Pub Date : 2025-09-03DOI: 10.1016/j.jmoldx.2025.08.004
Fei Dong
{"title":"Validating Fluorescence in Situ Hybridization with RNA Sequencing","authors":"Fei Dong","doi":"10.1016/j.jmoldx.2025.08.004","DOIUrl":"10.1016/j.jmoldx.2025.08.004","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1037-1038"},"PeriodicalIF":3.4,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145006752","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}
Pub Date : 2025-09-01DOI: 10.1016/j.jmoldx.2025.08.003
Krupa Jani , Taheefa Stephen , Cindy Lee , Arianna Pinto , Tracy McMillen , June L. Chan , N. Esther Babady
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 <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.
{"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 , Taheefa Stephen , Cindy Lee , Arianna Pinto , Tracy McMillen , June L. Chan , 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 <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":"2025-09-01","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}
Pub Date : 2025-09-01DOI: 10.1016/j.jmoldx.2025.08.002
Tom Bisson , Stefan Kaluziak , Norman Zerbe , Jochen K. Lennerz
Following recommendations from various consortia and professional societies, the double colon symbol (::) has become an integral part of gene fusion nomenclature (eg, EML4::ALK). 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 (>) 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.
{"title":"Solving the :: Fusion Nomenclature Challenge for File and Directory Naming","authors":"Tom Bisson , Stefan Kaluziak , Norman Zerbe , 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 (>) 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":"2025-09-01","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}
Pub Date : 2025-09-01DOI: 10.1016/j.jmoldx.2025.07.008
Beth A. Pitel , Saba Alvand , Mark A. Montanari , Daniel R. Sill , Carlos Sosa , Matthew J. Petersen , Christopher D. Hofich , Ganesh P. Pujari , Reid G. Meyer , Sounak Gupta , William R. Sukov , Jorge Torres-Mora , Kevin C. Halling , Katherine B. Geiersbach
Fluorescence in situ 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.
{"title":"Evaluation of Atypical Fluorescence in Situ Hybridization Findings by RNA Sequencing","authors":"Beth A. Pitel , Saba Alvand , Mark A. Montanari , Daniel R. Sill , Carlos Sosa , Matthew J. Petersen , Christopher D. Hofich , Ganesh P. Pujari , Reid G. Meyer , Sounak Gupta , William R. Sukov , Jorge Torres-Mora , Kevin C. Halling , 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":"2025-09-01","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}
Pub Date : 2025-08-25DOI: 10.1016/j.jmoldx.2025.06.001
Rohan Gnanaolivu , Neiladri Saha , Noemi Vidal-Folch , Jiayu Tan , Feng Li , Shawn McClelland , Zhiyv Niu , Devin Oglesbee , Chen Wang
Short-read next-generation sequencing is widely used for clinical diagnosis but faces limitations in accurately detecting variants in complex genomic regions, such as segmental duplications, guanine-cytosine rich areas, and repeat sequences. These challenging regions comprise only 3% to 5% of the exome, yet their stochastic library preparation and bioinformatics analysis impacts variant detection reproducibility. Evaluating reproducibility is time-consuming, and variants in these regions require validation through sequencing replicates and using orthogonal methods like long-range PCR or Sanger sequencing, thereby increasing costs and turnaround times for clinical laboratories. To address these challenges, ClinRay was developed; it is a novel, generalizable bioinformatics method that uses the concept of digital twins to synthetically enhance the data distribution for variants in regions with suspected poor reproducibility. ClinRay predicts the reproducibility of detected variants by short-read next-generation sequencing probes in these difficult-to-sequence genomic regions. The model was developed using alignment data from the binary format of the sequence alignment/map files of eight replicates of the Genome in a Bottle HG002 Coriell cell and publicly available genomic context annotation resources. The model achieved an area under the receiver-operating characteristic curve of 0.89 (95% CI, 0.88–0.90) on the test data and 0.85 (95% CI, 0.84–0.86) on an independent validation data set.
{"title":"Clinical Assessment of Next-Generation Sequencing Probe Reproducibility in Short-Read Sequencing (ClinRay) Using Digital Twins","authors":"Rohan Gnanaolivu , Neiladri Saha , Noemi Vidal-Folch , Jiayu Tan , Feng Li , Shawn McClelland , Zhiyv Niu , Devin Oglesbee , Chen Wang","doi":"10.1016/j.jmoldx.2025.06.001","DOIUrl":"10.1016/j.jmoldx.2025.06.001","url":null,"abstract":"<div><div>Short-read next-generation sequencing is widely used for clinical diagnosis but faces limitations in accurately detecting variants in complex genomic regions, such as segmental duplications, guanine-cytosine rich areas, and repeat sequences. These challenging regions comprise only 3% to 5% of the exome, yet their stochastic library preparation and bioinformatics analysis impacts variant detection reproducibility. Evaluating reproducibility is time-consuming, and variants in these regions require validation through sequencing replicates and using orthogonal methods like long-range PCR or Sanger sequencing, thereby increasing costs and turnaround times for clinical laboratories. To address these challenges, ClinRay was developed; it is a novel, generalizable bioinformatics method that uses the concept of digital twins to synthetically enhance the data distribution for variants in regions with suspected poor reproducibility. ClinRay predicts the reproducibility of detected variants by short-read next-generation sequencing probes in these difficult-to-sequence genomic regions. The model was developed using alignment data from the binary format of the sequence alignment/map files of eight replicates of the Genome in a Bottle HG002 Coriell cell and publicly available genomic context annotation resources. The model achieved an area under the receiver-operating characteristic curve of 0.89 (95% CI, 0.88–0.90) on the test data and 0.85 (95% CI, 0.84–0.86) on an independent validation data set.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 9","pages":"Pages 899-912"},"PeriodicalIF":3.4,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144902425","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}
Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.004
Douglas A. Mata , Angela A. Kou , Andreas M. Heilmann , Julius Honecker , Benjamin R. Kroger , Thomas Wieland , Erik A. Williams , Soo-Ryum Yang , Jamal K. Benhamida , Chad Vanderbilt , Chelsea Marcus , Irene Shyu , Caleb Ho , Matthew C. Hiemenz , Tyler Janovitz , Ethan S. Sokol , Zoe Fleischmann , Dexter Jin , Ryon P. Graf , Richard A. Hickman , Brennan Decker
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.
{"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 , Angela A. Kou , Andreas M. Heilmann , Julius Honecker , Benjamin R. Kroger , Thomas Wieland , Erik A. Williams , Soo-Ryum Yang , Jamal K. Benhamida , Chad Vanderbilt , Chelsea Marcus , Irene Shyu , Caleb Ho , Matthew C. Hiemenz , Tyler Janovitz , Ethan S. Sokol , Zoe Fleischmann , Dexter Jin , Ryon P. Graf , Richard A. Hickman , 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":"2025-08-20","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}
Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.007
Xueting Zhu, Shuang Yao, Jun Zhang, Lili Pan, Guanghua Luo, Lu Zheng
This study aimed to establish a two-dimensional PCR (2D-PCR) methodology capable of simultaneously identifying HLA-A∗31:01, HLA-B∗15:02, HLA-B∗57:01, and HLA-B∗58:01 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 HLA alleles: c.243T and c.290T for HLA-A∗31:01; c.206C, c.256G, and c.285G for HLA-B∗58:01; c.362T and c.419C for HLA-B∗57:01; and c.1012 + 104T (rs144012689) for HLA-B∗15:02. 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.
{"title":"Two-Dimensional PCR for Identifying the HLA Alleles Associated with Adverse Drug Reactions","authors":"Xueting Zhu, Shuang Yao, Jun Zhang, Lili Pan, Guanghua Luo, 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 10<sup>1</sup> 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":"2025-08-20","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}
Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.006
Somak Roy , Martine W. Tremblay , Edward Lockhart , Swaroop Aradhya , Pinar Bayrak-Toydemir , Mark Bowser , Jeana DaRe , Kristin Gibson , Michael Kennemer , Christopher Krueger , Matt Lebo , Rong Mao , Robert Nussbaum , Brendan O'Fallon , Andrew Rosato , Lisa V. Kalman , Birgit Funke
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 in silico 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 in silico 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 in silico reference materials, and provides guidance for generating in silico reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.
{"title":"From Expert Knowledge to Validation Resources","authors":"Somak Roy , Martine W. Tremblay , Edward Lockhart , Swaroop Aradhya , Pinar Bayrak-Toydemir , Mark Bowser , Jeana DaRe , Kristin Gibson , Michael Kennemer , Christopher Krueger , Matt Lebo , Rong Mao , Robert Nussbaum , Brendan O'Fallon , Andrew Rosato , Lisa V. Kalman , 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":"2025-08-20","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}
Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.005
He Fang , Stephen M. Eacker , Yu Wu , Cate Paschal , Mary Wood , Brad Nelson , Alexander Muratov , Yajuan J. Liu
Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence in situ hybridization, and chromosome microarray analysis. These methods often lack the resolution and sensitivity needed for comprehensive characterization of chromosomal aberrations. To address these limitations, genomic proximity mapping (GPM), a genome-wide chromosome conformation capture (Hi-C) technology, was implemented in a clinical setting. Here, GPM was applied to a cohort of 123 individuals with constitutional disorders, achieving a 100% concordance in detecting 110 copy number variants (>25 kb) and 27 structural rearrangements, in addition to novel findings. GPM demonstrated unique advantages, such as resolving chromosomal rearrangements with precise (<5 kb) breakpoint resolution, maintaining robust performance with challenging samples, including formalin-fixed, paraffin-embedded tissues, and detecting mosaicism with high sensitivity. Furthermore, GPM reliably provided copy number and loss-of-heterozygosity profiles, streamlining workflows. GPM also uncovered 12 novel structural variants missed by standard methods, highlighting its superior detection capability. This analysis revealed that cases with more than two chromosomal rearrangements identified by traditional cytogenetics often harbor additional, cryptic rearrangements that remain undetected by standard-of-care methods. GPM represents a transformative tool for genomic diagnostics, offering a high-resolution, comprehensive approach to detecting genomic alterations. Its ability to address limitations of conventional cytogenetics methods positions GPM as a needed advance in the diagnosis, prognosis, and therapeutic management of genetic disorders.
{"title":"Evaluation of Genomic Proximity Mapping for Detecting Genomic and Chromosomal Structural Variants in Constitutional Disorders","authors":"He Fang , Stephen M. Eacker , Yu Wu , Cate Paschal , Mary Wood , Brad Nelson , Alexander Muratov , Yajuan J. Liu","doi":"10.1016/j.jmoldx.2025.07.005","DOIUrl":"10.1016/j.jmoldx.2025.07.005","url":null,"abstract":"<div><div>Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence <em>in situ</em> hybridization, and chromosome microarray analysis. These methods often lack the resolution and sensitivity needed for comprehensive characterization of chromosomal aberrations. To address these limitations, genomic proximity mapping (GPM), a genome-wide chromosome conformation capture (Hi-C) technology, was implemented in a clinical setting. Here, GPM was applied to a cohort of 123 individuals with constitutional disorders, achieving a 100% concordance in detecting 110 copy number variants (>25 kb) and 27 structural rearrangements, in addition to novel findings. GPM demonstrated unique advantages, such as resolving chromosomal rearrangements with precise (<5 kb) breakpoint resolution, maintaining robust performance with challenging samples, including formalin-fixed, paraffin-embedded tissues, and detecting mosaicism with high sensitivity. Furthermore, GPM reliably provided copy number and loss-of-heterozygosity profiles, streamlining workflows. GPM also uncovered 12 novel structural variants missed by standard methods, highlighting its superior detection capability. This analysis revealed that cases with more than two chromosomal rearrangements identified by traditional cytogenetics often harbor additional, cryptic rearrangements that remain undetected by standard-of-care methods. GPM represents a transformative tool for genomic diagnostics, offering a high-resolution, comprehensive approach to detecting genomic alterations. Its ability to address limitations of conventional cytogenetics methods positions GPM as a needed advance in the diagnosis, prognosis, and therapeutic management of genetic disorders.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1054-1069"},"PeriodicalIF":3.4,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976902","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}
Pub Date : 2025-08-07DOI: 10.1016/j.jmoldx.2025.07.002
Cassandra Michel , Alizée Simon , Andréa Witz , Idrissia Hanriot , Julie Dardare , Margaux Betz , Maxime Brun , Pauline Gilson , Jean Louis Merlin , Alexandre Harlé
Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. BRCA1 and RAD51C promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.
{"title":"A Novel Droplet Digital PCR Assay for BRCA1 and RAD51C Methylation","authors":"Cassandra Michel , Alizée Simon , Andréa Witz , Idrissia Hanriot , Julie Dardare , Margaux Betz , Maxime Brun , Pauline Gilson , Jean Louis Merlin , Alexandre Harlé","doi":"10.1016/j.jmoldx.2025.07.002","DOIUrl":"10.1016/j.jmoldx.2025.07.002","url":null,"abstract":"<div><div>Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious <em>BRCA1/2</em> mutation, these mutations are not the sole cause of HRD. Promoter methylation of <em>BRCA1</em> and <em>RAD51C</em> contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. <em>BRCA1</em> and <em>RAD51C</em> promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious <em>BRCA1</em>/2 mutation, and 2 (2.6%) a <em>RAD51C</em> mutation. Methylation analysis revealed <em>BRCA1</em> promoter methylation in 32 patients (41.6%) and <em>RAD51C</em> promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond <em>BRCA1</em>/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 1026-1036"},"PeriodicalIF":3.4,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812620","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号