Evaluation of a next-generation sequencing assay for BRCA1 and BRCA2 mutation detection
The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end-users, 48 samples with previously identified 444 small-size variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. (Source: Jou...
Source: Journal of Molecular Diagnostics - October 20, 2017 Category: Pathology Authors: Gabriele Lorenzo Capone, Anna Laura Putignano, Sharon Trujillo Saavedra, Irene Paganini, Roberta Sestini, Francesca Gensini, Irene De Rienzo, Laura Papi, Berardino Porfirio Tags: Regular Article Source Type: research

Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors
We have developed a next-generation sequencing assay to quantify biomarkers of the host immune response in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. This assay aims to provide clinicians with a comprehensive characterization of the immunological tumor microenvironment as a guide for therapeutic decisions on patients with solid tumors. The assay relies on RNA-seq to semi-quantitatively measure the levels of 43 transcripts related to anticancer immune responses and 11 transcripts reflecting the relative abundance of tumor-infiltrating lymphocytes, as well as on DNA-seq to estimate mutational burden. (Source: ...
Source: Journal of Molecular Diagnostics - October 20, 2017 Category: Pathology Authors: Jeffrey M. Conroy, Sarabjot Pabla, Sean T. Glenn, Blake Burgher, Mary Nesline, Antonios Papanicolau-Sengos, Jonathan Andreas, Vincent Giamo, Felicia L. Lenzo, Fiona C.L. Hyland, Angela Omilian, Wiam Bshara, Moachun Qin, Ji He, Igor Puzanov, Marc S. Ernsto Tags: Regular Article Source Type: research

Heterogeneity Of Braf, Nras, And Tert-Promoter Mutational Status In Multiple Melanomas And Association With Mc1r Genotype: Findings From Molecular And Immunohistochemical Analysis
Data on somatic heterogeneity and germline-somatic interaction in multiple primary melanoma (MPM) patients are limited. We investigated the mutational status of BRAF, NRAS, and TERT-promoter genes, the most common genetic alterations in melanoma, in 97 melanomas of 44 MPM patients and compared molecular and immunohistochemical findings. We further evaluated the association of somatic alterations with the germline MC1R genotype. Mutations in the BRAF gene were identified in 41.2% (40/97) of melanomas, in NRAS in 2.1% (2/97) and, in TERT-promoter in 19.6% (19/97). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 20, 2017 Category: Pathology Authors: Cristina Pellegrini, Lucia Di Nardo, Gianluca Cipolloni, Claudia Martorelli, Marina De Padova, Ambra Antonini, Maria Giovanna Maturo, Laura Del Regno, Sara Strafella, Tamara Micantonio, Pietro Leocata, Ketty Peris, Maria Concetta Fargnoli Tags: Regular Article Source Type: research

Noncoding RNA Expression and Targeted Next-Generation Sequencing Distinguish Tubulocystic Renal Cell Carcinoma (TC-RCC) from Other Renal Neoplasms
Tubulocystic renal cell carcinoma (TC-RCC) is a rare recently described renal neoplasm characterized by gross, microscopic, and immunohistochemical differences from other renal tumor types and was recently classified as a distinct entity. However, this distinction remains controversial particularly because some genetic studies suggest a close relationship with papillary RCC (PRCC). The molecular basis of this disease remains largely unexplored. We therefore performed noncoding (nc) RNA/miRNA expression analysis and targeted next-generation sequencing mutational profiling on 13 TC-RCC cases (11 pure, two mixed TC-RCC/PRCC) ...
Source: Journal of Molecular Diagnostics - October 19, 2017 Category: Pathology Authors: Charles H. Lawrie, Mar ía Armesto, Marta Fernandez-Mercado, María Arestín, Lorea Manterola, Ibai Goicoechea, Erika Larrea, María M. Caffarel, Angela M. Araujo, Carla Sole, Maris Sperga, Isabel Alvarado-Cabrero, Michal Michal, Ondrej Hes, José I. Lóp Tags: Regular article Source Type: research

Non-coding RNA expression and targeted next-generation sequencing distinguish Tubulocystic Renal Cell Carcinoma (TC-RCC) from other renal neoplasms
Tubulocystic renal cell carcinoma (TC-RCC) is a rare recently described renal neoplasm characterized by gross, microscopic and immunohistochemical differences from other renal tumor types that was recently classified as a distinct entity. However, this distinction remains controversial particularly as some genetic studies suggest a close relationship with papillary renal cell carcinoma (PRCC). The molecular basis of this disease remains largely unexplored. We therefore performed non-coding (nc)RNA/miRNA expression analysis and targeted next-generation sequencing mutational profiling on 13 TC-RCC cases (11 pure, two mixed T...
Source: Journal of Molecular Diagnostics - October 19, 2017 Category: Pathology Authors: Charles H. Lawrie, Mar ía Armesto, Marta Fernandez-Mercado, María Arestín, Lorea Manterola, Ibai Goicoechea, Erika Larrea, María M. Caffarel, Angela M. Araujo, Carla Sole, Maris Sperga, Isabel Alvarado-Cabrero, Michal Michal, Ondrej Hes, José I. Lóp Tags: Regular Article Source Type: research

Identification of Enteric Viruses in Oral Swabs from Children with Acute Gastroenteritis
Stool is the diagnostic specimen of choice to identify enteropathogens in pediatric gastroenteritis. However, stool collection is challenging and its diagnostic characteristics in patients with isolated vomiting are unknown. Therefore, we evaluated if oral swabs are a suitable alternative specimen to stool specimens. Seven-hundred and thirty-eight oral swabs and 577 stool specimens were collected from 738 children with vomiting and/or diarrhea. All specimens were tested by a laboratory-developed RT-qPCR Gastroenteritis Virus Panel (GVP) for virus identification; 150 oral swabs and 577 stool specimens were tested by the Lum...
Source: Journal of Molecular Diagnostics - October 19, 2017 Category: Pathology Authors: Ran Zhuo, Brendon D. Parsons, Bonita E. Lee, Steven J. Drews, Linda Chui, Marie Louie, Bryanne Crago, Stephen B. Freedman, Samina Ali, Xiaoli Pang Tags: Regular Article Source Type: research

Plasmodium detection and differentiation by direct-on-blood PCR Nucleic Acid Lateral Flow Immunoassay (db-PCR-NALFIA): development, validation, and evaluation
Declining malaria transmission warrants the search for highly sensitive point-of-care diagnostics, especially in resource-limiting settings. Db-PCR-NALFIA is a simplified PCR-based technique with a lateral flow readout that does not require any sample preparation. Two duplex db-PCR-NALFIAs were developed: a pan-Plasmodium/P. falciparum and a pan-Plasmodium/P. vivax assay. Confirmed positive samples from returning travelers (n=61) and negative controls (n=40) were used for laboratory validations. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 19, 2017 Category: Pathology Authors: Johanna M. Roth, Laura de Bes, Patrick Sawa, George Omweri, Victor Osoti, Boris Oberheitmann, Henk D.F.H. Schallig, P ètra F. Mens Tags: Regular Article Source Type: research

Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier
Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It includes three major subtypes termed germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. With the emergence of novel targeted therapies, accurate methods capable of interrogating this cell-of-origin classification should soon become essential in the clinics. To address this issue, we developed a novel gene expression profiling DLBCL classifier based on reverse transcriptase multiplex ligation-dependent probe amplification. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 17, 2017 Category: Pathology Authors: Victor Bob ée, Philippe Ruminy, Vinciane Marchand, Pierre-Julien Viailly, Ahmad Abdel Sater, Liana Veresezan, Fanny Drieux, Caroline Bérard, Elodie Bohers, Sylvain Mareschal, Sydney Dubois, Jean-Philippe Jais, Karen Leroy, Martin Figeac, Jean-Michel Pic Tags: Regular article Source Type: research

Hepatitis C and the Impact of Host Genetics
This commentary highlights the article by Enache et  al that describes a diagnostic test for simultaneous genotyping of specific single nucleotide polymorphisms in chronic hepatitis C patients. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 26, 2017 Category: Pathology Authors: Linda Cook Tags: Commentary Source Type: research

HCV and the Impact of Host Genetics
This commentary highlights the article by Enache et al that describes a diagnostic test for simultaneous genotyping of specific single nucleotide polymorphisms in chronic hepatitis C patients. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 26, 2017 Category: Pathology Authors: Linda Cook Tags: Commentary Source Type: research

Molecular Signatures for Tumor Classification
Cancer classification in the clinic is primarily based on histological analysis in the proper clinical context, often supplemented by immunohistochemical and molecular studies. Recent genomic studies have shown the potential of integrated multiomics platforms for molecular classification. We performed unsupervised analyses of molecular platforms in The Cancer Genome Atlas data (n  = 6216 samples) in comparison with tumor type. Our data showed that mRNA signatures and DNA methylation signatures mapped to histological diagnosis with high accuracy (95% and 88%, respectively) as individual platforms. (Source: Journal...
Source: Journal of Molecular Diagnostics - September 1, 2017 Category: Pathology Authors: Yasin Mamatjan, Sameer Agnihotri, Anna Goldenberg, Peter Tonge, Sheila Mansouri, Gelareh Zadeh, Kenneth Aldape Tags: Regular Article Source Type: research

Molecular Signatures for Tumor Classification An Analysis of TCGA Data
Cancer classification in the clinic is primarily based on histological analysis in the proper clinical context, often supplemented by immunohistochemical and molecular studies. Recent genomic studies have shown the potential of integrated multiomics platforms for molecular classification. We performed unsupervised analyses of molecular platforms in TCGA data (n  = 6216 samples) in comparison with tumor type. Our data showed that mRNA signatures and DNA methylation signatures mapped to histological diagnosis with high accuracy (95% and 88%, respectively) as individual platforms. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 1, 2017 Category: Pathology Authors: Yasin Mamatjan, Sameer Agnihotri, Anna Goldenberg, Peter Tonge, Sheila Mansouri, Gelareh Zadeh, Kenneth Aldape Tags: Regular Article Source Type: research

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic-Based Multiplex PCR and Next-Generation Sequencing
Circulating tumor DNA (ctDNA) has potential to serve as a biomarker for noninvasive monitoring of treatment response and disease progression. However, broad clinical applicability of ctDNA has been limited by the low sensitivity, throughput and patient coverage offered by existing ctDNA detection methods. Here we report the adaptation and characterization of the MMP-Seq technology for high-throughput and sensitive quantitation of ctDNA. A multiplex PCR preamplification (PreAmp) step was developed and incorporated into the MMP-seq workflow to enable low input ctDNA analysis with enhanced sensitivity. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 1, 2017 Category: Pathology Authors: Yinghui Guan, Oleg Mayba, Thomas Sandmann, Shan Lu, Younjeong Choi, Walter C. Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga Wan Rachel Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Mark R. Lackner, Yulei Wang Tags: Regular Article Source Type: research

Characterization and Genomic Localization of a SMAD4 Processed Pseudogene
Like many clinical diagnostic laboratories, we undertake routine investigation of cancer-predisposed individuals by high-throughput sequencing of patient DNA that has been target-enriched for genes associated with hereditary cancer. Accurate diagnosis using such reagents requires alertness against rare nonpathogenic variants that may interfere with variant calling. In a cohort of 2042 such cases, we identified five that initially appeared to be carriers of a 95-bp deletion of SMAD4 intron 6. More detailed analysis indicated that these individuals all carried one copy of a SMAD4 processed gene. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 1, 2017 Category: Pathology Authors: Christopher M. Watson, Nick Camm, Laura A. Crinnion, Agne Antanaviciute, Julian Adlard, Alexander F. Markham, Ian M. Carr, Ruth Charlton, David T. Bonthron Tags: Regular Article Source Type: research

Next-Generation Sequencing
This study evaluated whether next-generation sequencing (NGS) using the 50-gene AmpliSeq Cancer Hotspot Panel version 2 can help facilitate this distinction. NGS was performed on known primary-metastatic pairs (8 patients) and multiple lung adenocarcinomas (11 patients). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 30, 2017 Category: Pathology Authors: Snehal B. Patel, Wendy Kadi, Ann E. Walts, Alberto M. Marchevsky, Andy Pao, Angela Aguiluz, Tudor Mudalige, Zhenqui Liu, Nan Deng, Jean Lopategui Tags: Regular Article Source Type: research

Next Generation Sequencing A Novel Approach to Distinguish Multifocal Primary Lung Adenocarcinomas from Intrapulmonary Metastases
Distinguishing between multiple lung primaries and intrapulmonary metastases is imperative for accurate staging. The American Joint Committee on Cancer (AJCC) criteria are routinely used for this purpose but can yield equivocal conclusions. We evaluated whether next generation sequencing (NGS) using the 50 gene AmpliSeq Cancer Hotspot Panel v2 can be used to facilitate this distinction. NGS was performed on known primary-metastatic pairs (8 patients) and multiple lung adenocarcinomas (11 patients). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 30, 2017 Category: Pathology Authors: Snehal B. Patel, Wendy Kadi, Ann E. Walts, Alberto M. Marchevsky, Andy Pao, Angela Aguiluz, Tudor Mudalige, Zhenqui Liu, Nan Deng, Jean Lopategui Tags: Regular Article Source Type: research

Triplex High-Resolution Melting Assay for the Simultaneous Assessment of IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720 Genotypes in Chronic Hepatitis C Patients
Chronic hepatitis C (CHC) is a leading cause of liver disease. Despite the improved efficacy of new antivirals, their high costs preclude their adoption in resource-limited settings, where CHC prevalence is highest. We developed a triplex high-resolution melting assay for the simultaneous assessment of three genetic polymorphisms related to the response to treatment and development of advanced fibrosis in CHC: IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720. We validated the assay in clinical samples from 130 CHC patients treated with classic therapy. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2017 Category: Pathology Authors: Elena L. Enache, Anca Sin, Liviu S. Enache, Ligia Bancu Tags: Regular Article Source Type: research

Triplex HRM assay for the simultaneous assessment of IL28B rs12979860, ABCB11 rs2287622, and RNF7 rs16851720 genotypes in chronic hepatitis C patients
Chronic hepatitis C (CHC) is a leading cause of liver disease. Despite the improved efficacy of new antivirals, their high costs preclude their adoption in resource-limited settings, where CHC prevalence is highest. We developed a triplex high resolution melting assay for the simultaneous assessment of three genetic polymorphisms related to the response to treatment and development of advanced fibrosis in CHC: IL28B rs12979860, ABCB11 rs2287622, and RNF7 rs16851720. We validated the assay in clinical samples from 130 CHC patients treated with classical therapy. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2017 Category: Pathology Authors: Elena Luminita Enache, Anca Sin, Liviu Sorin Enache, Ligia Bancu Tags: Regular Article Source Type: research

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