simple Targeted Next-Generation Sequencing
Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. (Source: Journal...
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Authors: Simon Garinet, Mario N éou, Bruno de La Villéon, Simon Faillot, Julien Sakat, Juliana P. Da Fonseca, Anne Jouinot, Christophe Le Tourneau, Maud Kamal, Windy Luscap-Rondof, Valentina Boeva, Sebastien Gaujoux, Michel Vidaud, Eric Pasmant, Franck Letourneu Tags: Regular article Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 19, 2017 Category: Pathology Source Type: research

Next-Generation Sequencing –Based Detection of Germline Copy Number Variations in BRCA1/BRCA2
We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 16, 2017 Category: Pathology Authors: Ane Y. Schmidt, Thomas v.O. Hansen, Lise B. Ahlborn, Lars J ønson, Christina W. Yde, Finn C. Nielsen Tags: Technical Advance Source Type: research

Next-Generation Sequencing –Based Detection of Germline Copy Number Variations in BRCA1/BRCA2: Validation of a one-step diagnostic work-flow
We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 16, 2017 Category: Pathology Authors: Ane Y. Schmidt, Thomas v. O. Hansen, Lise B. Ahlborn, Lars J ønson, Christina W. Yde, Finn C. Nielsen Tags: Technical Advance Source Type: research

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels
Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) greater than approximately 50bp. Since these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required resulting in increased labor, costs, and turnaround times. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 14, 2017 Category: Pathology Authors: Jennifer Kerkhof, Laila C. Schenkel, Jack Reilly, Sheri McRobbie, Erfan Aref-Eshghi, Alan Stuart, C. Anthony Rupar, Paul Adams, Robert A. Hegele, Hanxin Lin, David Rodenhiser, Joan Knoll, Peter J. Ainsworth, Bekim Sadikovic Tags: Regular Article Source Type: research

Improved assays for AGG interruptions in Fragile X premutation carriers
The learning disability fragile X syndrome results from the presence of>200 CGG/CCG-repeats in exon 1 of the X-linked gene FMR1. Such alleles arise by expansion from maternally transmitted FMR1 premutation alleles, alleles having 55 to 200 repeats. Expansion risk is directly related to maternal repeat number. However, AGG interruptions to the repeat tract are important modifiers of expansion risk. Thus, the ability to identify such interruptions is crucial for the appropriate genetic counseling of women who are premutation carriers. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 14, 2017 Category: Pathology Authors: Bruce E. Hayward, Karen Usdin Tags: Regular Article Source Type: research

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA
We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type –bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 14, 2017 Category: Pathology Authors: Christina Wood-Bouwens, Billy T. Lau, Christine M. Handy, HoJoon Lee, Hanlee P. Ji Tags: Regular Article Source Type: research

Evaluation and Clinical Validation of Two Field –Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome Coronavirus
Middle East respiratory syndrome (MERS) is an emerging zoonotic viral respiratory disease that was first identified in Saudi Arabia in 2012. In 2015, the largest MERS outbreak outside of the Middle East region occurred in the Republic of Korea. The rapid nosocomial transmission of MERS coronavirus (MERS-CoV) in Korean health care settings highlighted the importance and urgent need for a rapid and reliable on-site diagnostic assay to implement effective control and preventive measures. Here, the evaluation and validation of two newly developed reverse transcription-insulated isothermal PCR (RT-iiPCR) methods targeting the O...
Source: Journal of Molecular Diagnostics - August 11, 2017 Category: Pathology Authors: Yun Young Go, Yeon-Sook Kim, Shinhye Cheon, Sangwoo Nam, Keun Bon Ku, Meehyein Kim, Nam Hyuk Cho, Hyun Park, Pei-Yu Alison Lee, Yu-Chun Lin, Yun-Long Tsai, Hwa-Tang Thomas Wang, Udeni B.R. Balasuriya Tags: Regular Article Source Type: research

Norovirus Loads in Stool Specimens of Cancer Patients with Norovirus Gastroenteritis
In this study, a validated real-time quantitative PCR (qPCR) assay was used to determine viral loads NoV genogroup I and II (GI and GII) in NoV positive stool specimens of cancer patients. A total of 234 specimens from 152 patients were positive for NoV, including 201 of GII and 33 of GI. Geometric mean of logarithmic copies (GMLC) per gram of stool (w/w) of NoV-GII were 9.03 ±1.71 (mean ± standard deviation), which was significantly higher than that of NoV-GI (7.87±1.49) (OR=3.22; 95% CI = 1.33 to 7.76, P =0.009). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 11, 2017 Category: Pathology Authors: Taojun He, Tracy A. McMillen, Yuanyuan Qiu, Liang Hua Chen, Xuedong Lu, Xiao-Li Pang, Mini Kamboj, Yi-Wei Tang Tags: Regular Article Source Type: research

Development of a chromosomal microarray test for the detection of abnormalities in formalin-fixed, paraffin-embedded products of conception specimens
Testing the products of conception (POC) provides information regarding the cause of fetal loss, and helps determine the recurrence risk for future losses and chromosome abnormalities in subsequent pregnancies. Historically, the Mayo Clinic Cytogenetics Laboratory performed targeted fluorescent in situ hybridization (FISH) testing to identify aneuploidy of only certain chromosomes in formalin-fixed, paraffin-embedded (FFPE) POC samples. Chromosomal microarray (CMA) studies utilizing the Affymetrix OncoScan ™ FFPE Assay can detect copy number changes across the genome. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 11, 2017 Category: Pathology Authors: Troy J. Gliem, Umut Aypar Tags: Regular Article Source Type: research

Evaluation and Clinical Validation of Two Field-deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome Coronavirus
Middle East respiratory syndrome (MERS) is an emerging zoonotic viral respiratory disease that was first identified in Saudi Arabia in 2012. In 2015, the largest MERS outbreak outside of the Middle East region occurred in the Republic of Korea. The rapid nosocomial transmission of MERS-coronavirus (MERS-CoV) in Korean healthcare settings highlighted the importance and urgent need for a rapid and reliable on-site diagnostic assay to implement effective control and preventive measures. Here, we describe the evaluation and validation of two newly developed reverse transcription-insulated isothermal PCR (RT-iiPCR) methods targ...
Source: Journal of Molecular Diagnostics - August 11, 2017 Category: Pathology Authors: Yun Young Go, Yeon-Sook Kim, Shinhye Cheon, Sangwoo Nam, Keun Bon Ku, Meehyein Kim, Nam Hyuk Cho, Hyun Park, Pei-Yu Alison Lee, Yu-Chun Lin, Yun-Long Tsai, Hwa-Tang Thomas Wang, Udeni B.R. Balasuriya Tags: Regular Article Source Type: research

Clinical Validation of a Genome-Wide DNA Methylation Assay for Molecular Diagnosis of Imprinting Disorders
Genomic imprinting involves a DNA methylation –dependent and parent-of-origin–specific regulation of gene expression. Clinical assays for imprinting disorders are genomic locus–, disorder-, and molecular defect–specific. We aimed to clinically validate a genome-wide approach for simultaneous testing of common imprinting disorders in a s ingle assay. Using genome-wide DNA methylation arrays, epigenetic profiles from peripheral blood of patients with Angelman, Prader-Willi, Beckwith-Wiedemann, and Silver-Russell syndrome were compared to a reference cohort of 361 unaffected individuals. (Source: Journ...
Source: Journal of Molecular Diagnostics - August 11, 2017 Category: Pathology Authors: Erfan Aref-Eshghi, Laila C. Schenkel, Hanxin Lin, Cindy Skinner, Peter Ainsworth, Guillaume Par é, Victoria Siu, David Rodenhiser, Charles Schwartz, Bekim Sadikovic Tags: Regular Article Source Type: research

Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors
Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 9, 2017 Category: Pathology Authors: Julie W. Reeser, Dorrelyn Martin, Jharna Miya, Esko A. Kautto, Ezra Lyon, Eliot Zhu, Michele R. Wing, Amy Smith, Matthew Reeder, Eric Samorodnitsky, Hannah Parks, Karan R. Naik, Joseph Gozgit, Nicholas Nowacki, Kurtis D. Davies, Marileila Varella-Garcia, Tags: Regular Article Source Type: research

A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single-Nucleotide Variant Detection
Customized gene-panel tests, based on next-generation sequencing, have demonstrated their usefulness in a plethora of clinical settings. As with other clinical diagnostic techniques, gene-panel sequencing for clinical purposes requires precise quality control (QC) measures to ensure its reliability. Only detected variants are currently recorded in clinical reports; however, identifying whether a nondetected variant is a true or false negative is regarded essential in a clinical setting and, thus, a comprehensive QC measure is in demand. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 24, 2017 Category: Pathology Authors: Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung K.D. Kim, Donghyun Park, Jongsuk Chung, Sungkyu Kyung, Je-Gun Joung, Hyun-Tae Shin, Seung-Ho Shin, Younglan Kim, Byung S. Kim, Hojun Lee, Kyoung-Mee Kim, Jung-Sun Kim, Woong-Yang Park, Dae-Soon Son Tags: Technical Advance Source Type: research

Digital Multiplex Ligation-Dependent Probe Amplification for Detection of Key Copy Number Alterations in T- and B-Cell Lymphoblastic Leukemia
Recurrent and clonal genetic alterations are characteristic of different subtypes of T- and B-cell lymphoblastic leukemia (ALL), and several subtypes are strong independent predictors of patient outcome. A next-generation sequencing –based multiplex ligation-dependent probe amplification variant (digitalMLPA) has been developed enabling simultaneous detection of copy number alterations (CNAs) of up to 1000 target sequences. This novel digitalMLPA assay was designed and optimized to detect CNAs of 56 key target genes and regio ns in ALL. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 19, 2017 Category: Pathology Authors: Anne Benard-Slagter, Ilse Zondervan, Karel de Groot, Farzaneh Ghazavi, Virinder Sarhadi, Pieter Van Vlierberghe, Barbara De Moerloose, Claire Schwab, Kim Vettenranta, Christine J. Harrison, Sakari Knuutila, Jan Schouten, Tim Lammens, Suvi Savola Tags: Regular Article Source Type: research

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology
Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we tested the clinical and laboratory validity of an extended search for mutations in CFTR using a next-generation sequencing –based method, with a panel of 188 CFTR mutations customized for the Italian population. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 19, 2017 Category: Pathology Authors: Marco Lucarelli, Luigi Porcaro, Alice Biffignandi, Lucy Costantino, Valentina Giannone, Luisella Alberti, Sabina Maria Bruno, Carlo Corbetta, Erminio Torresani, Carla Colombo, Manuela Seia Tags: Regular Article Source Type: research

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design
HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe –based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 18, 2017 Category: Pathology Authors: Hou-Sung Jung, Gregory J. Tsongalis, Joel A. Lefferts Tags: Regular Article Source Type: research

CRISPR/Cas9 Technology –Based Xenograft Tumors as Candidate Reference Materials for Multiple EML4-ALK Rearrangements Testing
The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non –small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 18, 2017 Category: Pathology Authors: Rongxue Peng, Rui Zhang, Guigao Lin, Xin Yang, Ziyang Li, Kuo Zhang, Jiawei Zhang, Jinming Li Tags: Regular Article Source Type: research

Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing
The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 18, 2017 Category: Pathology Authors: Christina Alidousty, Danielle Brandes, Carina Heydt, Svenja Wagener, Maike Wittersheim, Stephan Christian Sch äfer, Barbara Holz, Sabine Merkelbach-Bruse, Reinhard Büttner, Jana Fassunke, Anne Maria Schultheis Tags: Regular Article Source Type: research

A Method for Next-Generation Sequencing of Paired Diagnostic and Remission Samples to Detect Mitochondrial DNA Mutations Associated with Leukemia
Somatic mitochondrial DNA (mtDNA) mutations have been identified in many human cancers, including leukemia. To identify somatic mutations, it is necessary to have a control tissue from the same individual for comparison. When patients with leukemia achieve remission, the remission peripheral blood may be a suitable and easily accessible control tissue, but this approach has not previously been applied to the study of mtDNA mutations. We have developed and validated a next-generation sequencing approach for the identification of leukemia-associated mtDNA mutations in 26 chronic myeloid leukemia patients at diagnosis using e...
Source: Journal of Molecular Diagnostics - July 18, 2017 Category: Pathology Authors: Ilaria S. Pagani, Chung H. Kok, Verity A. Saunders, Mark B. Van der Hoek, Susan L. Heatley, Anthony P. Schwarer, Christopher N. Hahn, Timothy P. Hughes, Deborah L. White, David M. Ross Tags: Regular Article Source Type: research

GBA Analysis in Next-Generation Era
Mutations in the gene encoding the lysosomal enzyme acid β-glucosidase (GBA) are responsible for Gaucher disease and represent the main genetic risk factor for developing Parkinson disease. In past years, next-generation sequencing (NGS) technology has been applied for the molecular analysis of the GBA gene, both as a single gene or as part of gene panel s. However, the presence of complex gene-pseudogene rearrangements, resulting from the presence of a highly homologous pseudogene (GBAP1) located downstream of the GBA gene, makes NGS analysis of GBA challenging. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 17, 2017 Category: Pathology Authors: Stefania Zampieri, Silvia Cattarossi, Bruno Bembi, Andrea Dardis Tags: Regular Article Source Type: research

Comparative Study and Analytical Verification of PCR Methods for the Diagnosis of Congenital Chagas Disease
Congenital infection is currently the first cause of new cases of Chagas disease in Argentina and nonendemic areas worldwide. Its diagnosis is of utmost importance to guarantee curative treatment. To improve such diagnosis, a transfer process of PCR tests to the national laboratory network has been initiated. We performed a comparative study of four PCR assays [two end-point PCR and two duplex real-time quantitative PCR (qPCR) procedures] to detect Trypanosoma cruzi DNA in blood samples. Because satellite DNA and kinetoplastid DNA qPCR methods have the best performance and the use of two different molecular targets for con...
Source: Journal of Molecular Diagnostics - July 17, 2017 Category: Pathology Authors: Carolina In és Cura, Juan Carlos Ramírez, Marcelo Rodríguez, Constanza Lopez-Albízu, Lucía Irazu, Karenina Scollo, Sergio Sosa-Estani Tags: Regular Article Source Type: research

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice
Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non –small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its meta stases. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 16, 2017 Category: Pathology Authors: Stephan Bartels, Sascha Persing, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann Tags: Regular article Source Type: research

Molecular Analysis of Circulating Free DNA from Lung Cancer Patients in Routine Laboratory Practice
Cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non –small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its meta stases. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 16, 2017 Category: Pathology Authors: Stephan Bartels, Sascha Persing, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann Tags: Regular Article Source Type: research

NPM1 for MRD? Droplet Like It's Hot!
This commentary highlights the article by Mencia-Trinchant et  al that describes a novel digital PCR assay for sensitive detection of minimal residual disease in NPM1 mutated acute myeloid leukemia. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2017 Category: Pathology Authors: Gerald B.W. Wertheim, Adam Bagg Tags: Commentary Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2017 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2017 Category: Pathology Source Type: research

Highly Multiplex Real-Time PCR –Based Screening for Blood-Borne Pathogens on an OpenArray Platform
Molecular diagnostics are increasingly used in the blood bank industry. A device that can combine simultaneous detection of multiple targets with the flexibility of inclusion of emerging pathogens is desirable for testing blood products. A highly multiplexed blood-borne pathogen panel (BBPP) using dual-label probe chemistry (TaqMan assays) was developed for simultaneous detection and discrimination of 17 viral pathogens in human plasma samples and 13 bacterial and protozoan pathogens in human blood samples on the OpenArray platform. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 14, 2017 Category: Pathology Authors: Elena Grigorenko, Carolyn Fisher, Sunali Patel, Valerie Winkelman, Phillip Williamson, Caren Chancey, Germ án Añez, Maria Rios, Victoria Majam, Sanjai Kumar, Robert Duncan Tags: Regular Article Source Type: research

PheoSeq
This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed, paraffin-embedded tissue. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 25, 2017 Category: Pathology Authors: Maria Curr ás-Freixes, Elena Piñeiro-Yañez, Cristina Montero-Conde, María Apellániz-Ruiz, Bruna Calsina, Veronika Mancikova, Laura Remacha, Susan Richter, Tonino Ercolino, Natalie Rogowski-Lehmann, Timo Deutschbein, María Calatayud, Sonsoles Guadali Tags: Regular Article Source Type: research

Validation of a Next-Generation Sequencing Pipeline for the Molecular Diagnosis of Multiple Inherited Cancer Predisposing Syndromes
Despite the growing knowledge of the genetic background behind the cancers that occur in a context of hereditary predisposition, personal or family cancer history may not be clear enough to support directional gene testing. Defined targeted next-generation sequencing gene panels allow identification of the causative disease mutations of multigene syndromes and differential diagnosis for syndromes with phenotypically overlapping characteristics. Herein, we established a next-generation sequencing analysis pipeline for the molecular diagnosis of multiple inherited cancer predisposing syndromes using the commercially availabl...
Source: Journal of Molecular Diagnostics - May 18, 2017 Category: Pathology Authors: Paula Paulo, Pedro Pinto, Ana Peixoto, Catarina Santos, Carla Pinto, Patr ícia Rocha, Isabel Veiga, Gabriela Soares, Catarina Machado, Fabiana Ramos, Manuel R. Teixeira Tags: Regular Article Source Type: research

Minimal Residual Disease Monitoring of Acute Myeloid Leukemia by Massively Multiplex Digital PCR in Patients with NPM1 Mutations
The presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 16, 2017 Category: Pathology Authors: Nuria Mencia-Trinchant, Yang Hu, Maria Antonina Alas, Fatima Ali, Bas J. Wouters, Sangmin Lee, Ellen K. Ritchie, Pinkal Desai, Monica L. Guzman, Gail J. Roboz, Duane C. Hassane Tags: Regular Article Source Type: research

Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis
We report a novel real-time PCR–based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolo r melting curve analysis. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 12, 2017 Category: Pathology Authors: Qiuying Huang, Xudong Wang, Ning Tang, Tizhen Yan, Ping Chen, Qingge Li Tags: Regular Article Source Type: research

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid
Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 12, 2017 Category: Pathology Authors: Meenakshi Mehrotra, Rajesh R. Singh, Wei Chen, Richard S.P. Huang, Alaa A. Almohammedsalim, Bedia A. Barkoh, Crystal M. Simien, Marcos Hernandez, Carmen Behrens, Keyur P. Patel, Mark J. Routbort, Russell R. Broaddus, L. Jeffrey Medeiros, Ignacio I. Wistub Tags: Regular Article Source Type: research

Concordance between Research Sequencing and Clinical Pharmacogenetic Genotyping in the eMERGE-PGx Study
There has been extensive debate about both the necessity of orthogonal confirmation of next-generation sequencing (NGS) results in Clinical Laboratory Improvement Amendments –approved laboratories and return of research NGS results to participants enrolled in research studies. In eMERGE-PGx, subjects underwent research NGS using PGRNseq and orthogonal targeted genotyping in clinical laboratories, which prompted a comparison of genotyping results between platforms. Con cordance (percentage agreement) was reported for 4077 samples tested across nine combinations of research and clinical laboratories. (Source: Journal o...
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Laura J. Rasmussen-Torvik, Berta Almoguera, Kimberly F. Doheny, Robert R. Freimuth, Adam S. Gordon, Hakon Hakonarson, Jared B. Hawkins, Ammar Husami, Lynn Ivacic, Iftikhar J. Kullo, Michael D. Linderman, Teri A. Manolio, Aniwaa O. Obeng, Renata Pellegrino Tags: Regular Article Source Type: research

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test
The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of an NGS workflow. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Bennett O.V. Shum, Ilya Henner, Daniele Belluoccio, Marcus J. Hinchcliffe Tags: Regular Article Source Type: research

Improving Mutation Screening in Patients with Colorectal Cancer Predisposition Using Next-Generation Sequencing
Identification of genetic alterations is important for family risk assessment in colorectal cancers. Next-generation sequencing (NGS) technologies provide useful tools for single-nucleotide and copy number variation (CNV) identification in many genes and samples simultaneously. Herein, we present the validation of current Multiplicom MASTR designs of mismatch repair combined to familial adenomatous polyposis genes in a single PCR reamplification test for eight DNA samples simultaneously on a MiSeq apparatus. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Jean-Marc Rey, Vincent Ducros, Pascal Pujol, Qing Wang, Marie-Pierre Buisine, Hanaa Aissaoui, Thierry Maudelonde, Sylviane Olschwang Tags: Regular Article Source Type: research

Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer –Related Single-Nucleotide Variants and Copy Number Variants Using Simulated Cell-Free DNA
Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, r...
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Xin Yang, Yuxing Chu, Rui Zhang, Yanxi Han, Lucheng Zhang, Yu Fu, Dan Li, Rongxue Peng, Dongdong Li, Jiansheng Ding, Ziyang Li, Meiru Zhao, Kuo Zhang, Tian Lu, Lang Yi, Qisheng Wu, Guigao Lin, Jiehong Xie, Tao Liu, Ling Yang, Xin Yi, Jinming Li Tags: Regular Article Source Type: research

Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next-Generation Sequencing Panel
Recent studies have discovered a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, Proteus syndrome, and megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, are caused by somatic activating variants in the genes involved in the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway. Because of the low-abundance nature of these pathogenic variants, Sanger sequencing often yields negative results. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Fengqi Chang, Liu Liu, Erica Fang, Guangcheng Zhang, Tiansheng Chen, Kajia Cao, Yanchun Li, Marilyn M. Li Tags: Regular Article Source Type: research

Overgrowth Syndromes Caused by Somatic Variants in the Phosphatidylinositol 3-Kinase/AKT/Mammalian Target of Rapamycin Pathway
Somatic variants have been well described in tumorigenesis; however, they are only recently appreciated in other human disorders, such as mosaic overgrowth syndromes. Although overgrowth is a manifestation in many genetic syndromes, not all overgrowth syndromes are inherited. Mosaic somatic variants have been lately described in several overgrowth disorders, such as Proteus syndrome, CLOVES (congenital, lipomatous, overgrowth, vascular malformations, epidermal nevi, and spinal/skeletal anomalies and/or scoliosis) syndrome, megalencephaly –polymicrogyria-polydactyly-hydrocephalus syndrome, and megalencephaly–cap...
Source: Journal of Molecular Diagnostics - May 11, 2017 Category: Pathology Authors: Gozde Akgumus, Fengqi Chang, Marilyn M. Li Tags: Review Source Type: research

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non –Small Cell Lung Carcinoma
This study validates a blood-based genome-testing service that provides accurate results within 72 hours. We focused on targetable variants in advanced non –small cell lung carcinoma—epidermal growth factor receptor gene (EGFR) variant L858R, exon 19 deletion (ΔE746−A750), and T790M; GTPase Kirsten ras gene (KRAS) variants G12C/D/V; and echinoderm microtubule associated protein like and 4 anaplastic lymphoma receptor tyrosine kinase fusion (EM L4-ALK) transcripts 1/2/3. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Authors: Hestia Mellert, Trudi Foreman, Leisa Jackson, Dianna Maar, Scott Thurston, Kristina Koch, Amanda Weaver, Samantha Cooper, Nicholas Dupuis, Ubaradka G. Sathyanarayana, Jakkie Greer, Westen Hahn, Dawne Shelton, Paula Stonemetz, Gary A. Pestano Tags: Regular article Source Type: research

Haplotype Counting for Sensitive Chimerism Testing
Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic in...
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Authors: Marija Debeljak, Evelina Mocci, Max C. Morrison, Aparna Pallavajjalla, Katie Beierl, Marie Amiel, Micha ël Noë, Laura D. Wood, Ming-Tseh Lin, Christopher D. Gocke, Alison P. Klein, Ephraim J. Fuchs, Richard J. Jones, James R. Eshleman Tags: Regular article Source Type: research

Identification of NTRK3 Fusions in Childhood Melanocytic Neoplasms
We report herein the discovery of recurrent NTRK3 gene rearrangements in childhood melanocytic neoplasms with spitzoid and/or atypical features, based on genome-wide copy number analysis by single-nucleotide polymorphism array, which showed intragenic copy number changes in NTRK3. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Authors: Lu Wang, Klaus J. Busam, Ryma Benayed, Robert Cimera, Jiajing Wang, Ryan Denley, Mamta Rao, Ruth Aryeequaye, Kerry Mullaney, Long Cao, Marc Ladanyi, Meera Hameed Tags: Regular article Source Type: research

Utilization of Whole-Exome Next-Generation Sequencing Variant Read Frequency for Detection of Lesion-Specific, Somatic Loss of Heterozygosity in a Neurofibromatosis Type 1 Cohort with Tibial Pseudarthrosis
A subset of neurofibromatosis type 1 patients develop tibial dysplasia, which can lead to pseudarthrosis. The tissue from the tibial pseudarthrosis region commonly has a somatic second hit in NF1: single-nucleotide variants, small deletions, or loss of heterozygosity (LOH). We used exome next-generation sequencing (NGS) variant frequency data (allelic imbalance analysis) to detect somatic LOH in pseudarthrosis tissue from three individuals with clinically and diagnostically confirmed neurofibromatosis type 1, and verified the results with microarray. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Authors: Rebecca L. Margraf, Chad VanSant-Webb, David Sant, John Carey, Heather Hanson, Jacques D'Astous, Dave Viskochil, David A. Stevenson, Rong Mao Tags: Regular article Source Type: research

Correction
In the article entitled, “Microfluidic Platform for Single Nucleotide Polymorphism Genotyping of the Thiopurine S-Methyltransferase Gene to Evaluate Risk for Adverse Drug Events” (Volume 9, pages 521–529 of the September 2007 issue of The Journal of Molecular Diagnostics; http://dx.doi.org/10.2353/jmoldx.2007.070014), the second author's named was misspelled. The correct name is Govind V. Kaigala. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Tags: Correction Source Type: research

Correction
In the article entitled, “Amplicon Indel Hunter Is a Novel Bioinformatics Tool to Detect Large Somatic Insertion/Deletion Mutations in Amplicon-Based Next-Generation Sequencing Data” (Volume 17, 635–643 of the November 2015 issue of The Journal of Molecular Diagnostics; http://dx.doi.org/10.1016/j.jmoldx.2015.06.005) there is an update to the location of the AIH/AID toolkit. The software can now be found at https://github.com/skadri01/aiHunter. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Tags: Correction Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 19, 2017 Category: Pathology Source Type: research