Toward Rare Blood Cell Preservation for RNA Sequencing
Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues—a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. (Source: Journ...
Source: Journal of Molecular Diagnostics - May 16, 2015 Category: Pathology Authors: Sanja Vickovic, Afshin Ahmadian, Rolf Lewensohn, Joakim Lundeberg Tags: Short Communication Source Type: research

Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3–Positive Tumors
Melanoma antigen family A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti–MAGE-A3 immunotherapy treatment. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 15, 2015 Category: Pathology Authors: Olivier Gruselle, Thierry Coche, Jamila Louahed Tags: Regular Article Source Type: research

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma
Personalized management of diffuse large B-cell lymphoma (DLBCL) requires codevelopment of a companion diagnostic assay for activated B-cell and germinal center B-cell subtyping. Current classification methods are costly/complex (microarray expression profiling) or lacking in reproducibility/diagnostic precision (immunophenotyping). We investigated the potential of QuantiGene Plex (QGP)—a branched DNA signal amplification assay with high-detection sensitivity from formalin-fixed, paraffin-embedded tissue—for DLBCL subtyping. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 14, 2015 Category: Pathology Authors: John S. Hall, Suzanne Usher, Richard J. Byers, Rebekah C. Higgins, Danish Memon, John A. Radford, Kim M. Linton Tags: Regular Article Source Type: research

Annotation of Sequence Variants in Cancer Samples
As DNA sequencing of multigene panels becomes routine for cancer samples in the clinical laboratory, an efficient process for classifying variants has become more critical. Determining which germline variants are significant for cancer disposition and which somatic mutations are integral to cancer development or therapy response remains difficult, even for well-studied genes such as BRCA1 and TP53. We compare and contrast the general principles and lines of evidence commonly used to distinguish the significance of cancer-associated germline and somatic genetic variants. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2015 Category: Pathology Authors: Lobin A. Lee, Kevin J. Arvai, Dan Jones Tags: Review Source Type: research

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing
Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Pr...
Source: Journal of Molecular Diagnostics - May 7, 2015 Category: Pathology Authors: Linnea M. Baudhuin, Susan A. Lagerstedt, Eric W. Klee, Numrah Fadra, Devin Oglesbee, Matthew J. Ferber Tags: Regular Article Source Type: research

Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non–Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue
A 15-gene prognostic signature for early-stage, completely resected, non–small-cell lung carcinoma, that distinguishes between patients with good and poor prognoses was clinically validated in prior studies. To achieve operational efficiencies, this study was designed to evaluate the assay's performance in RNA-stabilized tissue as an alternative to the fresh-frozen tissue format, originally used to develop the assay. The percent concordance between matched tissue formats was 84% (95% Wilson CI, 70%–92%), a level of agreement comparable to the inherent reproducibility of the assay observed within biological repl...
Source: Journal of Molecular Diagnostics - May 7, 2015 Category: Pathology Authors: Shuguang Huang, Nicholas J. Reitze, Amy L. Ewing, Suzanne McCreary, Arlette H. Uihlein, Stacey L. Brower, Dakun Wang, Tianhua Wang, Michael J. Gabrin, Katherine E. Keating, Jude Mulligan, Claire Wilson, Timothy Davison, Stuart McKenzie, Ming-Sound Tsao, F Tags: Regular Article Source Type: research

Evaluation of Gene Status in Breast Cancer Samples with Indeterminate Fluorescence Hybridization by Quantitative Real-Time PCR
Administration of drugs targeting HER2 (official name ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2015 Category: Pathology Authors: Vladimira Koudelakova, Jitka Berkovcova, Radek Trojanec, Jana Vrbkova, Lenka Radova, Jiri Ehrmann, Zdenek Kolar, Bohuslav Melichar, Marian Hajduch Tags: Regular Article Source Type: research

The c.1364C>A (p.A455E) Mutation in the Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform
Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2015 Category: Pathology Authors: Kristin K. Deeb, James D. Metcalf, Kaitlin M. Sesock, Junqing Shen, Christine A. Wensel, Larisa I. Rippel, Michelle Smith, Mark S. Chapman, Shulin Zhang Tags: Regular Article Source Type: research

Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence Hybridization by Quantitative Real-Time PCR Method
Administration of drugs targeting HER2 (official name ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2015 Category: Pathology Authors: Vladimira Koudelakova, Jitka Berkovcova, Radek Trojanec, Jana Vrbkova, Lenka Radova, Jiri Ehrmann, Zdenek Kolar, Bohuslav Melichar, Marian Hajduch Tags: Regular Article Source Type: research

Next-Generation Genotyping by Digital PCR to Detect and Quantify the V600E Mutation in Melanoma Biopsies
The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedd...
Source: Journal of Molecular Diagnostics - May 4, 2015 Category: Pathology Authors: Pierre-Jean Lamy, Florence Castan, Nicolas Lozano, Cécile Montélion, Patricia Audran, Frédéric Bibeau, Sylvie Roques, Frédéric Montels, Anne-Claire Laberenne Tags: Regular Article Source Type: research

A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing
In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT2; 19 positive...
Source: Journal of Molecular Diagnostics - April 29, 2015 Category: Pathology Authors: Théo G.M. Oliveira, Miguel Mitne-Neto, Louise T. Cerdeira, Julia D.C. Marsiglia, Edmundo Arteaga-Fernandez, José E. Krieger, Alexandre C. Pereira Tags: Regular article Source Type: research

A Variant Detection Pipeline for Inherited Cardiomyopathies–Associated Genes Using Ion Torrent PGM Platform
In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT2; 19 positive...
Source: Journal of Molecular Diagnostics - April 29, 2015 Category: Pathology Authors: Théo G.M. Oliveira, Miguel Mitne-Neto, Louise T. Cerdeira, Julia D.C. Marsiglia, Edmundo Arteaga-Fernandez, José E. Krieger, Alexandre C. Pereira Tags: Regular Article Source Type: research

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues
Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profiling–based biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Af...
Source: Journal of Molecular Diagnostics - April 29, 2015 Category: Pathology Authors: Svitlana Tyekucheva, Neil E. Martin, Edward C. Stack, Wei Wei, Vinod Vathipadiekal, Levi Waldron, Michelangelo Fiorentino, Rosina T. Lis, Meir J. Stampfer, Massimo Loda, Giovanni Parmigiani, Lorelei A. Mucci, Michael Birrer Tags: Regular Article Source Type: research

A Variant Detection Pipeline for Inherited Cardiomyopathies–Associated Genes Using Ion Torrent PGM Platform
In inherited cardiomyopathies, the investigation of genetics is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT...
Source: Journal of Molecular Diagnostics - April 29, 2015 Category: Pathology Authors: Théo G. Mimary Oliveira, Miguel Mitne-Neto, Louise Teixeira Cerdeira, Julia D. Carneiro Marsiglia, Edmundo Arteaga-Fernandez, José E. Krieger, Alexandre C. Pereira Tags: Regular Article Source Type: research

Evaluation of the Real-Q V600E Detection Assay in Fine-Needle Aspiration Samples of Thyroid Nodules
Recently, several molecular assays for detecting the BRAF V600E mutation in fine-needle aspiration (FNA) specimens have been developed. Herein, we tested 294 consecutive FNA samples from patients with thyroid nodules with the Real-Q BRAF V600E detection assay (Real-Q). These results were compared with an allele-specific PCR-based kit using dual-priming oligonucleotides (AS-PCR). Any discordant results between the two tests were also analyzed by mutant enrichment with 3′-modified oligonucleotide sequencing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 29, 2015 Category: Pathology Authors: Kyung S. Park, Young L. Oh, Chang-Seok Ki, Jong-Won Kim Tags: Regular Article Source Type: research

Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility?
Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disea...
Source: Journal of Molecular Diagnostics - April 20, 2015 Category: Pathology Authors: Bert Gold, Milena Cankovic, Larissa V. Furtado, Frederick Meier, Christopher D. Gocke Tags: Special article Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 20, 2015 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 20, 2015 Category: Pathology Source Type: research

Accurate Classification of Germinal Center B-Cell–Like/Activated B-Cell–Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase–Multiplex Ligation-Dependent Probe Amplification Assay
Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell–like and activated B-cell–like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation-dependent probe amplification assay and 14 gene signatures. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 17, 2015 Category: Pathology Authors: Sylvain Mareschal, Philippe Ruminy, Cristina Bagacean, Vinciane Marchand, Marie Cornic, Jean-Philippe Jais, Martin Figeac, Jean-Michel Picquenot, Thierry Jo Molina, Thierry Fest, Gilles Salles, Corinne Haioun, Karen Leroy, Hervé Tilly, Fabrice Jardin Tags: Regular article Source Type: research

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)
The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of...
Source: Journal of Molecular Diagnostics - March 22, 2015 Category: Pathology Authors: Donavan T. Cheng, Talia N. Mitchell, Ahmet Zehir, Ronak H. Shah, Ryma Benayed, Aijazuddin Syed, Raghu Chandramohan, Zhen Yu Liu, Helen H. Won, Sasinya N. Scott, A. Rose Brannon, Catherine O'Reilly, Justyna Sadowska, Jacklyn Casanova, Angela Yannes, Jaclyn Tags: Regular article Source Type: research

Msk-impact
The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed MSK-IMPACT, a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-em...
Source: Journal of Molecular Diagnostics - March 22, 2015 Category: Pathology Authors: Donavan T. Cheng, Talia Mitchell, Ahmet Zehir, Ronak H. Shah, Ryma Benayed, Aijazuddin Syed, Raghu Chandramohan, Zhen Yu Liu, Helen H. Won, Sasinya N. Scott, A. Rose Brannon, Catherine O'Reilly, Justyna Sadowska, Jacklyn Casanova, Angela Yannes, Jaclyn He Tags: Regular Article Source Type: research

Panels of Cytokines and Other Secretory Proteins as Potential Biomarkers of Ovarian Endometriosis
Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 health...
Source: Journal of Molecular Diagnostics - March 20, 2015 Category: Pathology Authors: Vida Kocbek, Katja Vouk, Nick A. Bersinger, Michael D. Mueller, Tea Lanišnik Rižner Tags: Regular Article Source Type: research

Evaluation of HPV Genotyping Assays for Archival Clinical Samples
Human papillomavirus (HPV) testing and genotyping of FFPE tissue samples is important in epidemiological investigations. Here, we compare four different HPV genotyping methods for use in FFPE clinical samples. Comparative testing was performed on 99 samples with a clinical suspicion of HPV. Specimens were analyzed with Anyplex II HPV28 detecting 28 genotypes using real-time PCR and melting curve analysis, CLART HPV2 detecting 35 genotypes using PCR and microarray detection, and MGP5+/6+ consensus primer system together with pyrosequencing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 16, 2015 Category: Pathology Authors: Gabriella Lillsunde Larsson, Jessica Carlsson, Mats G. Karlsson, Gisela Helenius Tags: Regular Article Source Type: research

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in
Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 14, 2015 Category: Pathology Authors: Grace X.Y. Lim, Yu Ling Loo, Farmaditya E.P. Mundhofir, Ferdy K. Cayami, Sultana M.H. Faradz, Indhu-Shree Rajan-Babu, Samuel S. Chong, Yvonne Y. Koh, Ming Guan Tags: Regular Article Source Type: research

Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Authors: Alicia Edin, Susanne Granholm, Satu Koskiniemi, Annika Allard, Anders Sjöstedt, Anders Johansson Tags: Regular Article Source Type: research

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection
The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within...
Source: Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Authors: Elena Castellanos-Rizaldos, Cloud Paweletz, Chen Song, Geoffrey R. Oxnard, Harvey Mamon, Pasi A. Jänne, G. Mike Makrigiorgos Tags: Regular Article Source Type: research

Development and Laboratory Evaluation of a Quantitative Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Authors: Alicia Edin, Susanne Granholm, Satu Koskiniemi, Annika Allard, Anders Sjöstedt, Anders Johansson Tags: Regular Article Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of -Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 11, 2015 Category: Pathology Authors: Guanshan Zhu, Xin Ye, Zhengwei Dong, Ya Chao Lu, Yun Sun, Yi Liu, Rose McCormack, Yi Gu, Xiaoqing Liu Tags: Regular article Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 11, 2015 Category: Pathology Authors: Guanshan Zhu, Xin Ye, Zhengwei Dong, Yachao Lu, Yun Sun, Yi Liu, Rose McCormack, Yi Gu, Xiaoqing Liu Tags: Regular Article Source Type: research

Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues
DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5′ untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples a...
Source: Journal of Molecular Diagnostics - March 3, 2015 Category: Pathology Authors: Shenli Zhang, Iain BeeHuat Tan, Nur S. Sapari, Heike Grabsch, Alicia Okines, Elizabeth C. Smyth, Toru Aoyama, Lindsay C. Hewitt, Imran Inam, Dan Bottomley, Matthew Nankivell, Sally P. Stenning, David Cunningham, Andrew Wotherspoon, Akira Tsuburaya, Takaki Tags: Technical Advance Source Type: research

A Systematic Approach to Novel Virus Discovery in Emerging Infectious Disease Outbreaks
The discovery of novel viruses is of great importance to human health—both in the setting of emerging infectious disease outbreaks and in disease syndromes of unknown etiology. Despite the recent proliferation of many efficient virus discovery methods, careful selection of a combination of methods is important to demonstrate a novel virus, its clinical associations, and its relevance in a timely manner. The identification of a patient or an outbreak with distinctive clinical features and negative routine microbiological workup is often the starting point for virus hunting. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 3, 2015 Category: Pathology Authors: Siddharth Sridhar, Kelvin K.W. To, Jasper F.W. Chan, Susanna K.P. Lau, Patrick C.Y. Woo, Kwok-Yung Yuen Tags: Review Source Type: research

Improving Molecular Genetic Test Utilization through Order Restriction, Test Review, and Guidance
The ordering of molecular genetic tests by health providers not well trained in genetics may have a variety of untoward effects. These include the selection of inappropriate tests, the ordering of panels when the assessment of individual or fewer genes would be more appropriate, inaccurate result interpretation and inappropriate patient guidance, and significant unwarranted cost expenditure. We sought to improve the utilization of molecular genetic tests by requiring providers without specialty training in genetics to use genetic counselors and molecular genetic pathologists to assist in test selection. (Source: Journal of...
Source: Journal of Molecular Diagnostics - February 28, 2015 Category: Pathology Authors: Jacquelyn D. Riley, Gary W. Procop, Kandice Kottke-Marchant, Robert Wyllie, Felicitas L. Lacbawan Tags: Special Article Source Type: research

A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing Buffer
We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Authors: Chang Hee Kim, Majid Abedi, Yenbou Liu, Sree Panuganti, Francisco Flores, Kevin R. Shah, Hannah Catterall, Krishna S. Morampudi, Robert Terbrueggen Tags: Technical advance Source Type: research

Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay
Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell lines. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Authors: Mulias Lian, Indhu-Shree Rajan-Babu, Kunal Singh, Caroline G. Lee, Hai-Yang Law, Samuel S. Chong Tags: Regular article 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, 2014. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Tags: Reviewer acknowledgment Source Type: research

Correction
In the article entitled, “A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing: Report of a Large Series of Myeloid Neoplasms” (Volume 17, pages 76–84 of the January 2015 issue of The Journal of Molecular Diagnostics), reference 24 on page 78 was incorrect. The correct citations are as follows: (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Tags: Correction Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 14, 2015 Category: Pathology Source Type: research

Reporting Incidental Findings in Genomic Scale Clinical Sequencing—A Clinical Laboratory Perspective
Advances in sequencing technologies have facilitated concurrent testing for many disorders, and the results generated may provide information about a patient's health that is unrelated to the clinical indication, commonly referred to as incidental findings. This is a paradigm shift from traditional genetic testing in which testing and reporting are tailored to a patient's specific clinical condition. Clinical laboratories and physicians are wrestling with this increased complexity in genomic testing and reporting of the incidental findings to patients. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - February 12, 2015 Category: Pathology Authors: Madhuri Hegde, Sherri Bale, Pinar Bayrak-Toydemir, Jane Gibson, Linda Jo Bone Jeng, Loren Joseph, Jordan Laser, Ira M. Lubin, Christine E. Miller, Lainie F. Ross, Paul G. Rothberg, Alice K. Tanner, Patrik Vitazka, Rong Mao Tags: Special article Source Type: research

Relationship between and Variants and the Clinical Phenotype in Late-Onset Cystic Fibrosis Disease with Chronic Pancreatitis
Cystic fibrosis (CF), the most common autosomal recessive disease in whites, is caused by mutations in the CF transmembrane conductance regulator (CFTR). So far,>1900 mutations have been described, most of which are nonsense, missense, and frameshift, and can lead to severe phenotypes, reducing the level of function of the CFTR protein. Synonymous variations are usually considered silent without pathogenic effects. However, synonymous mutations exhibiting exon skipping as a consequence of aberrant splicing of pre-mRNA differ. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - January 27, 2015 Category: Pathology Authors: Anna C. Tomaiuolo, Valentina M. Sofia, Cecilia Surace, Fabio Majo, Silvia Genovese, Stefano Petrocchi, Simona Grotta, Federico Alghisi, Vincenzina Lucidi, Adriano Angioni Tags: Regular Article Source Type: research

NADf Chip, a Two-Color Microarray for Simultaneous Screening of Multigene Mutations Associated with Hearing Impairment in North African Mediterranean Countries
Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - January 2, 2015 Category: Pathology Authors: Imen Chakchouk, Mariem Ben Said, Fida Jbeli, Riadh Benmarzoug, Salma Loukil, Ibtihel Smeti, Amine Chakroun, Abdullah Ahmed Yousef Gibriel, Abdelmonem Ghorbel, Hassen Hadjkacem, Saber Masmoudi Tags: Regular Article Source Type: research

Next-Generation Sequencing of the and Genes for the Genetic Diagnostics of Hereditary Breast and/or Ovarian Cancer
Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 hereditary breast and/or ovarian cancer patients. We first validated this approach in a cohort of 115 samples with previously known BRCA1 and BRCA2 mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq BRCA1 and BRCA2 panel. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - December 31, 2014 Category: Pathology Authors: Daniel Trujillano, Maximilian E.R. Weiss, Juliane Schneider, Julia Köster, Efstathios B. Papachristos, Viatcheslav Saviouk, Tetyana Zakharkina, Nahid Nahavandi, Lejla Kovacevic, Arndt Rolfs Tags: Regular Article Source Type: research

miR-210 Is a Prognostic Marker in Clear Cell Renal Cell Carcinoma
Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - December 30, 2014 Category: Pathology Authors: Sara Samaan, Heba W.Z. Khella, Andrew Girgis, Andreas Scorilas, Evi Lianidou, Manal Gabril, Sergey N. Krylov, Michael Jewett, Georg A. Bjarnason, Hala El-said, George M. Yousef Tags: Regular Article Source Type: research

A DNA Real-Time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia
The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Paul A. Bartley, Susan Latham, Bradley Budgen, David M. Ross, Elizabeth Hughes, Susan Branford, Deborah White, Timothy P. Hughes, Alexander A. Morley Tags: Regular article Source Type: research

Two Novel Methods for Rapid Detection and Quantification of R882 Mutations in Acute Myeloid Leukemia
DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at amino acid R882 in the methyltransferase domain of the gene. DNMT3A mutations have been reported to be stable during disease progression and are associated with unfavorable outcome in acute myeloid leukemia patients with normal karyotype. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarker...
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Melissa Mancini, Syed Khizer Hasan, Tiziana Ottone, Serena Lavorgna, Claudia Ciardi, Daniela F. Angelini, Francesca Agostini, Adriano Venditti, Francesco Lo-Coco Tags: Regular article Source Type: research

Report of New Haplotype for Gene
The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laboratory–developed assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F...
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Victoria M. Pratt, Brittany N. Beyer, Daniel L. Koller, Todd C. Skaar, David A. Flockhart, Kenneth D. Levy, Gail H. Vance Tags: Regular Article Source Type: research

A DNA Real-time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia
The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Paul A. Bartley, Susan Latham, Bradley Budgen, David M. Ross, Elizabeth Hughes, Susan Branford, Deborah White, Timothy P. Hughes, Alexander A. Morley Tags: Regular Article Source Type: research

Two Novel Methods for Rapid Detection and Quantification of R882 Mutations in Acute Myeloid Leukemia
DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at amino acid R882 in the methyltransferase domain of the gene. DNMT3A mutations have been reported to be stable during disease progression and are associated with unfavorable outcome in acute myeloid leukemia patients with normal karyotype. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarker...
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Melissa Mancini, Syed Khizer Hasan, Tiziana Ottone, Serena Lavorgna, Claudia Ciardi, Daniela F. Angelini, Francesca Agostini, Adriano Venditti, Francesco Lo-Coco Tags: Regular Article Source Type: research

High-Throughput Sequencing Using the Ion Torrent Personal Genome Machine for Clinical Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia
For patients with chronic lymphocytic leukemia, an important prognostic indicator is the somatic hypermutation status of immunoglobulin heavy chain variable region nucleic acid within the clonal cell population. Current clinical assays for this evaluation rely on Sanger sequencing and are complex, such that availability of testing for patients is limited and costly. However, advances in high-throughput sequencing provide an opportunity to improve complex clinical sequencing applications. Our goal was to determine whether high-throughput sequencing technology could reliably perform the sequencing required for somatic hyperm...
Source: Journal of Molecular Diagnostics - December 29, 2014 Category: Pathology Authors: Rebecca McClure, Ming Mai, Scott McClure Tags: Regular Article Source Type: research

Target-Enriched Next-Generation Sequencing Reveals Differences between Primary and Secondary Ovarian Tumors in Formalin-Fixed, Paraffin-Embedded Tissue
Differentiating primary endometrioid or mucinous ovarian tumors from secondary ovarian tumors can be challenging. We compared somatic mutation profiles of primary and secondary ovarian cancers to investigate if these profiles can help diagnose ovarian tumors. Cancer-related genes (n = 115) were screened by target-enriched next-generation sequencing in formalin-fixed, paraffin-embedded tumor tissue from 43 primary endometrioid and mucinous ovarian carcinomas and 28 proven colorectal cancer metastases to the ovary. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - December 26, 2014 Category: Pathology Authors: Stijn Crobach, Dina Ruano, Ronald van Eijk, Gert Jan Fleuren, Ivonne Minderhout, Ronelle Snowdowne, Carli Tops, Tom van Wezel, Hans Morreau Tags: Regular Article Source Type: research