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. This assay successfully detected all three polymorphisms on DNA extracted by the automated platform easyMAG from all samples when compared to sequenced amplicons. Analysis of 126 participants with recent HCV infection from the Australian Trial in Acute Hepatitis C study demonstrated the prevalence of favorable single-nucleotide polymorphisms were 62%, 51%, and 45% for rs8099917 TT, rs12979860 CC, and rs36...
Source: The Journal of Molecular Diagnostics - July 4, 2015 Category: Pathology Source Type: research

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma
Publication date: Available online 2 July 2015 Source:The Journal of Molecular Diagnostics Author(s): Qiushi Wang , Xin Yang , Yong He , Qiang Ma , Li Lin , Ping Fu , Hualiang Xiao 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 fr...
Source: The Journal of Molecular Diagnostics - July 2, 2015 Category: Pathology Source Type: research

Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma
Publication date: Available online 29 June 2015 Source:The Journal of Molecular Diagnostics Author(s): David A. Moore , Gerald Saldanha , Abdlrzag Ehdode , Mohamed Z. Mughal , Linda Potter , Lovesh Dyall , James H. Pringle 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 cal...
Source: The Journal of Molecular Diagnostics - June 30, 2015 Category: Pathology Source Type: research

A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing
Publication date: July 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 4 Author(s): Théo G.M. Oliveira , Miguel Mitne-Neto , Louise T. Cerdeira , Julia D.C. Marsiglia , Edmundo Arteaga-Fernandez , José E. Krieger , Alexandre C. Pereira 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 constr...
Source: The Journal of Molecular Diagnostics - June 23, 2015 Category: Pathology Source Type: research

Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique
Publication date: July 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 4 Author(s): Yi Wang , Yan Wang , Ruiting Lan , Huaqing Xu , Aijing Ma , Dongxun Li , Hang Dai , Xuejiao Yuan , Jianguo Xu , Changyun Ye 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...
Source: The Journal of Molecular Diagnostics - June 23, 2015 Category: Pathology Source Type: research

cDNA Hybrid Capture Improves Transcriptome Analysis on Low-Input and Archived Samples
In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 19, 2015 Category: Pathology Source Type: research

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing
We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing. (Source: The Journal of Molecular Diagnostics)
Source: The 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
Publication date: Available online 8 June 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 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...
Source: The Journal of Molecular Diagnostics - June 9, 2015 Category: Pathology Source Type: research

Evaluation of the Real-Q BRAF V600E Detection Assay in Fine-Needle Aspiration Samples of Thyroid Nodules
In conclusion, the detection of BRAF V600E mutations in PTC by Real-Q is compatible to that of AS-PCR. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues
Publication date: Available online 30 April 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 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 degradati...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

A Variant Detection Pipeline for Inherited Cardiomyopathies–Associated Genes Using Ion Torrent PGM Platform
Publication date: Available online 30 April 2015 Source:The Journal of Molecular Diagnostics Author(s): Théo G.M. Oliveira , Miguel Mitne-Neto , Louise T. Cerdeira , Julia D.C. Marsiglia , Edmundo Arteaga-Fernandez , José E. Krieger , Alexandre C. Pereira 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 const...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform
We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies
In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR
Publication date: Available online 5 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Vladimira Koudelakova , Jitka Berkovcova , Radek Trojanec , Jana Vrbkova , Lenka Radova , Jiri Ehrmann , Zdenek Kolar , Bohuslav Melichar , Marian Hajduch 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 ...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing
In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non–Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue
Publication date: Available online 8 May 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 , Frances A. Shepherd , Victoria Plamadeala 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...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Annotation of Sequence Variants in Cancer Samples Processes and Pitfalls for Routine Assays in the Clinical Laboratory
Publication date: Available online 11 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Lobin A. Lee , Kevin J. Arvai , Dan Jones 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...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma
Publication date: Available online 14 May 2015 Source:The Journal of Molecular Diagnostics Author(s): John S. Hall , Suzanne Usher , Richard J. Byers , Rebekah C. Higgins , Danish Memon , John A. Radford , Kim M. Linton 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 D...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3–Positive Tumors
In conclusion, we have developed a MAGE-A3–specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Toward Rare Blood Cell Preservation for RNA Sequencing
Publication date: Available online 16 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Sanja Vickovic , Afshin Ahmadian , Rolf Lewensohn , Joakim Lundeberg 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 larges...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

The Evolving Role of the Laboratory Professional in the Age of Genome Sequencing A Vision of the Association for Molecular Pathology
Publication date: Available online 2 June 2015 Source:The Journal of Molecular Diagnostics Author(s): Iris Schrijver , Daniel H. Farkas , Jane S. Gibson , Elaine LyonTeaser 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: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): Guanshan Zhu , Xin Ye , Zhengwei Dong , Ya Chao Lu , Yun Sun , Yi Liu , Rose McCormack , Yi Gu , Xiaoqing Liu 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 sensi...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): 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 F. Hechtman , Jinjuan Yao , Wei Song , Dara S. Ross , Alifya Oultache , Snjezana Dogan , Laetitia Borsu , Meera Hameed , Khedoudja Nafa , Maria E. Arcila , Marc Ladanyi , Michael F. Berger The identification of specific genetic alterations as key oncogeni...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility? A Report of the Association for Molecular Pathology
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): Bert Gold , Milena Cankovic , Larissa V. Furtado , Frederick Meier , Christopher D. Gocke 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 ...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices
Publication date: May 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 3 Author(s): Scott M. Berry , Alex J. LaVanway , Hannah M. Pezzi , David J. Guckenberger , Meghan A. Anderson , Jennifer M. Loeb , David J. Beebe The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitatio...
Source: The Journal of Molecular Diagnostics - April 19, 2015 Category: Pathology Source Type: research

Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Alicia Edin , Susanne Granholm , Satu Koskiniemi , Annika Allard , Anders Sjöstedt , Anders Johansson 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 line...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

MSK-IMPACT A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology
Publication date: Available online 20 March 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 Hechtman , Jinjuan Yao , Wei Song , Dara Ross , Alifya Oultache , Snjezana Dogan , Laetitia Borsu , Meera Hameed , Khedoudja Nafa , Maria E. Arcila , Marc Ladanyi , Michael F. Berger The identification of specific genetic alterations as key oncogenic drive...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

Panels of Cytokines and Other Secretory Proteins as Potential Biomarkers of Ovarian Endometriosis
Publication date: Available online 20 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Vida Kocbek , Katja Vouk , Nick A. Bersinger , Michael D. Mueller , Tea Lanišnik Rižner 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 d...
Source: The Journal of Molecular Diagnostics - April 18, 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 A CALYM Study
Publication date: Available online 16 April 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 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, impe...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

Evaluation of HPV Genotyping Assays for Archival Clinical Samples
Publication date: Available online 16 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Gabriella Lillsunde Larsson , Jessica Carlsson , Mats G. Karlsson , Gisela Helenius 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 genotyp...
Source: The Journal of Molecular Diagnostics - March 16, 2015 Category: Pathology Source Type: research

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1
Publication date: Available online 14 March 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 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 ...
Source: The Journal of Molecular Diagnostics - March 14, 2015 Category: Pathology 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
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Alicia Edin , Susanne Granholm , Satu Koskiniemi , Annika Allard , Anders Sjöstedt , Anders Johansson 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 line...
Source: The Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Source Type: research

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Elena Castellanos-Rizaldos , Cloud Paweletz , Chen Song , Geoffrey R. Oxnard , Harvey Mamon , Pasi A. Jänne , G. Mike Makrigiorgos 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 se...
Source: The Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Publication date: Available online 11 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Guanshan Zhu , Xin Ye , Zhengwei Dong , Yachao Lu , Yun Sun , Yi Liu , Rose McCormack , Yi Gu , Xiaoqing Liu 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 sens...
Source: The Journal of Molecular Diagnostics - March 11, 2015 Category: Pathology Source Type: research

Improving Molecular Genetic Test Utilization through Order Restriction, Test Review, and Guidance
Publication date: Available online 27 February 2015 Source:The Journal of Molecular Diagnostics Author(s): Jacquelyn D. Riley , Gary W. Procop , Kandice Kottke-Marchant , Robert Wyllie , Felicitas L. Lacbawan 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...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology 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
Publication date: March 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 2 Author(s): Paul A. Bartley , Susan Latham , Bradley Budgen , David M. Ross , Elizabeth Hughes , Susan Branford , Deborah White , Timothy P. Hughes , Alexander A. Morley 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 ...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Two Novel Methods for Rapid Detection and Quantification of DNMT3A R882 Mutations in Acute Myeloid Leukemia
We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H muta...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay
Publication date: March 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 2 Author(s): Mulias Lian , Indhu-Shree Rajan-Babu , Kunal Singh , Caroline G. Lee , Hai-Yang Law , Samuel S. Chong 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&n...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology 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. Gene expression measured on stabilized blood sa...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Reporting Incidental Findings in Genomic Scale Clinical Sequencing—A Clinical Laboratory Perspective A Report of the Association for Molecular Pathology
This report by the Association of Molecular Pathology workgroup discusses the pros and cons of next-generation sequencing technology, potential benefits, and harms for reporting of incidental findings, including the effect on both the laboratory and the patient, and compares those with other areas of medicine. The importance of genetic counseling to preserve patient autonomy is also reviewed. The discussion and recommendations presented by the workgroup underline the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues
Publication date: Available online 4 March 2015 Source:The Journal of Molecular Diagnostics Author(s): 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 Yoshikawa , Richie Soong , Patrick Tan 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 polymo...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

A Systematic Approach to Novel Virus Discovery in Emerging Infectious Disease Outbreaks
Publication date: Available online 4 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Siddharth Sridhar , Kelvin K.W. To , Jasper F.W. Chan , Susanna K.P. Lau , Patrick C.Y. Woo , Kwok-Yung Yuen 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...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Lisa V. Kalman , Jack C. Tarleton , Alan K. Percy , Swaroop Aradhya , Sherri Bale , Shannon D. Barker , Pinar Bayrak-Toydemir , Christina Bridges , Arlene M. Buller-Burckle , Soma Das , Ramaswamy K. Iyer , Timothy D. Vo , Val V. Zvereff , Lorraine H. Toji Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett s...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Gene Expression Ratio Test Distinguishes Normal Lung from Lung Tumors in Solid Tissue and FNA Biopsies
This study supports the hypothesis that the gene-ratio approach reliably distinguishes normal lung from cancerous tissues in FNA samples and can be optimized to diagnose benign nodules. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Sensitive Detection and Serovar Differentiation of Typhoidal and Nontyphoidal Salmonella enterica Species Using 16S rRNA Gene PCR Coupled with High-Resolution Melt Analysis
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Billie J. Masek , Justin Hardick , Helen Won , Samuel Yang , Yu-Hsiang Hsieh , Richard E. Rothman , Charlotte A. Gaydos Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that c...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

A Novel Subtyping Assay for Detection of Clostridium difficile Virulence Genes
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Stephanie L. Angione , Aartik A. Sarma , Aleksey Novikov , Leah Seward , Jennifer H. Fieber , Leonard A. Mermel , Anubhav Tripathi This proof-of-concept study demonstrates the application of a novel nucleic acid detection platform to detect Clostridium difficile in subjects presenting with acute diarrheal symptoms. This method amplifies three genes associated with C. difficile infection, including genes and deletions (cdtB and tcdC) associated with hypervirulence attributed to the NAP1/027/BI strain. Amplification of D...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next-Generation Sequencing
In conclusion, the NGS method was superior to Sanger sequencing for detecting PKD gene mutations, achieving high sensitivity and improved gene coverage. These characteristics suggest that NGS would be an appropriate new standard for clinical genetic testing of ADPKD. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Gerald B.W. Wertheim , Catherine Smith , Maria E. Figueroa , Michael Kalos , Adam Bagg , Martin Carroll , Stephen R. Master Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Relationship between CFTR and CTRC Variants and the Clinical Phenotype in Late-Onset Cystic Fibrosis Disease with Chronic Pancreatitis
Publication date: Available online 28 January 2015 Source:The Journal of Molecular Diagnostics Author(s): Anna C. Tomaiuolo , Valentina M. Sofia , Cecilia Surace , Fabio Majo , Silvia Genovese , Stefano Petrocchi , Simona Grotta , Federico Alghisi , Vincenzina Lucidi , Adriano Angioni 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 prot...
Source: The Journal of Molecular Diagnostics - January 28, 2015 Category: Pathology Source Type: research

Target-Enriched Next-Generation Sequencing Reveals Differences between Primary and Secondary Ovarian Tumors in Formalin-Fixed, Paraffin-Embedded Tissue
Publication date: Available online 26 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Stijn Crobach , Dina Ruano , Ronald van Eijk , Gert Jan Fleuren , Ivonne Minderhout , Ronelle Snowdowne , Carli Tops , Tom van Wezel , Hans Morreau 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, par...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research