Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 11, 2015 Category: Pathology Source Type: research

Title Page
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 11, 2015 Category: Pathology Source Type: research

Abstract Disclosures
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 11, 2015 Category: Pathology Source Type: research

Abstracts
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 11, 2015 Category: Pathology Source Type: research

Author Index
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - October 11, 2015 Category: Pathology Source Type: research

Aberrant Levels of miRNAs in Bone Marrow Microenvironment and Peripheral Blood of Myeloma Patients and Disease Progression
In this study, we found that the extracellular BM microenvironment in MM contains a unique miRNA signature detectable by miRNA microarray and quantitative real-time PCR, which is partially represented in the peripheral blood. Eleven miRNAs were significantly decreased in both BM and serum of MM patients in comparison with controls. Evaluation of these miRNAs in plasma of a separate cohort of MM patients and controls confirmed significantly aberrant levels of let-7a, let-7b, let-7i, miR-15b, miR-16, and miR-20a in both serum and plasma. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 30, 2015 Category: Pathology Authors: Weixin Wang, Meghan Corrigan-Cummins, Emily A. Barber, Layla M. Saleh, Adriana Zingone, Azam Ghafoor, Rene Costello, Yong Zhang, Roger J. Kurlander, Neha Korde, Aldo M. Roccaro, Irene M. Ghobrial, Ola Landgren, Katherine R. Calvo Tags: Regular article Source Type: research

Aberrant Level of miRNAs in Bone Marrow Microenvironment and Peripheral Blood of Myeloma Patients and Disease Progression
In this study, we found that the extracellular BM microenvironment in MM contains a unique miRNA signature detectable by miRNA microarray and quantitative real-time PCR, which is partially represented in the peripheral blood. Eleven miRNAs were significantly decreased in both BM and serum of MM patients in comparison with controls. Evaluation of these miRNAs in plasma of a separate cohort of MM patients and controls confirmed significantly aberrant levels of let-7a, let-7b, let-7i, miR-15b, miR-16, and miR-20a in both serum and plasma. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - September 30, 2015 Category: Pathology Authors: Weixin Wang, Meghan Corrigan-Cummins, Emily A. Barber, Layla M. Saleh, Adriana Zingone, Azam Ghafoor, Rene Costello, Yong Zhang, Roger J. Kurlander, Neha Korde, Aldo M. Roccaro, Irene M. Ghobrial, Ola Landgren, Katherine R. Calvo Tags: Regular Article Source Type: research

Next-Generation Sequencing for Infectious Disease Diagnosis and Management
Next-generation sequencing (NGS) technologies are increasingly being used for diagnosis and monitoring of infectious diseases. Herein, we review the application of NGS in clinical microbiology, focusing on genotypic resistance testing, direct detection of unknown disease-associated pathogens in clinical specimens, investigation of microbial population diversity in the human host, and strain typing. We have organized the review into three main sections: i) applications in clinical virology, ii) applications in clinical bacteriology, mycobacteriology, and mycology, and iii) validation, quality control, and maintenance of pro...
Source: Journal of Molecular Diagnostics - September 30, 2015 Category: Pathology Authors: Martina I. Lefterova, Carlos J. Suarez, Niaz Banaei, Benjamin A. Pinsky Tags: Special Article Source Type: research

HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples
In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. In combination with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular...
Source: Journal of Molecular Diagnostics - September 3, 2015 Category: Pathology Authors: Lotte N.J. Moens, Elin Falk-Sörqvist, Viktor Ljungström, Johanna Mattsson, Magnus Sundström, Linnéa La Fleur, Lucy Mathot, Patrick Micke, Mats Nilsson, Johan Botling Tags: Regular Article Source Type: research

Haplotype Determination Using Long Range Allele-Specific Amplification
Cytochrome P450 (CYP) 2D6, a major contributor to the metabolism and bioactivation of many clinically used drugs, is encoded by a complex, highly polymorphic gene locus. To aid in the characterization of CYP2D6 allelic variation, we developed allele-specific long-range PCR (ASXL-PCR) to amplify only the allele of interest for further characterization by PCR. This development was achieved utilizing single-nucleotide polymorphisms in the upstream region of CYP2D6 and a universal CYP2D6-specific reverse primer. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 31, 2015 Category: Pathology Authors: Andrea Gaedigk, Amanda K. Riffel, J. Steven Leeder Tags: Regular Article Source Type: research

Haplotype Determination Using Long Range Allele-Specific Amplification and Real-Time PCR
Cytochrome P450 (CYP) 2D6, a major contributor to the metabolism and bioactivation of many clinically used drugs, is encoded by a complex, highly polymorphic gene locus. To aid in the characterization of CYP2D6 allelic variation, we developed allele-specific long-range PCR (ASXL-PCR) to amplify only the allele of interest for further characterization by real-time PCR. This development was achieved utilizing single-nucleotide polymorphisms in the upstream region of CYP2D6 and a universal CYP2D6-specific reverse primer. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 31, 2015 Category: Pathology Authors: Andrea Gaedigk, Amanda K. Riffel, J. Steven Leeder Tags: Regular Article Source Type: research

Haplotype Determination Using Allele-Specific Amplification Combined with Long-Range Real-Time PCR
Cytochrome P450 (CYP) 2D6, a major contributor to the metabolism and bioactivation of many clinically used drugs, is encoded by a complex, highly polymorphic gene locus. To aid in the characterization of CYP2D6 allelic variation, we developed allele-specific amplification combined with real-time long-range (ASXL)-PCR to amplify only the allele of interest for further characterization. This development was achieved utilizing single-nucleotide polymorphisms in the upstream region of CYP2D6 and a universal CYP2D6-specific reverse primer. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 31, 2015 Category: Pathology Authors: Andrea Gaedigk, Amanda K. Riffel, J. Steven Leeder Tags: Regular Article Source Type: research

Detection of Dual and Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes
Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 30, 2015 Category: Pathology Authors: Mia Y. Platt, Amir T. Fathi, Darrell R. Borger, Andrew M. Brunner, Robert P. Hasserjian, Leonora Balaj, Amy Lum, Stephen Yip, Dora Dias-Santagata, Zongli Zheng, Long P. Le, Timothy A. Graubert, A. John Iafrate, Valentina Nardi Tags: Regular Article Source Type: research

Development of a Center for Personalized Cancer Care at a Regional Cancer Center
We report the feasibility of a tumor sequencing advisory board at a regional cancer center. Specimens were analyzed for approximately 2800 mutations in 50 genes. Outcomes of interest included tumor sequencing advisory board function and processes, timely discussion of results, and proportion of reports having potentially actionable mutations. NGS results were successfully generated for 15 patients, with median time from tissue processing to reporting of 11.6 days (range, 5 to 21 days), and presented at a biweekly multidisciplinary tumor sequencing advisory board. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 30, 2015 Category: Pathology Authors: Brian R. Lane, Jeffrey Bissonnette, Tracy Waldherr, Deborah Ritz-Holland, Dave Chesla, Sandra L. Cottingham, Sheryl Alberta, Cong Liu, Amanda Bartenbaker Thompson, Carrie Graveel, Jeffrey P. MacKeigan, Sabrina L. Noyes, Judy Smith, Nehal Lakhani, Matthew Tags: Regular Article Source Type: research

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous
Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Jianli Li, Hongzheng Dai, Yanming Feng, Jia Tang, Stella Chen, Xia Tian, Elizabeth Gorman, Eric S. Schmitt, Terah A.A. Hansen, Jing Wang, Sharon E. Plon, Victor Wei Zhang, Lee-Jun C. Wong Tags: Regular article Source Type: research

Cancer Driver Log (CanDL)
Massively parallel sequencing technologies have enabled characterization of genomic alterations across multiple tumor types. Efforts have focused on identifying driver mutations because they represent potential targets for therapy. However, because of the presence of driver and passenger mutations, it is often challenging to assign the clinical relevance of specific mutations observed in patients. Currently, there are multiple databases and tools that provide in silico assessment for potential drivers; however, there is no comprehensive resource for mutations with functional characterization. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Senthilkumar Damodaran, Jharna Miya, Esko Kautto, Eliot Zhu, Eric Samorodnitsky, Jharna Datta, Julie W. Reeser, Sameek Roychowdhury Tags: Regular article Source Type: research

Mutations in the Kinase Domain of the / Gene Identified in a Wide Variety of Human Cancers
We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non–small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Wenhsiang Wen, Wangjuh (Sting) Chen, Nick Xiao, Ryan Bender, Anatole Ghazalpour, Zheng Tan, Jeffrey Swensen, Sherri Z. Millis, Gargi Basu, Zoran Gatalica, Michael F. Press Tags: Regular article Source Type: research

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of DNA in Blood Samples from Chagas Disease Patients
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Juan Carlos Ramírez, Carolina Inés Cura, Otacilio da Cruz Moreira, Eliane Lages-Silva, Natalia Juiz, Elsa Velázquez, Juan David Ramírez, Anahí Alberti, Paula Pavia, María Delmans Flores-Chávez, Arturo Muñoz-Calderón, Deyanira Pérez-Morales, Jos Tags: Regular article Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Source Type: research

MSIplus for Integrated Colorectal Cancer Molecular Testing by Next-Generation Sequencing
Molecular analysis of colon cancers currently requires multiphasic testing that uses various assays with different performance characteristics, adding cost and time to patient care. We have developed a single, next-generation sequencing assay to simultaneously evaluate colorectal cancers for mutations in relevant cancer genes (KRAS, NRAS, and BRAF) and for tumor microsatellite instability (MSI). In a sample set of 61 cases, the assay demonstrated overall sensitivity of 100% and specificity of 100% for identifying cancer-associated mutations, with a practical limit of detection at 2% mutant allele fraction. (Source: Journal...
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Jennifer A. Hempelmann, Sheena M. Scroggins, Colin C. Pritchard, Stephen J. Salipante Tags: Regular article Source Type: research

One-Step Ligation on RNA Amplification for the Detection of Point Mutations
The detection of point mutations is required in the diagnosis of many human diseases. The conformal specificity of DNA ligases was elegantly used to distinguish single-nucleotide mismatches. However, to detect point mutations in RNA retroviruses, conventional ligase-mediated approaches require the reverse transcription of viral genomes before separate ligation and amplification steps. We developed one-step ligation on RNA amplification (LRA) for the direct detection of RNA point mutations. The process combines the ligase-mediated joining of two oligonucleotides and subsequent hot start amplification into a single-tube reac...
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Lei Zhang, Jingjing Wang, Mia Coetzer, Stephanie Angione, Rami Kantor, Anubhav Tripathi Tags: Regular Article Source Type: research

MSIplus
Molecular analysis of colon cancers currently requires multiphasic testing that uses various assays with different performance characteristics, adding cost and time to patient care. We have developed a single, next-generation sequencing assay to simultaneously evaluate colorectal cancers for mutations in relevant cancer genes (KRAS, NRAS, and BRAF) and for tumor microsatellite instability (MSI). In a sample set of 61 cases, the assay demonstrated overall sensitivity of 100% and specificity of 100% for identifying cancer-associated mutations, with a practical limit of detection at 2% mutant allele fraction. (Source: Journal...
Source: Journal of Molecular Diagnostics - August 28, 2015 Category: Pathology Authors: Jennifer A. Hempelmann, Sheena M. Scroggins, Colin C. Pritchard, Stephen J. Salipante Tags: Regular Article Source Type: research

Comparative Evaluation of Broad-Panel PCR Assays for the Detection of Gastrointestinal Pathogens in Pediatric Oncology Patients
Broadly multiplexed molecular amplification assays offer an unprecedented ability to diagnose gastrointestinal infection in immunocompromised patients. However, little data are available to compare the performance of such systems in this population. A total of 436 stool samples were collected from 199 patients, predominantly immunocompromised pediatric oncology patients. Remnant samples were tested in parallel with the use of the premarket (investigational use only) versions of two broadly multiplexed PCR assays (BioFire and Luminex), and the results of samples corresponding to the first episode per patient were compared w...
Source: Journal of Molecular Diagnostics - August 27, 2015 Category: Pathology Authors: Zhengming Gu, Haiqing Zhu, Alicia Rodriguez, Mohammad Mhaissen, Stacey Schultz-Cherry, Elisabeth Adderson, Randall T. Hayden Tags: Regular Article Source Type: research

Full-Gene Sequencing Identifies Rare Mutations Not Detected in Targeted Mutation Analysis
Genetic α-1 antitrypsin (AAT) deficiency is characterized by low serum AAT levels and the identification of causal mutations or an abnormal protein. It needs to be distinguished from deficiency because of nongenetic causes, and diagnostic delay may contribute to worse patient outcome. Current routine clinical testing assesses for only the most common mutations. We wanted to determine the proportion of unexplained cases of AAT deficiency that harbor causal mutations not identified through current standard allele-specific genotyping and isoelectric focusing (IEF). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 27, 2015 Category: Pathology Authors: Rondell P. Graham, Michelle A. Dina, Sarah C. Howe, Malinda L. Butz, Kurt S. Willkomm, David L. Murray, Melissa R. Snyder, Kandelaria M. Rumilla, Kevin C. Halling, W. Edward Highsmith Tags: Regular Article Source Type: research

Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma
In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 26, 2015 Category: Pathology Authors: Daniela Drandi, Lenka Kubiczkova-Besse, Simone Ferrero, Nadia Dani, Roberto Passera, Barbara Mantoan, Manuela Gambella, Luigia Monitillo, Elona Saraci, Paola Ghione, Elisa Genuardi, Daniela Barbero, Paola Omedè, Davide Barberio, Roman Hajek, Umberto Vito Tags: Regular Article Source Type: research

Amplicon Indel Hunter Is a Novel Bioinformatics Tool to Detect Large Somatic Insertion/Deletion Mutations in Amplicon-Based Next-Generation Sequencing Data
Amplicon-based targeted next-generation sequencing assays are used widely to test for clinically relevant somatic mutations in cancer. However, accurate detection of large insertions and deletions (indels) via these assays remains challenging. Sequencing reads that cover these anomalies are, by definition, different from the reference sequence, and lead to variable performance of alignment algorithms. Reads with large indels may be aligned incorrectly, causing incorrect calls, or may remain unmapped and essentially ignored by downstream informatics pipeline sub-processes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 25, 2015 Category: Pathology Authors: Sabah Kadri, Chao J. Zhen, Michelle N. Wurst, Bradley C. Long, Zi-Feng Jiang, Y. Lynn Wang, Larissa V. Furtado, Jeremy P. Segal Tags: Technical advance Source Type: research

Amplicon Indel Hunter
Amplicon-based targeted next-generation sequencing assays are used widely to test for clinically relevant somatic mutations in cancer. However, accurate detection of large insertions and deletions (indels) via these assays remains challenging. Sequencing reads that cover these anomalies are, by definition, different from the reference sequence, and lead to variable performance of alignment algorithms. Reads with large indels may be aligned incorrectly, causing incorrect calls, or may remain unmapped and essentially ignored by downstream informatics pipeline sub-processes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 25, 2015 Category: Pathology Authors: Sabah Kadri, Chao J. Zhen, Michelle N. Wurst, Bradley C. Long, Zi-Feng Jiang, Y. Lynn Wang, Larissa V. Furtado, Jeremy P. Segal Tags: Technical Advance Source Type: research

Mutational Analysis of BRAF Inhibitor–Associated Squamoproliferative Lesions
In recent years, there has been increasing use of BRAF-inhibiting drugs for the treatment of various malignancies, including melanoma. However, these agents are associated with the development of other nonmelanoma skin lesions, in particular squamoproliferative lesions such as keratoacanthomas (KAs), squamous cell carcinomas, and BRAF inhibitor–associated verrucous keratoses. The molecular pathogenesis of these lesions is of interest, not only for therapeutic reasons, but also for the insight it might provide into the development of similar lesions in a sporadic setting. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - August 25, 2015 Category: Pathology Authors: Britt Clynick, Tania Tabone, Kathryn Fuller, Wendy Erber, Katie Meehan, Michael Millward, Benjamin A. Wood, Nathan T. Harvey Tags: Regular Article Source Type: research

A Proof-of-Concept Case Study for Personalized Noninvasive Prenatal Diagnosis
This commentary highlights the article by van den Oever et al that describes a new method of prenatal diagnosis of single-mutation disorders. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 23, 2015 Category: Pathology Authors: Heather G. LaBreche, Siby Sebastian Tags: Commentary Source Type: research

A Proof-of -Concept Case Study for Personalized Noninvasive Prenatal Diagnosis
This commentary highlights the article by van den Oever et al that describes a new method of prenatal diagnosis of single-mutation disorders. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 23, 2015 Category: Pathology Authors: Heather G. LaBreche, Siby Sebastian Tags: Commentary Source Type: research

A Systematic Comparison of Traditional and Multigene Panel Testing for Hereditary Breast and Ovarian Cancer Genes in More Than 1000 Patients
Gene panels for hereditary breast and ovarian cancer risk assessment are gaining acceptance, even though the clinical utility of these panels is not yet fully defined. Technical questions remain, however, about the performance and clinical interpretation of gene panels in comparison with traditional tests. We tested 1105 individuals using a 29-gene next-generation sequencing panel and observed 100% analytical concordance with traditional and reference data on>750 comparable variants. These 750 variants included technically challenging classes of sequence and copy number variation that together represent a significant fr...
Source: Journal of Molecular Diagnostics - July 21, 2015 Category: Pathology Authors: Stephen E. Lincoln, Yuya Kobayashi, Michael J. Anderson, Shan Yang, Andrea J. Desmond, Meredith A. Mills, Geoffrey B. Nilsen, Kevin B. Jacobs, Federico A. Monzon, Allison W. Kurian, James M. Ford, Leif W. Ellisen Tags: Regular Article Source Type: research

Processed Pseudogene Confounding Deletion/Duplication Assays for
Mutations in SMAD4 have been associated with juvenile polyposis syndrome and combined juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome. SMAD4 is part of the SMAD gene family. To date, there has been no report in the literature of a SMAD4 pseudogene. An unusual SMAD4 duplication pattern was seen in multiple patient samples using two different duplication/deletion platforms: multiplex ligation–dependent probe amplification and chromosomal microarray. Follow-up confirmatory testing included real-time quantitative PCR and sequencing of an exon/exon junction, all results leading to the conclusion of the e...
Source: Journal of Molecular Diagnostics - July 9, 2015 Category: Pathology Authors: Alison Millson, Tracey Lewis, Tina Pesaran, David Salvador, Katrina Gillespie, Chia-Ling Gau, Genevieve Pont-Kingdon, Elaine Lyon, Pinar Bayrak-Toydemir Tags: Regular Article Source Type: research

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing
High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. (Source: Journal o...
Source: Journal of Molecular Diagnostics - July 9, 2015 Category: Pathology Authors: Ming Wang, Leire Escudero-Ibarz, Sarah Moody, Naiyan Zeng, Alexandra Clipson, Yuanxue Huang, Xuemin Xue, Nicholas F. Grigoropoulos, Sharon Barrans, Lisa Worrillow, Tim Forshew, Jing Su, Andrew Firth, Howard Martin, Andrew Jack, Kim Brugger, Ming-Qing Du Tags: Regular Article Source Type: research

Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues
The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinical usefulness of PCR for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection in lung tissue that show chronic granulomatous inflammation. A total of 199 formalin-fixed, paraffin-embedded specimens, including 137 Mycobacterium tuberculosis (MTB), 17 NTM cases, and 45 other than mycobacterial cases were collected. We performed acid-fast staining, MTB and NTM nested PCRs, and MTB and NTM real-time PCRs. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 8, 2015 Category: Pathology Authors: Yo Na Kim, Kyoung Min Kim, Ha Na Choi, Ju Hyung Lee, Ho Sung Park, Kyu Yun Jang, Woo Sung Moon, Myoung Jae Kang, Dong Geun Lee, Myoung Ja Chung Tags: Regular Article Source Type: research

Oligonucleotide PIK3CA/Chromosome 3 Dual Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA
The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 8, 2015 Category: Pathology Authors: Wenjun Zhang, Antony Hubbard, Leslie Baca-Parkinson, Stacey Stanislaw, Frank Vladich, Mark D. Robida, James G. Grille, Daniel Maxwell, Tsu-Shuen Tsao, William Carroll, Tracie Gardner, June Clements, Shalini Singh, Lei Tang Tags: Regular Article Source Type: research

Targeted Next-Generation Sequencing for Hereditary Cancer Syndromes
Lynch syndrome is a hereditary cancer syndrome that results from germline mutations in one of the DNA mismatch repair genes, leading to an increased lifetime risk of cancer. Colorectal cancer is most commonly identified with Lynch syndrome; however, women with Lynch syndrome have an increased risk of developing endometrial cancer (up to 60%), which is the sentinel diagnosis in approximately one-half of the cases. Current screening algorithms are developed on family history and laboratory-based tests, including immunohistochemistry for mismatch repair proteins and microsatellite instability testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 7, 2015 Category: Pathology Authors: Laura J. Tafe Tags: Review Source Type: research

Fragile X Syndrome
Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in 4000 for males and 1 in 5000 to 8000 for females. The American College of Medical Genetics and Genomics has recommended diagnostic testing for fragile X in symptomatic persons, women with ovarian dysfunction, and persons with tremor/ataxia syndrome. Although medical and scientific professionals do not currently recommend screening nonsymptomatic populations, improvements in current treatment approaches and ongoing clinical trials have generated growing interest in screening for fragile X. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 7, 2015 Category: Pathology Authors: Justine I. Lyons, Gregory Kerr, Patricia W. Mueller Tags: Special Article Source Type: research

A Novel Targeted Approach for Noninvasive Detection of Paternally Inherited Mutations in Maternal Plasma
The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 7, 2015 Category: Pathology Authors: Jessica M.E. van den Oever, Ivonne J.H.M. van Minderhout, Cornelis L. Harteveld, Nicolette S. den Hollander, Egbert Bakker, Nienke van der Stoep, Elles M.J. Boon Tags: Regular Article Source Type: research

Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity
Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity an...
Source: Journal of Molecular Diagnostics - July 4, 2015 Category: Pathology Authors: Pavel Lobachevsky, Lisa Woodbine, Kuang-Chih Hsiao, Sharon Choo, Chris Fraser, Paul Gray, Jai Smith, Nickala Best, Laura Munforte, Elena Korneeva, Roger F. Martin, Penny A. Jeggo, Olga A. Martin Tags: Regular Article Source Type: research

Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types
We describe a simple and rapid genotyping method for IFNL rs12979860, rs8099917, and rs368234815 using high-resolution melting analysis for DNA extracted from whole blood, buffy coat, plasma, serum, and dried blood spots. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 3, 2015 Category: Pathology Authors: François M.J. Lamoury, Sofia Bartlett, Brendan Jacka, Behzad Hajarizadeh, Jason Grebely, Gail V. Matthews, Gregory J. Dore, Tanya L. Applegate Tags: Regular article Source Type: research

Interferon Lambda 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types
We describe a simple and rapid genotyping method for IFNL rs12979860, rs8099917, and rs368234815 using high-resolution melting analysis for DNA extracted from whole blood, buffy coat, plasma, serum, and dried blood spots. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 3, 2015 Category: Pathology Authors: François M.J. Lamoury, Sofia Bartlett, Brendan Jacka, Behzad Hajarizadeh, Jason Grebely, Gail V. Matthews, Gregory J. Dore, Tanya L. Applegate Tags: Regular Article Source Type: research

Risk of Misdiagnosis Due to Allele Dropout and False-Positive PCR Artifacts in Molecular Diagnostics
Quality control is a complex issue for clinical molecular diagnostic applications. In the case of genotyping assays, artifacts such as allele dropout represent a risk of misdiagnosis for amplification-based methods. However, its frequency of occurrence in PCR-based diagnostic assays remains unknown. To maximize the likelihood of detecting allele dropout, our clinical genotyping PCR-based assays are designed with two independent assays for each allele (nonoverlapping primers on each DNA strand). To estimate the incidence of allelic dropout, we took advantage of the capacity of our clinical assays to detect such events. (Sou...
Source: Journal of Molecular Diagnostics - July 2, 2015 Category: Pathology Authors: Jonatan Blais, Sébastien B. Lavoie, Sylvie Giroux, Johanne Bussières, Carmen Lindsay, Jacqueline Dionne, Mélissa Laroche, Yves Giguère, François Rousseau Tags: Regular Article Source Type: research

Droplet Digital PCR for Absolute Quantification of  Gene Rearrangement in Lung Adenocarcinoma
Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive non–small-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - July 2, 2015 Category: Pathology Authors: Qiushi Wang, Xin Yang, Yong He, Qiang Ma, Li Lin, Ping Fu, Hualiang Xiao Tags: Regular Article Source Type: research

Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma
Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR–based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 29, 2015 Category: Pathology Authors: David A. Moore, Gerald Saldanha, Abdlrzag Ehdode, Mohamed Z. Mughal, Linda Potter, Lovesh Dyall, James H. Pringle Tags: Regular Article Source Type: research

Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time–LAMP assay. In this system, the LAMP forward or backward inner primers contain 5′ end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5′ end with a fluorophore and in the middle with a dark quencher. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 19, 2015 Category: Pathology Authors: Yi Wang, Yan Wang, Ruiting Lan, Huaqing Xu, Aijing Ma, Dongxun Li, Hang Dai, Xuejiao Yuan, Jianguo Xu, Changyun Ye Tags: Regular article Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 19, 2015 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 19, 2015 Category: Pathology Source Type: research

Evaluation of Mutational Testing of Preneoplastic Barrett's Mucosa by Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett's Esophagus
Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EA...
Source: Journal of Molecular Diagnostics - June 8, 2015 Category: Pathology Authors: Armando Del Portillo, Stephen M. Lagana, Yuan Yao, Takeshi Uehara, Nirag Jhala, Tapan Ganguly, Peter Nagy, Jorge Gutierrez, Aesis Luna, Julian Abrams, Yang Liu, Randall Brand, Jorge L. Sepulveda, Gary W. Falk, Antonia R. Sepulveda Tags: Regular Article Source Type: research

The Evolving Role of the Laboratory Professional in the Age of Genome Sequencing
The Association for Molecular Pathology emphasizes the need for proactive engagement of laboratory directors with clinicians, patients, and professional organizations as genomic sequence analysis gains importance in diagnostic medicine. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 2, 2015 Category: Pathology Authors: Iris Schrijver, Daniel H. Farkas, Jane S. Gibson, Elaine Lyon, AMP Executive Committee Tags: Perspectives Source Type: research