The Journal of Molecular Diagnostics RSS feed: Articles in Press. The Journal of Molecular Diagnostics , the official publication of the Association for Molecular Pathology (AMP) , co-published by the American Society for Investigative Pathology (ASIP) , seeks to publish high quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine. The editors welcome for review articles that contain: novel discoveries or clinicopathologic correlations including studies in oncology , infectious diseases , inherited diseases , predisposition to disease, or the description or polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods for diagnosis or monitoring of disease or disease predisposition. http://www.journals.elsevierhealth.com/periodicals/jmdi//inpress?rss=yesElsevier Inc.en © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. The Journal of Molecular Diagnostics1525-15782013-06-10 © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. healthpermissions@elsevier.comhttp://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000585/abstract?rss=yesAberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.Genome-Wide Identification and Validation of a Novel Methylation Biomarker, SDC2, for Blood-Based Detection of Colorectal Cancer - Corrected ProofTaeJeong Oh, Nayoung Kim, Youngho Moon, Myung Soon Kim, Benjamin D. Hoehn, Chan Hee Park, Tae Soo Kim, Nam Kyu Kim, Hyun Cheol Chung, Sungwhan An10.1016/j.jmoldx.2013.03.004The Journal of Molecular Diagnostics (2013)2013-06-10The Journal of Molecular Diagnostics2013-06-10REGULAR ARTICLEShttp://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000688/abstract?rss=yesLung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contraindications. Histologic diagnosis can be challenging owing to tumor characteristics, and it has been shown to have less-than-ideal agreement among pathologists reviewing the same specimens. Microarray profiling studies using frozen specimens have shown that histologies exhibit different gene expression trends; however, frozen specimens are not amenable to routine clinical application. Herein, we developed a gene expression–based predictor of lung cancer histology for FFPE specimens, which are routinely available in clinical settings. Genes predictive of lung cancer histologies were derived from published cohorts that had been profiled by microarrays. Expression of these genes was measured by quantitative RT-PCR (RT-qPCR) in a cohort of patients with FFPE lung cancer. A histology expression predictor (HEP) was developed using RT-qPCR expression data for adenocarcinoma, carcinoid, small cell carcinoma, and squamous cell carcinoma. In cross-validation, the HEP exhibited mean accuracy of 84% and κ = 0.77. In separate independent validation sets, the HEP was compared with pathologist diagnoses on the same tumor block specimens, and the HEP yielded similar accuracy and precision as the pathologists. The HEP also exhibited good performance in specimens with low tumor cellularity. Therefore, RT-qPCR gene expression from FFPE specimens can be effectively used to predict lung cancer histology.Prediction of Lung Cancer Histological Types by RT-qPCR Gene Expression in FFPE Specimens - Uncorrected ProofMatthew D. Wilkerson, Jason M. Schallheim, D. Neil Hayes, Patrick J. Roberts, Roy R.L. Bastien, Michael Mullins, Xiaoying Yin, C. Ryan Miller, Leigh B. Thorne, Katherine B. Geiersbach, Kenneth Muldrew, William K. Funkhouser, Cheng Fan, Michele C. Hayward, Steven Bayer, Charles M. Perou, Philip S. Bernard10.1016/j.jmoldx.2013.03.007The Journal of Molecular Diagnostics (2013)2013-05-22The Journal of Molecular Diagnostics2013-05-22http://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000573/abstract?rss=yesWe investigated the robustness of amplicon deep sequencing to study its utility in routine clinical applications offering patient-specific individualized assays for molecular disease characterization and monitoring. Amplicons were designed targeting RUNX1, CEBPA, CBL, NRAS, KRAS, DNMT3A, EZH2, and TP53 using different PCR amplification strategies and Roche GS FLX Titanium and Illumina MiSeq sequencing platforms. Thirty-three patients with leukemia were selected as an exemplary cohort representing heterogeneous cancer specimens. Both standard two-primer amplification and four-primer microfluidics PCRs yielded highly linear characteristics in detecting molecular alterations in series of dilution experiments. By fitting a linear mixed-effects model to the logarithmized data, a slope β of −1.000 (95% CI, ±0.046) was obtained for two-primer assays and of −0.998 (95% CI, ±0.105) was obtained for four-primer assays, which represented a near-perfect decrease of the mutation load. Furthermore, data are presented on technical precision, limit of detection, and occurrence of small subclones in TP53- and RUNX1-mutated patients to identify clonal disease progression and residual disease. We demonstrate that, depending on the local sequence context for each amplicon, the limit of detection of the assay cannot be lower than a range of 0.25% to 3.5%. In conclusion, amplicon deep sequencing enabled the assessment of mutations in a highly robust manner and across a broad range of relative frequencies of mutations. This assay detects residual disease or identifies mutations with predictive relevance to direct treatment strategies.Robustness of Amplicon Deep Sequencing Underlines Its Utility in Clinical Applications - Uncorrected ProofVera Grossmann, Andreas Roller, Hans-Ulrich Klein, Sandra Weissmann, Wolfgang Kern, Claudia Haferlach, Martin Dugas, Torsten Haferlach, Susanne Schnittger, Alexander Kohlmann10.1016/j.jmoldx.2013.03.003The Journal of Molecular Diagnostics (2013)2013-05-15The Journal of Molecular Diagnostics2013-05-15http://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS152515781300069X/abstract?rss=yesMyotonic dystrophy type 1 (DM1) is caused by expansion of a CTG triplet repeat in the 3′ untranslated region of the DMPK gene that encodes a serine-threonine kinase. Patients with larger repeats tend to have a more severe phenotype. Clinical laboratories require reference and quality control materials for DM1 diagnostic and carrier genetic testing. Well-characterized reference materials are not available. To address this need, the Centers for Disease Control and Prevention–based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community, the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members, and the Coriell Cell Repositories, has established and characterized cell lines from patients with DM1 to create a reference material panel. The CTG repeats in genomic DNA samples from 10 DM1 cell lines were characterized in three clinical genetic testing laboratories using PCR and Southern blot analysis. DMPK alleles in the samples cover four of five DM1 clinical categories: normal (5 to 34 repeats), mild (50 to 100 repeats), classical (101 to 1000 repeats), and congenital (>1000 repeats). We did not identify or establish Coriell cell lines in the premutation range (35 to 49 repeats). These samples are publicly available for quality control, proficiency testing, test development, and research and should help improve the accuracy of DM1 testing.Development of a Genomic DNA Reference Material Panel for Myotonic Dystrophy Type 1 (DM1) Genetic Testing - Uncorrected ProofLisa Kalman, Jack Tarleton, Monica Hitch, Madhuri Hegde, Nick Hjelm, Elizabeth Berry-Kravis, Lili Zhou, James E. Hilbert, Elizabeth A. Luebbe, Richard T. Moxley, Lorraine Toji10.1016/j.jmoldx.2013.03.008The Journal of Molecular Diagnostics (2013)2013-05-15The Journal of Molecular Diagnostics2013-05-15http://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000561/abstract?rss=yesOne of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry–based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, the human EGF Receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue–SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue–SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue - Uncorrected ProofTodd Hembrough, Sheeno Thyparambil, Wei-Li Liao, Marlene M. Darfler, Joseph Abdo, Kathleen M. Bengali, Stephen M. Hewitt, Richard A. Bender, David B. Krizman, Jon Burrows10.1016/j.jmoldx.2013.03.002The Journal of Molecular Diagnostics (2013)2013-05-13The Journal of Molecular Diagnostics2013-05-13TECHNICAL ADVANCEhttp://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000597/abstract?rss=yesSanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.The Development of Next-Generation Sequencing Assays for the Mitochondrial Genome and 108 Nuclear Genes Associated with Mitochondrial Disorders - Uncorrected ProofShale Dames, Lan-Szu Chou, Ye Xiao, Tyler Wayman, Jennifer Stocks, Marc Singleton, Karen Eilbeck, Rong Mao10.1016/j.jmoldx.2013.03.005The Journal of Molecular Diagnostics (2013)2013-05-13The Journal of Molecular Diagnostics2013-05-13http://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000676/abstract?rss=yesArray comparative hybridization has been used successfully to identify genomic alterations in stillbirth material; however, high DNA quantity and quality requirements may limit its utility in some fetal samples. Molecular inversion probe (MIP) array analysis of FFPE stillbirth autopsy samples circumvents the challenges associated with karyotype and short-term fetal cell culture, requires limited DNA input, and allows for retrospective evaluation of fetal loss. We performed MIP analysis on archival FFPE autopsy tissue to identify underlying genetic abnormalities not previously detected using traditional cytogenetic methods. Archival FFPE stillbirth cases (≥20 weeks gestation) were identified with the following characteristics: i) the phenotype suggested underlying genomic alterations; ii) the karyotype was either normal or not available and there were no other known genetic abnormalities; or iii) previous microarray testing was not performed. Genomic DNA (75 ng) was processed onto a 330,000-feature MIP array. Twenty-seven of 29 (93.1%) FFPE samples had passing MIP quality control scores. Abnormalities were seen in 3 of 27 (11%) archival samples (deletion of 17q12, trisomy 18, and a case of 4qter duplication and 13qter deletion arising from an unbalanced 4q;13q translocation), which, if identified at the time of autopsy, may have changed the course of medical management. This study highlights the benefits of using MIP array analysis for identification of genomic alterations in FFPE stillbirth autopsy tissue.Molecular Inversion Probe Array for the Genetic Evaluation of Stillbirth Using Formalin-Fixed, Paraffin-Embedded Tissue - Uncorrected ProofLeslie R. Rowe, Harshwardhan M. Thaker, John. M. Opitz, Joshua D. Schiffman, Zaid M. Haddadin, Lance K. Erickson, Sarah T. South10.1016/j.jmoldx.2013.03.006The Journal of Molecular Diagnostics (2013)2013-05-13The Journal of Molecular Diagnostics2013-05-13TECHNICAL ADVANCEhttp://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS1525157813000603/abstract?rss=yesFragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening.Fragile X Screening: Quantification of FMRP in Dried Blood Spots by a Luminex Immunoassay - Uncorrected ProofGiuseppe LaFauci, Tatyana Adayev, Richard Kascsak, Regina Kascsak, Sarah Nolin, Pankaj Mehta, William T. Brown, Carl Dobkin10.1016/j.jmoldx.2013.02.006The Journal of Molecular Diagnostics (2013)2013-05-09The Journal of Molecular Diagnostics2013-05-09http://www.journals.elsevierhealth.com/periodicals/jmdi/article/PIIS152515781300041X/abstract?rss=yesObjective: To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Participants: Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Evidence: Three unbiased literature searches of electronic databases were performed to capture published articles from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Consensus Process: Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). Conclusions: The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology - Corrected ProofNeal I. Lindeman, Philip T. Cagle, Mary Beth Beasley, Dhananjay Arun Chitale, Sanja Dacic, Giuseppe Giaccone, Robert Brian Jenkins, David J. Kwiatkowski, Juan-Sebastian Saldivar, Jeremy Squire, Erik Thunnissen, Marc Ladanyi10.1016/j.jmoldx.2013.03.001The Journal of Molecular Diagnostics (2013)2013-04-08The Journal of Molecular Diagnostics2013-04-08SPECIAL ARTICLE