Pub Date : 2025-09-23DOI: 10.1016/j.jmoldx.2025.08.005
Marzena Wojtaszewska , Monika Szelest , Marta Szarawarska , Jarosław Grzyb , Beata Blajer-Olszewska , Michał Gniot , Emilia Jaskuła , Jarosław Dybko , Michał Soin , Katarzyna Wasilewska , Sylwia Czekalska , Magdalena Zawada , Magdalena Wojtas , Iwona Solarska , Agnieszka Kwak , Piotr Wójcik , Ewelina Nowak-Ozimek , Artur Kowalik , Tomasz Stokłosa , Agnieszka Chudy , Mirosław Markiewicz
Somatic hypermutation (SHM) status of IGHV gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose between inefficient Sanger sequencing protocols and expensive, recently developed next-generation sequencing–based methods. The performance of both types of methods seemed incomparable, and concerted validation of different protocols between laboratories was inconsiderable. Here, a new tagmentation-based approach to sequencing of IGHV locus is presented, which is agnostic to the amplification protocol used and enables direct comparison of the amplicons and libraries dedicated to different platforms (Sanger, IonTorrent, and Illumina). To demonstrate its potential, the 12 associated molecular diagnostics laboratories were asked to amplify an artificially prepared oligoclonal DNA sample containing a near-equimolar mixture of IGHV clonotypes from six different classes. The PCR products collected from laboratories were then tagmented and sequenced according to the common TAG-CLL workflow. The productivity, degree of germline identity, and SHM status concordance between laboratories have been analyzed. Moreover, systematic biases toward uneven amplification of different clonotypes and the prevalence of accidental artifacts in vitro and in silico have been evaluated, providing a framework for future validation of IGHV SHM methods and next-generation sequencing immunoinformatic pipeline benchmarking.
{"title":"TAG-CLL, a Novel Tagmentation-Based Approach to Somatic Hypermutation Testing of IGHV Reveals the Weak Points of Both Sanger and Next-Generation Sequencing Methods","authors":"Marzena Wojtaszewska , Monika Szelest , Marta Szarawarska , Jarosław Grzyb , Beata Blajer-Olszewska , Michał Gniot , Emilia Jaskuła , Jarosław Dybko , Michał Soin , Katarzyna Wasilewska , Sylwia Czekalska , Magdalena Zawada , Magdalena Wojtas , Iwona Solarska , Agnieszka Kwak , Piotr Wójcik , Ewelina Nowak-Ozimek , Artur Kowalik , Tomasz Stokłosa , Agnieszka Chudy , Mirosław Markiewicz","doi":"10.1016/j.jmoldx.2025.08.005","DOIUrl":"10.1016/j.jmoldx.2025.08.005","url":null,"abstract":"<div><div>Somatic hypermutation (SHM) status of <em>IGHV</em> gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose between inefficient Sanger sequencing protocols and expensive, recently developed next-generation sequencing–based methods. The performance of both types of methods seemed incomparable, and concerted validation of different protocols between laboratories was inconsiderable. Here, a new tagmentation-based approach to sequencing of <em>IGHV</em> locus is presented, which is agnostic to the amplification protocol used and enables direct comparison of the amplicons and libraries dedicated to different platforms (Sanger, IonTorrent, and Illumina). To demonstrate its potential, the 12 associated molecular diagnostics laboratories were asked to amplify an artificially prepared oligoclonal DNA sample containing a near-equimolar mixture of <em>IGHV</em> clonotypes from six different classes. The PCR products collected from laboratories were then tagmented and sequenced according to the common TAG-CLL workflow. The productivity, degree of germline identity, and SHM status concordance between laboratories have been analyzed. Moreover, systematic biases toward uneven amplification of different clonotypes and the prevalence of accidental artifacts <em>in vitro</em> and <em>in silico</em> have been evaluated, providing a framework for future validation of <em>IGHV</em> SHM methods and next-generation sequencing immunoinformatic pipeline benchmarking.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1137-1153"},"PeriodicalIF":3.4,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145151680","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}
Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. The goal was to develop and optimize a sensitive and specific mass spectrometry–based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated, and their corresponding heavy synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the most common mass spectrometer used in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal patients and patients with β-thalassemia, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous, or compound heterozygous states. The intra-assay and interassay precision CV were both <20%. We envision that such mass spectrometry–based assays could be used as first-line screening assay for hemoglobin variants, including sickle cell disease as well as thalassemias.
{"title":"A Mass Spectrometry–Based Multiplexed Targeted Assay for Detection of Hemoglobinopathies from Dried Blood Spots","authors":"Anikha Bellad , Kannan Rangiah , Sandip Chavan , Jayesh Warade , Barnali Das , Akhilesh Pandey","doi":"10.1016/j.jmoldx.2025.07.003","DOIUrl":"10.1016/j.jmoldx.2025.07.003","url":null,"abstract":"<div><div>Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. The goal was to develop and optimize a sensitive and specific mass spectrometry–based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated, and their corresponding heavy synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the most common mass spectrometer used in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal patients and patients with β-thalassemia, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous, or compound heterozygous states. The intra-assay and interassay precision CV were both <20%. We envision that such mass spectrometry–based assays could be used as first-line screening assay for hemoglobin variants, including sickle cell disease as well as thalassemias.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 1003-1016"},"PeriodicalIF":3.4,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145121098","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 : 2025-09-03DOI: 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}
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