Pub Date : 2025-11-19DOI: 10.1016/j.jmoldx.2025.10.009
Shuyuan Li , Bailing Liu , Jingfan Zhang , Ning Tang , Renyi Hua , Jinling Yang , Xingling Huang , Haoxian Li , Aiping Mao , Libao Chen , Jiwei Huang , Yanlin Wang
Conventional methods for spinal muscular atrophy (SMA) screening have been challenging in detecting SMN1/2 single-nucleotide variants (SNVs) and small insertions and deletions, SMN1 2 + 0 silent carrier, and the copy number (CN) of SMN2. To address these limitations, a long-read sequencing (LRS)-based approach termed comprehensive analysis of SMA 2 (CASMA2) was developed. CASMA2 was used to perform CN analysis by integrating Poisson distribution with an endogenous reference gene, the first such method developed for LRS platforms. The performance and clinical feasibility of CASMA2 were evaluated by using 414 retrospective peripheral blood samples and 303 prospective dried blood spot samples. CASMA2 displayed 100% accuracy in SMN1/2 CN analysis and identified the SNVs/insertions and deletions in SMN1/2. CASMA2 also showed the capability of screening for the SMN1 2 + 0 silent carrier with family-trio haplotype analysis. It achieved a 99.0% (410 of 414) first-attempt success rate for long-term peripheral blood samples and a 98.7% (299 of 303) rate for dried blood spot samples. CASMA2 offers a clinically feasible, precise, and efficient method for SMA carrier and newborn screening.
{"title":"A More Clinically Effective Long-Read Sequencing–Based Approach for Comprehensive Analysis of Spinal Muscular Atrophy","authors":"Shuyuan Li , Bailing Liu , Jingfan Zhang , Ning Tang , Renyi Hua , Jinling Yang , Xingling Huang , Haoxian Li , Aiping Mao , Libao Chen , Jiwei Huang , Yanlin Wang","doi":"10.1016/j.jmoldx.2025.10.009","DOIUrl":"10.1016/j.jmoldx.2025.10.009","url":null,"abstract":"<div><div>Conventional methods for spinal muscular atrophy (SMA) screening have been challenging in detecting <em>SMN1/2</em> single-nucleotide variants (SNVs) and small insertions and deletions, <em>SMN1</em> 2 + 0 silent carrier, and the copy number (CN) of <em>SMN2</em>. To address these limitations, a long-read sequencing (LRS)-based approach termed comprehensive analysis of SMA 2 (CASMA2) was developed. CASMA2 was used to perform CN analysis by integrating Poisson distribution with an endogenous reference gene, the first such method developed for LRS platforms. The performance and clinical feasibility of CASMA2 were evaluated by using 414 retrospective peripheral blood samples and 303 prospective dried blood spot samples. CASMA2 displayed 100% accuracy in <em>SMN1</em>/<em>2</em> CN analysis and identified the SNVs/insertions and deletions in <em>SMN1/2</em>. CASMA2 also showed the capability of screening for the <em>SMN1</em> 2 + 0 silent carrier with family-trio haplotype analysis. It achieved a 99.0% (410 of 414) first-attempt success rate for long-term peripheral blood samples and a 98.7% (299 of 303) rate for dried blood spot samples. CASMA2 offers a clinically feasible, precise, and efficient method for SMA carrier and newborn screening.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 2","pages":"Pages 136-146"},"PeriodicalIF":3.4,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145566110","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 : 2025-11-19DOI: 10.1016/j.jmoldx.2025.10.007
Huayue Yang , Dan Long , Hang Lei , Liujun Guo , Chengyan Gao , Haoyu Hao , Jiaming Li , Xuefeng Wang , Xi Wu , Jing Dai , Can Lou , Xiaohong Cai
Splice-site variants within the ABO gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized how four intron 6 splice-site variants—three in the A1 allele (c.374+4A>T, c.374+4A>G, and c.374+5G>A) and one in the ABO∗B.01 allele (c.374+2_374+3insT)—affect ABO pre-mRNA splicing. A combination of serologic, molecular genetic, bioinformatic, and minigene assays established a genotype-splicing-phenotype association model. Bioinformatics predictions showed that all variants impaired the recognition of the 5′ splice site. Specifically, the A allele variant c.374+5G>A induced approximately 2.8% exon 6 retention, whereas the c.374+4A>G (Afinn/Aweak) and c.374+4A>T (Aweak) variants preserved approximately 6.9% and 10.2% functional transcripts, respectively. The novel B allele insertion variant c.374+2_374+3insT (Bel subtype) retained approximately 4.7% exon 6–containing transcripts, sufficient for trace B glycosyltransferase expression. Quantitative real-time PCR analysis confirmed that the transcriptional levels of the ABO gene in the ABel subtype individual carrying the c.374+2_374+3insT variant were approximately 51.7% of those in the ABO∗A1.02/ABO∗B.01 control. This study demonstrated that splice-site variants in the ABO gene reduce the abundance of functional transcripts via altered transcriptional regulation, which, in turn, leads to decreased ABO glycosyltransferase expression, ultimately resulting in weak antigen phenotypes of varying intensity.
{"title":"Functional Analysis of Four Splice-Site Variants, including a Novel Variant, on Antigen Expression and ABO Subgroup Formation","authors":"Huayue Yang , Dan Long , Hang Lei , Liujun Guo , Chengyan Gao , Haoyu Hao , Jiaming Li , Xuefeng Wang , Xi Wu , Jing Dai , Can Lou , Xiaohong Cai","doi":"10.1016/j.jmoldx.2025.10.007","DOIUrl":"10.1016/j.jmoldx.2025.10.007","url":null,"abstract":"<div><div>Splice-site variants within the <em>ABO</em> gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized how four intron 6 splice-site variants—three in the <em>A1</em> allele (c.374+4A>T, c.374+4A>G, and c.374+5G>A) and one in the <em>ABO∗B.01</em> allele (c.374+2_374+3insT)—affect <em>ABO</em> pre-mRNA splicing. A combination of serologic, molecular genetic, bioinformatic, and minigene assays established a genotype-splicing-phenotype association model. Bioinformatics predictions showed that all variants impaired the recognition of the 5′ splice site. Specifically, the <em>A</em> allele variant c.374+5G>A induced approximately 2.8% exon 6 retention, whereas the c.374+4A>G (A<sub>finn</sub>/A<sub>weak</sub>) and c.374+4A>T (A<sub>weak</sub>) variants preserved approximately 6.9% and 10.2% functional transcripts, respectively. The novel <em>B</em> allele insertion variant c.374+2_374+3insT (B<sub>el</sub> subtype) retained approximately 4.7% exon 6–containing transcripts, sufficient for trace B glycosyltransferase expression. Quantitative real-time PCR analysis confirmed that the transcriptional levels of the <em>ABO</em> gene in the AB<sub>el</sub> subtype individual carrying the c.374+2_374+3insT variant were approximately 51.7% of those in the <em>ABO∗A1.02/ABO∗B.01</em> control. This study demonstrated that splice-site variants in the <em>ABO</em> gene reduce the abundance of functional transcripts via altered transcriptional regulation, which, in turn, leads to decreased ABO glycosyltransferase expression, ultimately resulting in weak antigen phenotypes of varying intensity.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 2","pages":"Pages 170-178"},"PeriodicalIF":3.4,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145566107","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 : 2025-10-30DOI: 10.1016/j.jmoldx.2025.10.006
Yan Xu , Bin Hu , Xu Han , Yiyao Chen , Jingqi Lin , Yanlin Wang , Jian Wang , Niu Li , Shuyuan Li
Despite the availability of various in silico prediction tools, accurately assessing the pathogenicity of splice-region variants remains limited. In this study, 18 splice-region variants of uncertain significance (VUSs) were functionally characterized using either in vitro (minigene) or in vivo (RT-PCR) experiments. The impacts of in-frame mutants on the indicated protein were further analyzed by modeling the three-dimensional protein structures. The accuracy of different in silico prediction tools (SpliceAI, varSEAK, and Splicing Prediction Pipeline) was also compared. The 18 VUSs included 3 canonical and 15 noncanonical splicing variants. Splicing abnormalities were observed in 16 variants (88.9%), with exon skipping (33.3%, 6/18) being the most prevalent event. Among them, 11 variants were predicted to generate premature termination codons, 3 caused in-frame alterations, and 2 resulted in both outcomes. Structural modeling revealed significant disruption of protein secondary structure in four of five in-frame alterations. Integrating functional and structural evidence, 15 VUSs (83.3%) were reclassified as pathogenic/likely pathogenic, enabling definitive diagnoses and informed reproductive decisions for affected families. Among the in silico tools, SpliceAI demonstrated the highest accuracy in predicting splicing anomalies; however, all tools showed decreased accuracy in analyzing specific splicing patterns. This study establishes an integrated workflow for assessing the pathogenicity of splicing VUSs in clinical diagnostics, offering a practical approach to overcome this critical diagnostic challenge.
{"title":"Enhanced Comprehension of the Pathogenicity of Splicing Variants","authors":"Yan Xu , Bin Hu , Xu Han , Yiyao Chen , Jingqi Lin , Yanlin Wang , Jian Wang , Niu Li , Shuyuan Li","doi":"10.1016/j.jmoldx.2025.10.006","DOIUrl":"10.1016/j.jmoldx.2025.10.006","url":null,"abstract":"<div><div>Despite the availability of various <em>in silico</em> prediction tools, accurately assessing the pathogenicity of splice-region variants remains limited. In this study, 18 splice-region variants of uncertain significance (VUSs) were functionally characterized using either <em>in vitro</em> (minigene) or <em>in vivo</em> (RT-PCR) experiments. The impacts of in-frame mutants on the indicated protein were further analyzed by modeling the three-dimensional protein structures. The accuracy of different <em>in silico</em> prediction tools (SpliceAI, varSEAK, and Splicing Prediction Pipeline) was also compared. The 18 VUSs included 3 canonical and 15 noncanonical splicing variants. Splicing abnormalities were observed in 16 variants (88.9%), with exon skipping (33.3%, 6/18) being the most prevalent event. Among them, 11 variants were predicted to generate premature termination codons, 3 caused in-frame alterations, and 2 resulted in both outcomes. Structural modeling revealed significant disruption of protein secondary structure in four of five in-frame alterations. Integrating functional and structural evidence, 15 VUSs (83.3%) were reclassified as pathogenic/likely pathogenic, enabling definitive diagnoses and informed reproductive decisions for affected families. Among the <em>in silico</em> tools, SpliceAI demonstrated the highest accuracy in predicting splicing anomalies; however, all tools showed decreased accuracy in analyzing specific splicing patterns. This study establishes an integrated workflow for assessing the pathogenicity of splicing VUSs in clinical diagnostics, offering a practical approach to overcome this critical diagnostic challenge.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 75-84"},"PeriodicalIF":3.4,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426677","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 : 2025-10-30DOI: 10.1016/j.jmoldx.2025.09.010
Jinyong Kim , Shilpa Venkataraman , Sandip N. Chavan , Ramesh Bokka , Julie I. Tange , Rachel R. Leger , Rajiv K. Pruthi , Dong Chen , Jansen N. Seheult , Akhilesh Pandey
Factor XIII (FXIII) is a heterotetramer that plays a crucial role in the coagulation cascade, facilitating fibrin crosslinking to stabilize clots. Congenital or acquired deficiency of the FXIII A or B subunits can lead to prolonged bleeding. Existing enzyme-linked immunosorbent assay–based and latex agglutination assays for measuring FXIII are limited to detecting only the FXIIIA subunit of the complex. In this study, a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay was developed and validated to accurately quantify both FXIIIA and FXIIIB subunits in plasma. Peptides measured by this targeted assay were rigorously selected based on analytical robustness, with the method demonstrating excellent sensitivity (limit of detection, 1.56 to 3.67 mU/mL), linearity (r2 > 0.998), and precision (CV, <10%). Comparison with the conventional assay based on ammonia release demonstrated a strong correlation (r = 0.983) and agreement (Cohen κ = 0.846) for FXIIIA. On analysis of 98 clinical plasma samples, the assay accurately identified cases with FXIII deficiency based on significantly reduced FXIIIA with varying alterations in FXIIIB levels. These results establish the clinical utility of the LC-MS/MS assay, highlight its potential for improving diagnostic accuracy for FXIII deficiency, and lay the foundation for future research on FXIII dynamics in pathologic states.
{"title":"A Targeted LC-MS/MS–Based Quantitative Assay for Detecting Plasma Factor XIII A/B Subunit Deficiency","authors":"Jinyong Kim , Shilpa Venkataraman , Sandip N. Chavan , Ramesh Bokka , Julie I. Tange , Rachel R. Leger , Rajiv K. Pruthi , Dong Chen , Jansen N. Seheult , Akhilesh Pandey","doi":"10.1016/j.jmoldx.2025.09.010","DOIUrl":"10.1016/j.jmoldx.2025.09.010","url":null,"abstract":"<div><div>Factor XIII (FXIII) is a heterotetramer that plays a crucial role in the coagulation cascade, facilitating fibrin crosslinking to stabilize clots. Congenital or acquired deficiency of the FXIII A or B subunits can lead to prolonged bleeding. Existing enzyme-linked immunosorbent assay–based and latex agglutination assays for measuring FXIII are limited to detecting only the FXIIIA subunit of the complex. In this study, a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay was developed and validated to accurately quantify both FXIIIA and FXIIIB subunits in plasma. Peptides measured by this targeted assay were rigorously selected based on analytical robustness, with the method demonstrating excellent sensitivity (limit of detection, 1.56 to 3.67 mU/mL), linearity (<em>r</em><sup>2</sup> > 0.998), and precision (CV, <10%). Comparison with the conventional assay based on ammonia release demonstrated a strong correlation (<em>r</em> = 0.983) and agreement (Cohen κ = 0.846) for FXIIIA. On analysis of 98 clinical plasma samples, the assay accurately identified cases with FXIII deficiency based on significantly reduced FXIIIA with varying alterations in FXIIIB levels. These results establish the clinical utility of the LC-MS/MS assay, highlight its potential for improving diagnostic accuracy for FXIII deficiency, and lay the foundation for future research on FXIII dynamics in pathologic states.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 64-74"},"PeriodicalIF":3.4,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426607","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}
Fragile X syndrome (FXS) is the most common cause of intellectual disability. It is usually caused by the expansion of the CGG trinucleotide repeat (>200 repeats) in FMR1, resulting in DNA hypermethylation and gene silencing. Conventional FXS diagnosis is based on a combination of PCR and Southern blot (SB) analysis, which is technically challenging and labor-intensive. Methylation-specific triplet-primed PCR (msTP-PCR) has been proposed to overcome these limitations in detecting FMR1 expansion and methylation status. However, its performance in FXS diagnosis in clinical laboratory settings has not been extensively studied. An msTP-PCR assay was validated, implemented, and compared versus the conventional diagnostic protocol in clinical samples. A total of 420 clinical samples (339 male subjects and 81 female subjects) previously genotyped for FMR1 CGG repeat expansion using reference methods (PCR-based methods and/or SB analysis) were investigated using msTP-PCR. The results showed high concordance with the reference results for allele categorization, repeat number correlation, and methylation status. However, discordant results were observed in rare cases of female patients with complex mosaic normal, premutation, and full mutation alleles, which need further confirmation via SB analysis. Nonetheless, these results confirm that the msTP-PCR method is a useful alternative technique for FXS diagnosis and prenatal testing, as it is rapid, reliable, cost-effective, and can potentially reduce the requirement for SB analysis.
{"title":"Clinical Utility and Performance of Methylation-Specific Triplet-Primed PCR for Fragile X Syndrome Diagnosis","authors":"Areerat Hnoonual , Wipawan Arunthong , Oradawan Plong-on , Pornsiri Sangmanee , Pornprot Limprasert","doi":"10.1016/j.jmoldx.2025.10.001","DOIUrl":"10.1016/j.jmoldx.2025.10.001","url":null,"abstract":"<div><div>Fragile X syndrome (FXS) is the most common cause of intellectual disability. It is usually caused by the expansion of the CGG trinucleotide repeat (>200 repeats) in <em>FMR1</em>, resulting in DNA hypermethylation and gene silencing. Conventional FXS diagnosis is based on a combination of PCR and Southern blot (SB) analysis, which is technically challenging and labor-intensive. Methylation-specific triplet-primed PCR (msTP-PCR) has been proposed to overcome these limitations in detecting <em>FMR1</em> expansion and methylation status. However, its performance in FXS diagnosis in clinical laboratory settings has not been extensively studied. An msTP-PCR assay was validated, implemented, and compared versus the conventional diagnostic protocol in clinical samples. A total of 420 clinical samples (339 male subjects and 81 female subjects) previously genotyped for <em>FMR1</em> CGG repeat expansion using reference methods (PCR-based methods and/or SB analysis) were investigated using msTP-PCR. The results showed high concordance with the reference results for allele categorization, repeat number correlation, and methylation status. However, discordant results were observed in rare cases of female patients with complex mosaic normal, premutation, and full mutation alleles, which need further confirmation via SB analysis. Nonetheless, these results confirm that the msTP-PCR method is a useful alternative technique for FXS diagnosis and prenatal testing, as it is rapid, reliable, cost-effective, and can potentially reduce the requirement for SB analysis.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 112-123"},"PeriodicalIF":3.4,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145410638","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 : 2025-10-28DOI: 10.1016/j.jmoldx.2025.10.003
Elizabeth Thacker , Lanie Happ , Magdalena Czader , Lin Wang , Christopher Giauque , Jennifer Shingleton , Cassandra Love , Clay Parker , Devang Thakkar , Matt Sperling , Kemin Xu , Kenneth Ofori , Andrea Vrydaghs , Jen Loy-Flynn , Robert Erno , Callie Barker , Jordan Bouldin , Eric D. Hsi , Sandeep S. Dave
Next-generation sequencing (NGS) has become integral to the clinical workup of blood cancers. However, there remain barriers to implementing clinical NGS, including the separate workflows required for DNA and RNA sequencing, complex bioinformatics analyses, and long turnaround times. Duoseq has been developed as a comprehensive, kitted solution to these barriers and implemented as a genomic profiling tool for blood cancers to simultaneously detect single-nucleotide variants (SNVs), small insertions/deletions (indels), and structural variants (SVs; translocations and fusions). Additionally, Duoseq can evaluate numerous other blood cancer diagnostic markers, including gene expression, copy number alterations, cell of origin, oncogenic viral status (eg, Epstein-Barr virus), B-/T-cell receptor clonality, Ig heavy chain mutational status, and diffuse large B-cell lymphoma subtypes. Analytical and clinical validation of Duoseq was performed in parallel at two clinical institutions. Limit of detection was confirmed as 5% variant allele frequency for SNVs, 10% variant allele frequency for indels, and ≥20% tumor purity for SVs. Repeatability studies showed >99% intrarun and interrun positive predictive value and >96% percentage positive agreement. Duoseq was run on formalin-fixed, paraffin-embedded biopsy specimens (N = 197), using NGS and/or fluorescence in situ hybridization as orthogonal comparison. SNVs, indels, and SVs achieved accuracy of >95%. These results establish Duoseq as a genomic profiling tool for blood cancers that can enable laboratories to provide critical diagnostic information in a time- and cost-effective manner.
{"title":"Clinical Validation of Duoseq, a Novel Assay for Clinical DNA and RNA Sequencing","authors":"Elizabeth Thacker , Lanie Happ , Magdalena Czader , Lin Wang , Christopher Giauque , Jennifer Shingleton , Cassandra Love , Clay Parker , Devang Thakkar , Matt Sperling , Kemin Xu , Kenneth Ofori , Andrea Vrydaghs , Jen Loy-Flynn , Robert Erno , Callie Barker , Jordan Bouldin , Eric D. Hsi , Sandeep S. Dave","doi":"10.1016/j.jmoldx.2025.10.003","DOIUrl":"10.1016/j.jmoldx.2025.10.003","url":null,"abstract":"<div><div>Next-generation sequencing (NGS) has become integral to the clinical workup of blood cancers. However, there remain barriers to implementing clinical NGS, including the separate workflows required for DNA and RNA sequencing, complex bioinformatics analyses, and long turnaround times. Duoseq has been developed as a comprehensive, kitted solution to these barriers and implemented as a genomic profiling tool for blood cancers to simultaneously detect single-nucleotide variants (SNVs), small insertions/deletions (indels), and structural variants (SVs; translocations and fusions). Additionally, Duoseq can evaluate numerous other blood cancer diagnostic markers, including gene expression, copy number alterations, cell of origin, oncogenic viral status (eg, Epstein-Barr virus), B-/T-cell receptor clonality, Ig heavy chain mutational status, and diffuse large B-cell lymphoma subtypes. Analytical and clinical validation of Duoseq was performed in parallel at two clinical institutions. Limit of detection was confirmed as 5% variant allele frequency for SNVs, 10% variant allele frequency for indels, and ≥20% tumor purity for SVs. Repeatability studies showed >99% intrarun and interrun positive predictive value and >96% percentage positive agreement. Duoseq was run on formalin-fixed, paraffin-embedded biopsy specimens (<em>N</em> = 197), using NGS and/or fluorescence <em>in situ</em> hybridization as orthogonal comparison. SNVs, indels, and SVs achieved accuracy of >95%. These results establish Duoseq as a genomic profiling tool for blood cancers that can enable laboratories to provide critical diagnostic information in a time- and cost-effective manner.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 85-100"},"PeriodicalIF":3.4,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145410798","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 : 2025-10-28DOI: 10.1016/j.jmoldx.2025.10.004
Yaji Xu , Usha Singh , Katherine Bell , Lesley Farrington , Karen Urtishak , Michael Gormley , Won Kim , Jenny Zhang , Kristy Potts , Songbai Wang
This retrospective study was designed to demonstrate the clinical utility of two diagnostic tests, the FoundationOneCDx (F1CDx) and Exact Sciences Resolution homologous recombination deficiency (HRD; Resolution HRD assay) as clinical trial–enrollment assays to identify patients with metastatic castration-resistant prostate cancer harboring homologous recombination repair (HRR) gene alterations. Tumor tissue and plasma collected from patients with metastatic castration-resistant prostate cancer in the phase 3 MAGNITUDE study were tested using the F1CDx tissue assay and/or the Resolution HRD plasma assay. Patients with HRR alterations were randomized (1:1) to receive niraparib (NIRA) and abiraterone acetate (AA) + prednisone (NIRA + AAP) or placebo and AAP (PBO + AAP; NCT03748641). Efficacy was based on radiographic progression-free survival (primary end point), time tosymptomatic progression, time to cytotoxic chemotherapy, and overall survival as secondary end points (interim analysis 1). Of 423 HRR patients, 291 (68.8%) were HRR positive (HRR+) by F1CDx [BRCA+: 162 (38.2%)], and 38 (8.9%) were HRR negative by F1CDx but HRR+ by Resolution HRD assay. Also, 277 of 423 (65.5%) were HRR+ by Resolution HRD assay [BRCA+: 150 (35.5%)], and 124 (29.3%) were HRR negative by Resolution HRD assay but HRR+ by F1CDx assay. Clinically meaningful benefits for all end points were comparable for BRCA+ and HRR+ patients detected by either tissue or plasma assays. These results demonstrated the clinical utility of both tissue and plasma assays in identifying patients for NIRA + AAP treatment.
{"title":"Clinical Performance of Tissue- and Plasma-Based Diagnostic Assays in Identifying Homologous Recombination Repair Gene Alterations in Patients with Metastatic Castration-Resistant Prostate Cancer following Treatment with Niraparib with Abiraterone Acetate Plus Prednisone (Niraparib + AAP)","authors":"Yaji Xu , Usha Singh , Katherine Bell , Lesley Farrington , Karen Urtishak , Michael Gormley , Won Kim , Jenny Zhang , Kristy Potts , Songbai Wang","doi":"10.1016/j.jmoldx.2025.10.004","DOIUrl":"10.1016/j.jmoldx.2025.10.004","url":null,"abstract":"<div><div>This retrospective study was designed to demonstrate the clinical utility of two diagnostic tests, the FoundationOneCDx (F1CDx) and Exact Sciences Resolution homologous recombination deficiency (HRD; Resolution HRD assay) as clinical trial–enrollment assays to identify patients with metastatic castration-resistant prostate cancer harboring homologous recombination repair (HRR) gene alterations. Tumor tissue and plasma collected from patients with metastatic castration-resistant prostate cancer in the phase 3 MAGNITUDE study were tested using the F1CDx tissue assay and/or the Resolution HRD plasma assay. Patients with HRR alterations were randomized (1:1) to receive niraparib (NIRA) and abiraterone acetate (AA) + prednisone (NIRA + AAP) or placebo and AAP (PBO + AAP; NCT03748641). Efficacy was based on radiographic progression-free survival (primary end point), time tosymptomatic progression, time to cytotoxic chemotherapy, and overall survival as secondary end points (interim analysis 1). Of 423 HRR patients, 291 (68.8%) were HRR positive (HRR<sup>+</sup>) by F1CDx [<em>BRCA</em><sup><em>+</em></sup>: 162 (38.2%)], and 38 (8.9%) were HRR negative by F1CDx but HRR<sup>+</sup> by Resolution HRD assay. Also, 277 of 423 (65.5%) were HRR<sup>+</sup> by Resolution HRD assay [<em>BRCA</em><sup>+</sup>: 150 (35.5%)], and 124 (29.3%) were HRR negative by Resolution HRD assay but HRR<sup>+</sup> by F1CDx assay. Clinically meaningful benefits for all end points were comparable for <em>BRCA</em><sup><em>+</em></sup> and HRR<sup>+</sup> patients detected by either tissue or plasma assays. These results demonstrated the clinical utility of both tissue and plasma assays in identifying patients for NIRA + AAP treatment.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 101-111"},"PeriodicalIF":3.4,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145410655","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 : 2025-10-28DOI: 10.1016/j.jmoldx.2025.10.002
Liyona Kampel , Shlomo Tsuriel , Leonor L. Trejo , Yaniv Hadi , Gilad Horowitz , Anton Warshavsky , Dov Hershkovitz , Nidal Muhanna
Liquid biopsy offers a promising noninvasive alternative for tissue sampling in solid cancers. Saliva, an easily accessible biofluid, can harbor tumor DNA, yet its clinical utility compared with plasma circulating tumor DNA (ctDNA) in head and neck squamous cell carcinoma (HNSCC) remains uncertain. Tumor samples from patients with HNSCC underwent next-generation sequencing to identify TP53 mutations. Matched plasma and saliva cell-free DNA (cfDNA) samples were analyzed for the presence of tumor-specific mutations. Pathologic features and survival data were evaluated in relation to mutation detectability in biofluids. Overall, TP53 mutations were detected in 64 of 85 tumors (75%). Liquid biopsy analysis included 36 plasma and 21 saliva samples from 40 patients. Plasma ctDNA was detected in 68% of oral cavity squamous cell carcinoma compared with 29% of laryngeal cancer cases, and it was associated with nodal metastases (P = 0.034). Detection of ctDNA showed a trend toward worse progression-free survival (37.4 versus 68.5 months; P = 0.134). Tumor mutation was identified in saliva cfDNA in 57% of cases, irrespective of disease stage or presence of regional metastases. Plasma ctDNA emerged as a potential prognostic marker in HNSCC, whereas mutated saliva cfDNA, although frequently detectable, lacked prognostic value or correlation with adverse pathologic features. Further research is warranted to elucidate the mechanisms governing tumor DNA shedding into saliva and to define its clinical applications.
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