Journal of Molecular Diagnostics最新文献

{"title":"Evaluation of Long-Read Genome Sequencing for Genomic Profiling of Myeloid Cancers","authors":"Haley J. Abel ,&nbsp;Mohamed Mahgoub ,&nbsp;Nidhi Davarapalli ,&nbsp;Rohan Kodgule ,&nbsp;Christopher A. Miller ,&nbsp;Robert S. Fulton ,&nbsp;Catrina Fronick ,&nbsp;Christopher Markovic ,&nbsp;Sharon Heath ,&nbsp;Jacqueline E. Payton ,&nbsp;Meagan A. Jacoby ,&nbsp;Daniel C. Link ,&nbsp;Matthew J. Walter ,&nbsp;Eric J. Duncavage ,&nbsp;Timothy J. Ley ,&nbsp;David H. Spencer","doi":"10.1016/j.jmoldx.2025.09.001","DOIUrl":"10.1016/j.jmoldx.2025.09.001","url":null,"abstract":"<div><div>Whole-genome sequencing (WGS) is a comprehensive approach for the genomic evaluation of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We recently described a streamlined tumor-only WGS assay (ChromoSeq) that uses Illumina short-read sequencing with targeted analysis to detect the full range of clinically relevant somatic mutations. Here we sought to determine the performance of this targeted analysis approach using long-read sequencing data from Oxford Nanopore Technologies and Pacific Biosciences. Samples from 26 patients with AML and MDS were sequenced to a mean of 52× coverage. Head-to-head comparison of reportable somatic variants to standard WGS revealed more than 96% recall and 91% precision for single nucleotide variants for both long-read platforms. Performance was lower for insertion/deletions (66% recall and 42% precision), especially in regions with few phased reads that facilitate accurate variant detection. The long-read platforms were 95% accurate for copy number calls, and they detected all recurrent structural variants with no false-positive findings. In addition, long reads properly identified intronic insertions near repetitive elements that were incorrectly identified as interchromosomal structural rearrangements by standard WGS. These results indicate that targeted, tumor-only analysis of long-read sequence data is a feasible approach for the genomic evaluation of myeloid cancers, and they show the utility of incorporating variants discovered via long-read sequencing to improve variant interpretation in short-read WGS.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1242-1254"},"PeriodicalIF":3.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145187385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation and Clinical Utility of a Pan-Cancer Circulating Tumor DNA Assay as a First-Approach Test","authors":"Nisha Kanwar ,&nbsp;Michael B. Campion ,&nbsp;Amber R. Schneider ,&nbsp;Dragana Milosevic ,&nbsp;Carlos Sosa ,&nbsp;Antonina A. Wojcik ,&nbsp;Kevin C. Halling ,&nbsp;Kandelaria M. Rumilla ,&nbsp;Ying-Chun Lo ,&nbsp;Zhiyv Niu ,&nbsp;Katherine B. Geiersbach ,&nbsp;Margaret A. DiGuardo ,&nbsp;Benjamin R. Kipp ,&nbsp;Gang Zheng","doi":"10.1016/j.jmoldx.2025.08.009","DOIUrl":"10.1016/j.jmoldx.2025.08.009","url":null,"abstract":"<div><div>The feasibility of circulating tumor (ct)-DNA assays in first-approach pan-cancer genomic profiling is not well established. Furthermore, low ctDNA levels limit assay sensitivity, which challenges adaptation to clinical genomic profiling. In this study, a 33-gene next-generation sequencing–based ctDNA panel was validated, and these issues were investigated using real-world clinical data. The cohorts included 123 patients who underwent first-approach ctDNA testing, and 48 patients for whom matched tissue was tested at the same time-point. The overall ctDNA assay failure rate was 0%. Insufficient tumor tissue was the main reason for liquid biopsy (69%). The most common primary cancer profiled was lung (39.0%), followed by colon (13.8%), bile duct (8.9%), pancreas (8.1%), and breast and prostate (each 4.1%). Tier I variants were detected in 33.3% of patients, and Tier I or II variants were detected in 65.0% (including 54.5% cholangiocarcinomas, in which tissue biopsy may be challenging due to anatomic location). Compared with matched tissue, ctDNA showed 76% sensitivity for Tier I variants. Actionable variants were increased by 14.3% with ctDNA versus tissue testing alone. ctDNA results preceded tissue results by an average of 21 days. High feasibility, actionability, and sensitivity support ctDNA assays as a potential first-line genomic test, especially in specific tumor types for advanced tumors with insufficient or unavailable tissue.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1213-1223"},"PeriodicalIF":3.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Disclosure Statement","authors":"","doi":"10.1016/S1525-1578(25)00259-4","DOIUrl":"10.1016/S1525-1578(25)00259-4","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages A1-A2"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145425957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Title Page","authors":"","doi":"10.1016/S1525-1578(25)00258-2","DOIUrl":"10.1016/S1525-1578(25)00258-2","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Page OFC"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145425956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analytical and Clinical Evaluation of the AltoStar Adenovirus PCR Kit 1.5 and the AltoStar Automation System AM16 for Adenovirus Detection in Plasma and Stool Samples","authors":"Krupa Jani ,&nbsp;Taheefa Stephen ,&nbsp;Cindy Lee ,&nbsp;Arianna Pinto ,&nbsp;Tracy McMillen ,&nbsp;June L. Chan ,&nbsp;N. Esther Babady","doi":"10.1016/j.jmoldx.2025.08.003","DOIUrl":"10.1016/j.jmoldx.2025.08.003","url":null,"abstract":"<div><div>Human adenoviruses (HAdVs) can result in significant morbidity and mortality in immunocompromised patients. The AltoStar Adenovirus PCR Kit 1.5 (AltoStar HAdV PCR) is a real-time PCR test for the detection and quantification of HAdV DNA. In this study, the performance of the AltoStar HAdV PCR was evaluated compared with a laboratory-developed HAdV PCR based on MultiCode HAdV reagents (MultiCode HAdV PCR) using plasma and stool specimens. Performance of the AltoStar HAdV PCR was established by determining the linearity, lower limit of detection, precision, specificity, accuracy, and inclusivity of the assay. Accuracy was determined by testing plasma and stool clinical samples previously tested by the MultiCode HAdV PCR and inclusivity evaluated by using several HAdV genotypes. A time-and-motion study was performed to compare the workflows of both PCRs. The limit of detection was &lt;200 copies/mL for both sample types. Overall agreement with the MultiCode HAdV PCR was 91% for plasma samples and 85.5% for stool samples. Quantitative agreement between the two tests was moderate. Specificity and precision in both sample types were high. All major HAdV species tested were detected. Hands-on-time was significantly less for the semi-automated AltoStar HAdV PCR. The AltoStar HAdV PCR showed great performance for the detection and/or quantitation of HAdV in clinical samples.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1115-1122"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144993690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Specificity Detection of UV Mutational Signatures in Circulating Tumor DNA for Diagnostic Classification of Cutaneous and Unknown Primary Tumors Using FoundationOne Liquid CDx","authors":"Douglas A. Mata ,&nbsp;Angela A. Kou ,&nbsp;Andreas M. Heilmann ,&nbsp;Julius Honecker ,&nbsp;Benjamin R. Kroger ,&nbsp;Thomas Wieland ,&nbsp;Erik A. Williams ,&nbsp;Soo-Ryum Yang ,&nbsp;Jamal K. Benhamida ,&nbsp;Chad Vanderbilt ,&nbsp;Chelsea Marcus ,&nbsp;Irene Shyu ,&nbsp;Caleb Ho ,&nbsp;Matthew C. Hiemenz ,&nbsp;Tyler Janovitz ,&nbsp;Ethan S. Sokol ,&nbsp;Zoe Fleischmann ,&nbsp;Dexter Jin ,&nbsp;Ryon P. Graf ,&nbsp;Richard A. Hickman ,&nbsp;Brennan Decker","doi":"10.1016/j.jmoldx.2025.07.004","DOIUrl":"10.1016/j.jmoldx.2025.07.004","url":null,"abstract":"<div><div>This study evaluated a UV mutational signature classifier applied to circulating tumor DNA (ctDNA) in cell-free DNA liquid biopsies, using the FoundationOne Liquid CDx platform. Among 73,790 samples, 12.9% (9527) met the variant threshold for signature analysis, with UV signatures detected in 3.8% (365) of cases. Of these, 51.5% (188) were initially submitted as cutaneous, 8.5% (31) as unspecified primary, and 40.0% (146) as extracutaneous tumors. The UV classifier demonstrated high specificity (99.7%) and variable sensitivity, reaching up to 37.5% and 67.6% in samples with elevated ctDNA levels and mutational loads, respectively. Molecular pathologist review confirmed that 74.0% (270/365) of UV calls were true positives and enabled resolution of false positives and ambiguous findings through integration of genomic and clinicopathologic features. This refined sensitivity estimates to 41.9% and 70.2% in cases with elevated ctDNA levels and mutational loads, respectively. Confirmed UV-positive tumors exhibited genomic alterations characteristic of sun-exposed skin cancers. In 175 matched liquid and tissue biopsy pairs, the positive percent agreement for UV signature detection was 38.7% overall, increasing to 87.5% and 90.6% in subsets with elevated ctDNA levels and mutational load, respectively. These results underline the utility of FoundationOne Liquid CDx, complemented by molecular pathologist oversight, in identifying cancers of cutaneous origin to refine diagnoses and guide treatment for patients with advanced cancers.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1039-1053"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two-Dimensional PCR for Identifying the HLA Alleles Associated with Adverse Drug Reactions","authors":"Xueting Zhu,&nbsp;Shuang Yao,&nbsp;Jun Zhang,&nbsp;Lili Pan,&nbsp;Guanghua Luo,&nbsp;Lu Zheng","doi":"10.1016/j.jmoldx.2025.07.007","DOIUrl":"10.1016/j.jmoldx.2025.07.007","url":null,"abstract":"<div><div>This study aimed to establish a two-dimensional PCR (2D-PCR) methodology capable of simultaneously identifying <em>HLA-A∗31:01</em>, <em>HLA-B∗15:02</em>, <em>HLA-B∗57:01</em>, and <em>HLA-B∗58:01</em> alleles to prevent adverse drug reactions and guide safe clinical medication use. To achieve this goal, key nucleotide sites were first selected through multiple sequence alignment for the purpose of identifying <em>HLA</em> alleles: c.243T and c.290T for <em>HLA-A∗31:01</em>; c.206C, c.256G, and c.285G for <em>HLA-B∗58:01</em>; c.362T and c.419C for <em>HLA-B∗57:01</em>; and c.1012 + 104T (rs144012689) for <em>HLA-B∗15:02</em>. Using universal tag sequences linked to the 5′ end of primers and base-quenched probes, high-throughput identification of multiple target genes was achieved through PCR amplification and melting curve analysis, followed by methodologic optimization and evaluation. Results indicated that the 2D-PCR method has a detection limit of approximately 10¹ copies and achieves high specificity and accuracy. The entire detection process can be completed in approximately 100 minutes, with a cost of less than $1 (USD). Furthermore, 2D-PCR overcomes the limitations of traditional fluorescent channels, providing technical support for the identification of multiple target genes. In conclusion, this study shows that 2D-PCR offers a convenient and rapid approach for human leukocyte antigen allele identification to prevent adverse drug reactions and holds potential for clinical application.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1084-1097"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Solving the :: Fusion Nomenclature Challenge for File and Directory Naming","authors":"Tom Bisson ,&nbsp;Stefan Kaluziak ,&nbsp;Norman Zerbe ,&nbsp;Jochen K. Lennerz","doi":"10.1016/j.jmoldx.2025.08.002","DOIUrl":"10.1016/j.jmoldx.2025.08.002","url":null,"abstract":"<div><div>Following recommendations from various consortia and professional societies, the double colon symbol (::) has become an integral part of gene fusion nomenclature (eg, <em>EML4::ALK</em>). Although widely adopted, its use presents technical challenges, as many common operating systems restrict the use of colons (:) in file and directory names. Consequently, the double colon (::) is often replaced with an underscore (_) or other allowed characters, introducing ambiguity and inconsistency. The first objective of this work is to raise awareness of this issue. The second objective is to propose a simple technical solution, that is, replacing the command-carrying symbol with a visually similar and functionally distinct ASCII character. The technical solution includes confirmation of functional compatibility and visual compliance with the established fusion nomenclature. The proposal also includes using Unicode characters to replace the slash (/) for alternative variants, the greater than symbol (&gt;) for substitutions, and the asterisk (∗) for nonsense variants, for example. To support consistency, keyboard shortcuts or custom scripts may be used to automate these substitutions. The straightforward solution presented in this paper counterbalances unintended technical consequences and thereby promotes harmonization towards a unified genomic variant nomenclature.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1070-1073"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144994119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From Expert Knowledge to Validation Resources","authors":"Somak Roy ,&nbsp;Martine W. Tremblay ,&nbsp;Edward Lockhart ,&nbsp;Swaroop Aradhya ,&nbsp;Pinar Bayrak-Toydemir ,&nbsp;Mark Bowser ,&nbsp;Jeana DaRe ,&nbsp;Kristin Gibson ,&nbsp;Michael Kennemer ,&nbsp;Christopher Krueger ,&nbsp;Matt Lebo ,&nbsp;Rong Mao ,&nbsp;Robert Nussbaum ,&nbsp;Brendan O'Fallon ,&nbsp;Andrew Rosato ,&nbsp;Lisa V. Kalman ,&nbsp;Birgit Funke","doi":"10.1016/j.jmoldx.2025.07.006","DOIUrl":"10.1016/j.jmoldx.2025.07.006","url":null,"abstract":"<div><div>Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant genes and variants, there is an urgent need for reference materials to optimize, validate, and quality control NGS tests. This pilot study documents the paucity of physical reference materials for widely tested genes and demonstrates the utility of <em>in silico</em> mutagenized reference materials to supplement physical samples when developing NGS tests. We examined published, expert curated lists of clinically relevant variants for these widely tested genes and found that publicly available reference materials were available for only 29.4%. We outline the steps for generating <em>in silico</em> resources and used 49 curated variants to conduct a blinded proof-of-concept study with three experienced NGS laboratories. One laboratory detected all added variants, and two detected all but one. This study revealed common scenarios that could lead to false-negative results when common pathogenic variants cannot be tested during analytical validation. This work highlights the need to establish centralized knowledge bases for common, pathogenic variants, demonstrates the utility of <em>in silico</em> reference materials, and provides guidance for generating <em>in silico</em> reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1074-1083"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Atypical Fluorescence in Situ Hybridization Findings by RNA Sequencing","authors":"Beth A. Pitel ,&nbsp;Saba Alvand ,&nbsp;Mark A. Montanari ,&nbsp;Daniel R. Sill ,&nbsp;Carlos Sosa ,&nbsp;Matthew J. Petersen ,&nbsp;Christopher D. Hofich ,&nbsp;Ganesh P. Pujari ,&nbsp;Reid G. Meyer ,&nbsp;Sounak Gupta ,&nbsp;William R. Sukov ,&nbsp;Jorge Torres-Mora ,&nbsp;Kevin C. Halling ,&nbsp;Katherine B. Geiersbach","doi":"10.1016/j.jmoldx.2025.07.008","DOIUrl":"10.1016/j.jmoldx.2025.07.008","url":null,"abstract":"<div><div>Fluorescence <em>in situ</em> hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: to summarize typical and atypical BAP FISH results on 56,584 formalin-fixed, paraffin-embedded solid tumors over approximately one decade of clinical testing; and to investigate the clinical utility of RNA sequencing (RNA-seq) for tumors with equivocal FISH results. Of 8586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA-seq was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46 of 113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37 of 62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4 of 44 (9.1%) tumors with other atypical signal patterns. This study provides a useful framework for categorizing atypical BAP FISH results and demonstrates the clinical utility of follow-up RNA-seq testing on tumors with equivocal FISH results.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1098-1114"},"PeriodicalIF":3.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144994166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}