Pub Date : 2025-10-21DOI: 10.1016/j.jmoldx.2025.09.009
Krupa Jani , Tracy McMillen , Cindy Lee , Edwin Miranda , Stephanie Rizzo , Anoshe Aslam , Tania Bubb , Mini Kamboj , N. Esther Babady
Candida auris is a high-priority multidrug-resistant yeast. In New York, C. auris is reportable, and surveillance screening is recommended for high-risk patients. This study provides a retrospective review of the yield and characteristics of C. auris cases detected through active surveillance testing at a tertiary cancer care center. Testing for C. auris was performed using a laboratory-developed real-time PCR test on patients admitted to the intensive care unit. Sample sources included axilla, groin, and nares swabs. Candida auris–positive PCR samples were further tested by culture with recovered isolates identified and further characterized by whole-genome sequencing and antifungal susceptibility testing. From 2019 to 2023, 27,299 samples were tested, with 139 positive samples (0.5%) on 16 unique patients. Positive swabs included 40 of 139 (28.7%) axilla, 44 of 139 (31.7%) nares, and 55 of 139 (39.5%) groin. A total of 134 of 139 (96.4%) samples were cultured, and 76 of 134 (56.7%) were positive in culture. An increase in positive swabs was noted. Four patients developed disseminated infections following a positive surveillance swab. Whole-genome sequencing classified all isolates as clade I, except for one isolate identified as clade III. Resistance to fluconazole was detected in 80% of isolates. Although the positivity rate remained low in this patient population, the recent increase in cases of C. auris nationwide underlies the need for active surveillance to prevent spread of this multidrug-resistant organism.
{"title":"Yield of an Active Candida auris Molecular Surveillance Program at a Tertiary Care Cancer Center","authors":"Krupa Jani , Tracy McMillen , Cindy Lee , Edwin Miranda , Stephanie Rizzo , Anoshe Aslam , Tania Bubb , Mini Kamboj , N. Esther Babady","doi":"10.1016/j.jmoldx.2025.09.009","DOIUrl":"10.1016/j.jmoldx.2025.09.009","url":null,"abstract":"<div><div><em>Candida auris</em> is a high-priority multidrug-resistant yeast. In New York, <em>C. auris</em> is reportable, and surveillance screening is recommended for high-risk patients. This study provides a retrospective review of the yield and characteristics of <em>C. auris</em> cases detected through active surveillance testing at a tertiary cancer care center. Testing for <em>C. auris</em> was performed using a laboratory-developed real-time PCR test on patients admitted to the intensive care unit. Sample sources included axilla, groin, and nares swabs. <em>Candida auris–</em>positive PCR samples were further tested by culture with recovered isolates identified and further characterized by whole-genome sequencing and antifungal susceptibility testing. From 2019 to 2023, 27,299 samples were tested, with 139 positive samples (0.5%) on 16 unique patients. Positive swabs included 40 of 139 (28.7%) axilla, 44 of 139 (31.7%) nares, and 55 of 139 (39.5%) groin. A total of 134 of 139 (96.4%) samples were cultured, and 76 of 134 (56.7%) were positive in culture. An increase in positive swabs was noted. Four patients developed disseminated infections following a positive surveillance swab. Whole-genome sequencing classified all isolates as clade I, except for one isolate identified as clade III. Resistance to fluconazole was detected in 80% of isolates. Although the positivity rate remained low in this patient population, the recent increase in cases of <em>C. auris</em> nationwide underlies the need for active surveillance to prevent spread of this multidrug-resistant organism.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1224-1231"},"PeriodicalIF":3.4,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145356661","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 : 2025-10-17DOI: 10.1016/j.jmoldx.2025.09.006
Simona Adamusová , Anttoni Korkiakoski , Nea Laine , Anna Musku , Tuula Rantasalo , Jorma Kim , Juuso Blomster , Jukka Laine , Tatu Hirvonen , Juha-Pekka Pursiheimo , Manu Tamminen
Liquid biopsies quantifying mutations in circulating tumor DNA by targeted next-generation sequencing have been gaining popularity. They are performed by various library preparation methods, each with distinct advantages and limitations. This work introduces Bridge Capture, a novel technology that goes beyond the advantages of market-leading liquid biopsy technologies, eliminating the need to compromise between scalability, cost-efficiency, sensitivity, or panel size. Twenty-four matched contrived colorectal biospecimens mimicking circulating tumor DNA were analyzed by Bridge Capture, Archer LIQUIDPlex, and AmpliSeq CHP version 2 for Illumina to compare variant allele frequency (VAF) detection. Bridge Capture was evaluated for sequencing depth requirement, interlaboratory reproducibility, automatization, and panel scalability. Of all methods, Bridge Capture detected the lowest VAF, and all VAFs strongly correlated with Archer LIQUIDPlex (R2 = 0.995) and AmpliSeq CHPv2 for Illumina (R2 = 0.988). Owing to its unique design, the Bridge Capture is compatible with the commonly used next-generation sequencing platforms and effectively uses sequencing capacity, permitting affordable and sensitive variant detection. The method demonstrated high reproducibility across independent laboratories and between automated and manual workflow. The panel size was increased by 300% and had negligible impact on performance and cross-reactivity of the probes, implying high multiplexing capabilities. Taken together, Bridge Capture is a cost-efficient, simple, rapid, and sensitive cancer diagnostics tool that has a potential to significantly improve the detection of mutations.
{"title":"Bridge Capture Permits Cost-Efficient, Rapid, and Sensitive Molecular Precision Diagnostics","authors":"Simona Adamusová , Anttoni Korkiakoski , Nea Laine , Anna Musku , Tuula Rantasalo , Jorma Kim , Juuso Blomster , Jukka Laine , Tatu Hirvonen , Juha-Pekka Pursiheimo , Manu Tamminen","doi":"10.1016/j.jmoldx.2025.09.006","DOIUrl":"10.1016/j.jmoldx.2025.09.006","url":null,"abstract":"<div><div>Liquid biopsies quantifying mutations in circulating tumor DNA by targeted next-generation sequencing have been gaining popularity. They are performed by various library preparation methods, each with distinct advantages and limitations. This work introduces Bridge Capture, a novel technology that goes beyond the advantages of market-leading liquid biopsy technologies, eliminating the need to compromise between scalability, cost-efficiency, sensitivity, or panel size. Twenty-four matched contrived colorectal biospecimens mimicking circulating tumor DNA were analyzed by Bridge Capture, Archer LIQUIDPlex, and AmpliSeq CHP version 2 for Illumina to compare variant allele frequency (VAF) detection. Bridge Capture was evaluated for sequencing depth requirement, interlaboratory reproducibility, automatization, and panel scalability. Of all methods, Bridge Capture detected the lowest VAF, and all VAFs strongly correlated with Archer LIQUIDPlex (<em>R</em><sup>2</sup> = 0.995) and AmpliSeq CHPv2 for Illumina (<em>R</em><sup>2</sup> = 0.988). Owing to its unique design, the Bridge Capture is compatible with the commonly used next-generation sequencing platforms and effectively uses sequencing capacity, permitting affordable and sensitive variant detection. The method demonstrated high reproducibility across independent laboratories and between automated and manual workflow. The panel size was increased by 300% and had negligible impact on performance and cross-reactivity of the probes, implying high multiplexing capabilities. Taken together, Bridge Capture is a cost-efficient, simple, rapid, and sensitive cancer diagnostics tool that has a potential to significantly improve the detection of mutations.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 53-63"},"PeriodicalIF":3.4,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145330761","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}
Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in which routine histology review was implemented at a US molecular diagnostics laboratory. The effects of implementation on laboratory compliance and the analytical performance of a targeted POLE assay were measured as changes in tumor cellularity documentation, tumor sample enrichment (in samples with <40% tumor cellularity), POLE mutation rate, tumor signal intensity, and repeat-testing rate. Endometrial carcinoma samples (N = 1752) and tested for POLE mutations using a multiplex PCR assay. POLE mutation rates were 6.3% and 5.0% before and after intervention, respectively (P = 0.25), with the mutations most commonly detected being p.Pro286Arg (47%) and p.Val411Leu (21%). Documentation of tumor cellularity increased from 29% to 100%, and the rate of tumor enrichment increased from 1.4% to 31.5% (both, P < 0.0001). Mutation signal intensity increased from 0.32 to 0.58, and the repeat-testing rate decreased from 8.8% to 2.3% (P = 0.004 and <0.0001, respectively). Systematic preanalytical histology review was associated with improved analytical performance of a targeted POLE assay, accompanied by compliance in tumor cellularity documentation, increased tumor enrichment, and decreased repeated testing, supporting preanalytical assessment in improving somatic mutation detection in pathology specimens with low tumor content.
{"title":"Preanalytical Histology Review Improves POLE Mutation Detection in Endometrial Carcinomas","authors":"Hehua Huang, Sara Hartinger, Rachel L.P. Geisick, Chandler Ho, Fei Dong","doi":"10.1016/j.jmoldx.2025.09.008","DOIUrl":"10.1016/j.jmoldx.2025.09.008","url":null,"abstract":"<div><div>Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in which routine histology review was implemented at a US molecular diagnostics laboratory. The effects of implementation on laboratory compliance and the analytical performance of a targeted <em>POLE</em> assay were measured as changes in tumor cellularity documentation, tumor sample enrichment (in samples with <40% tumor cellularity), <em>POLE</em> mutation rate, tumor signal intensity, and repeat-testing rate. Endometrial carcinoma samples (<em>N</em> = 1752) and tested for <em>POLE</em> mutations using a multiplex PCR assay. <em>POLE</em> mutation rates were 6.3% and 5.0% before and after intervention, respectively (<em>P</em> = 0.25), with the mutations most commonly detected being p.Pro286Arg (47%) and p.Val411Leu (21%). Documentation of tumor cellularity increased from 29% to 100%, and the rate of tumor enrichment increased from 1.4% to 31.5% (both, <em>P</em> < 0.0001). Mutation signal intensity increased from 0.32 to 0.58, and the repeat-testing rate decreased from 8.8% to 2.3% (<em>P</em> = 0.004 and <0.0001, respectively). Systematic preanalytical histology review was associated with improved analytical performance of a targeted <em>POLE</em> assay, accompanied by compliance in tumor cellularity documentation, increased tumor enrichment, and decreased repeated testing, supporting preanalytical assessment in improving somatic mutation detection in pathology specimens with low tumor content.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 22-26"},"PeriodicalIF":3.4,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145330820","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 : 2025-10-17DOI: 10.1016/j.jmoldx.2025.09.007
Alexandra Saint-Charles , Julien Masliah-Planchon , Elnaz Saberi-Ansari , Angela Bellini , Marie Bernkopf , Jaime Font de Mora , Rosa Noguera , Nadine Van Roy , Angharad Goodman , Ales Vicha , Valery Attignon , Valérie Combaret , Klaus Beiske , Tommy Martinsson , Jacqueline Schoumans , Maria Rossing , Bastiaan Tops , Frank Westermann , Sophie Cotteret , Matthias Fischer , Gudrun Schleiermacher
In high-risk neuroblastoma, identification of ALK activating genetic alterations is considered for clinical decision-making at relapse or more recently in frontline treatment. The accurate diagnosis of genetic alterations requires harmonization of molecular techniques and reporting, especially when these concern inclusion criteria for clinical trials. Analysis and validation of 14 DNA samples harboring distinct ALK alterations were performed across the 21 SIOPEN (International Society of Paediatric Oncology Europe Neuroblastoma) molecular diagnostic laboratories. These included ALK mutations at or outside hotspots in the tyrosine kinase domain with variant allele frequencies (VAFs) of 1% to 91% or ALK genomic amplification. Each laboratory used their own techniques: ALK amplifications were detected by pan-genomic copy number techniques or fluorescence in situ hybridization, and ALK mutations were characterized by next-generation sequencing techniques. All laboratories correctly identified high-level ALK amplification and ALK mutations within the known hotspots with VAF >5%, with the exception of two cases. Differences in interpretation and reporting were apparent for samples harboring mutations with a VAF <5% or outside known hotspots. These results highlight the importance of standard operating procedures, standardized reporting, and the robustness of ALK genetic testing in the SIOPEN laboratories, and the need for expert discussions regarding atypical ALK alterations, to validate eligibility for ALK targeted treatment in clinical trials.
{"title":"Harmonization of Reporting of ALK Genetic Alterations in Neuroblastoma","authors":"Alexandra Saint-Charles , Julien Masliah-Planchon , Elnaz Saberi-Ansari , Angela Bellini , Marie Bernkopf , Jaime Font de Mora , Rosa Noguera , Nadine Van Roy , Angharad Goodman , Ales Vicha , Valery Attignon , Valérie Combaret , Klaus Beiske , Tommy Martinsson , Jacqueline Schoumans , Maria Rossing , Bastiaan Tops , Frank Westermann , Sophie Cotteret , Matthias Fischer , Gudrun Schleiermacher","doi":"10.1016/j.jmoldx.2025.09.007","DOIUrl":"10.1016/j.jmoldx.2025.09.007","url":null,"abstract":"<div><div>In high-risk neuroblastoma, identification of <em>ALK</em> activating genetic alterations is considered for clinical decision-making at relapse or more recently in frontline treatment. The accurate diagnosis of genetic alterations requires harmonization of molecular techniques and reporting, especially when these concern inclusion criteria for clinical trials. Analysis and validation of 14 DNA samples harboring distinct <em>ALK</em> alterations were performed across the 21 SIOPEN (International Society of Paediatric Oncology Europe Neuroblastoma) molecular diagnostic laboratories. These included <em>ALK</em> mutations at or outside hotspots in the tyrosine kinase domain with variant allele frequencies (VAFs) of 1% to 91% or <em>ALK</em> genomic amplification. Each laboratory used their own techniques: <em>ALK</em> amplifications were detected by pan-genomic copy number techniques or fluorescence <em>in situ</em> hybridization, and <em>ALK</em> mutations were characterized by next-generation sequencing techniques. All laboratories correctly identified high-level <em>ALK</em> amplification and <em>ALK</em> mutations within the known hotspots with VAF >5%, with the exception of two cases. Differences in interpretation and reporting were apparent for samples harboring mutations with a VAF <5% or outside known hotspots. These results highlight the importance of standard operating procedures, standardized reporting, and the robustness of <em>ALK</em> genetic testing in the SIOPEN laboratories, and the need for expert discussions regarding atypical <em>ALK</em> alterations, to validate eligibility for ALK targeted treatment in clinical trials.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 27-38"},"PeriodicalIF":3.4,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145330832","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 : 2025-10-10DOI: 10.1016/j.jmoldx.2025.09.004
Peter J.B. Sabatini , Josh Bridgers , Shujun Huang , Tong Zhang , Clare Sheen , Tracy Stockley , Robert Kridel , Ian Bosdet , Marco A. Marra , Christian Steidl , David W. Scott , Aly Karsan
Gene expression signatures are important for classifying lymphoid malignancies, although routine diagnostic workflows predominantly use immunohistochemical staining and fluorescence in situ hybridization. These traditional methods are labor intensive and may misclassify the underlying oncogenic signatures, leading to inaccurate prognostication. To address this issue, an RNA expression panel was developed, the Lymphoma Expression Analysis (LExA120) 120 gene expression panel, using the NanoString platform for rapid, modular analysis of various lymphoma subtypes. The LExA120 panel targets 95 genes and 25 housekeeping genes to evaluate aggressive B-cell lymphomas, including: diffuse large B-cell lymphoma cell-of-origin, dark zone, and primary mediastinal large B-cell lymphoma signatures; Epstein-Barr virus (EBV) status; and a classical Hodgkin lymphoma posttransplant risk. Fifty-four formalin-fixed, paraffin-embedded tissue samples were tested with known diagnoses and 51 samples with known EBV status. The panel showed high concordance with previously validated methods according to Pearson correlation coefficients of the signature scores. The assay also displayed high reproducibility in repeated tests and across different clinical laboratories. This study confirmed the panel’s ability to stratify EBV-positive and EBV-negative lymphomas with high diagnostic certainty. Although EBER in situ hybridization confirmation was needed in approximately 12% of cases, synergizing with traditional techniques may facilitate more rapid and cost-effective diagnoses. The LExA120 panel offers a multiplexed approach to lymphoma classification, enhancing the efficiency and accuracy for subtyping lymphomas.
{"title":"Validation of a Modular Gene Expression Assay for Risk Stratification and Subtyping Lymphomas","authors":"Peter J.B. Sabatini , Josh Bridgers , Shujun Huang , Tong Zhang , Clare Sheen , Tracy Stockley , Robert Kridel , Ian Bosdet , Marco A. Marra , Christian Steidl , David W. Scott , Aly Karsan","doi":"10.1016/j.jmoldx.2025.09.004","DOIUrl":"10.1016/j.jmoldx.2025.09.004","url":null,"abstract":"<div><div>Gene expression signatures are important for classifying lymphoid malignancies, although routine diagnostic workflows predominantly use immunohistochemical staining and fluorescence <em>in situ</em> hybridization. These traditional methods are labor intensive and may misclassify the underlying oncogenic signatures, leading to inaccurate prognostication. To address this issue, an RNA expression panel was developed, the Lymphoma Expression Analysis (LExA120) 120 gene expression panel, using the NanoString platform for rapid, modular analysis of various lymphoma subtypes. The LExA120 panel targets 95 genes and 25 housekeeping genes to evaluate aggressive B-cell lymphomas, including: diffuse large B-cell lymphoma cell-of-origin, dark zone, and primary mediastinal large B-cell lymphoma signatures; Epstein-Barr virus (EBV) status; and a classical Hodgkin lymphoma posttransplant risk. Fifty-four formalin-fixed, paraffin-embedded tissue samples were tested with known diagnoses and 51 samples with known EBV status. The panel showed high concordance with previously validated methods according to Pearson correlation coefficients of the signature scores. The assay also displayed high reproducibility in repeated tests and across different clinical laboratories. This study confirmed the panel’s ability to stratify EBV-positive and EBV-negative lymphomas with high diagnostic certainty. Although EBER <em>in situ</em> hybridization confirmation was needed in approximately 12% of cases, synergizing with traditional techniques may facilitate more rapid and cost-effective diagnoses. The LExA120 panel offers a multiplexed approach to lymphoma classification, enhancing the efficiency and accuracy for subtyping lymphomas.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 1-7"},"PeriodicalIF":3.4,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145281585","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 : 2025-10-10DOI: 10.1016/j.jmoldx.2025.09.003
Muneeza Maqsood , John Toubia , Carol Wadham , Naranie Shanmuganathan , Nur Hezrin Shahrin , Adelina Fernandes , Joe McConnell , Verity Saunders , Dominik Kaczorowski , Rosalie R. Kenyon , Ming Lin , Timothy P. Hughes , Chung Hoow Kok , Susan Branford
RNA-based targeted sequencing aids the detection of several types of variants in hematologic and other malignancies, including splice-altering variants. However, accurately identifying clinically relevant mis-splicing events remains challenging because of the inherent complexity of the human transcriptome and the high prevalence of false-positive splice junctions in deep RNA-sequencing data. To address these challenges, SpliceChaser and BreakChaser were developed, which are bioinformatics tools designed to enhance the detection and characterization of relevant splice-altering events. SpliceChaser improves the identification of clinically relevant atypical splicing by analyzing read length diversity within flanking sequences of the mapped reads around the splice junctions. BreakChaser processes soft-clipped sequences and alignment anomalies to enhance the detection of targeted deletion breakpoints associated with atypical splice isoforms generated from intrachromosomal gene deletions. These tools were developed and validated using a cohort of >1400 RNA-sequencing samples from patients with chronic myeloid leukemia. Collectively, SpliceChaser and BreakChaser achieved a positive percentage agreement of 98% and a positive predictive value of 91% for the detection of clinically relevant atypical splice-altering variants or gene deletions in the targeted regions. By integrating splicing and breakpoint detection with robust filtering strategies, these tools facilitate precise identification of clinically relevant variants, paving the way for improved diagnostics and therapeutic strategies in chronic myeloid leukemia and other malignancies.
{"title":"Enhanced Detection of Splice-Altering Variants in Hematologic Malignancies Using Targeted RNA-Sequencing Data","authors":"Muneeza Maqsood , John Toubia , Carol Wadham , Naranie Shanmuganathan , Nur Hezrin Shahrin , Adelina Fernandes , Joe McConnell , Verity Saunders , Dominik Kaczorowski , Rosalie R. Kenyon , Ming Lin , Timothy P. Hughes , Chung Hoow Kok , Susan Branford","doi":"10.1016/j.jmoldx.2025.09.003","DOIUrl":"10.1016/j.jmoldx.2025.09.003","url":null,"abstract":"<div><div>RNA-based targeted sequencing aids the detection of several types of variants in hematologic and other malignancies, including splice-altering variants. However, accurately identifying clinically relevant mis-splicing events remains challenging because of the inherent complexity of the human transcriptome and the high prevalence of false-positive splice junctions in deep RNA-sequencing data. To address these challenges, SpliceChaser and BreakChaser were developed, which are bioinformatics tools designed to enhance the detection and characterization of relevant splice-altering events. SpliceChaser improves the identification of clinically relevant atypical splicing by analyzing read length diversity within flanking sequences of the mapped reads around the splice junctions. BreakChaser processes soft-clipped sequences and alignment anomalies to enhance the detection of targeted deletion breakpoints associated with atypical splice isoforms generated from intrachromosomal gene deletions. These tools were developed and validated using a cohort of >1400 RNA-sequencing samples from patients with chronic myeloid leukemia. Collectively, SpliceChaser and BreakChaser achieved a positive percentage agreement of 98% and a positive predictive value of 91% for the detection of clinically relevant atypical splice-altering variants or gene deletions in the targeted regions. By integrating splicing and breakpoint detection with robust filtering strategies, these tools facilitate precise identification of clinically relevant variants, paving the way for improved diagnostics and therapeutic strategies in chronic myeloid leukemia and other malignancies.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"28 1","pages":"Pages 39-52"},"PeriodicalIF":3.4,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145281565","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 : 2025-10-08DOI: 10.1016/j.jmoldx.2025.08.012
Julien Corné , Florence Godey , Angelina Legros , Laurent Castéra , Sophie Krieger , Mathilde Chérel , Agathe Cochet , Fanny Le Du , Héloïse Bourien , Antoine Deleuze , Laurence Crouzet , Christophe Perrin , Claudia Lefeuvre-Plesse , Véronique Diéras , Thibault De la Motte Rouge
Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels, digital PCR analysis seems highly appropriate. However, new targeted next-generation sequencing solutions are now available, enabling highly sensitive multigene analysis that could benefit patients. This study compared the analytical performance of multiplex digital PCR and targeted next-generation sequencing in detecting somatic erb-b2 receptor tyrosine kinase 2 (ERBB2), estrogen receptor 1 (ESR1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutations in a series of 32 plasma samples from patients with metastatic breast cancer. Forty-four mutations were detected, with an overall concordance of 95% (90/95) and a high degree of correlation (R2 = 0.9786). Interestingly, two ESR1 mutations detected in multiplex drop-off digital PCR were also detected by targeted next-generation sequencing with comparable mutant allele frequencies, enabling the identification of these specific variants (p.D538N and p.536LYD>P). Moreover, an additional PIK3CA mutation (p.P539R) was first detected using targeted next-generation sequencing and later confirmed with a newly designed digital PCR assay. Although more expensive than multiplex digital PCR, these new types of small targeted next-generation sequencing gene panels could provide a rapid answer to a specific clinical question with a ready-to-use solution, which could benefit patients.
{"title":"Comparative Performance of a Targeted Next-Generation Sequencing Assay Against Multiplexed Digital PCR Assays in Detecting ERBB2, ESR1, and PIK3CA Mutations in Plasma Circulating Cell-Free DNA from Liquid Biopsies","authors":"Julien Corné , Florence Godey , Angelina Legros , Laurent Castéra , Sophie Krieger , Mathilde Chérel , Agathe Cochet , Fanny Le Du , Héloïse Bourien , Antoine Deleuze , Laurence Crouzet , Christophe Perrin , Claudia Lefeuvre-Plesse , Véronique Diéras , Thibault De la Motte Rouge","doi":"10.1016/j.jmoldx.2025.08.012","DOIUrl":"10.1016/j.jmoldx.2025.08.012","url":null,"abstract":"<div><div>Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels, digital PCR analysis seems highly appropriate. However, new targeted next-generation sequencing solutions are now available, enabling highly sensitive multigene analysis that could benefit patients. This study compared the analytical performance of multiplex digital PCR and targeted next-generation sequencing in detecting somatic erb-b2 receptor tyrosine kinase 2 (<em>ERBB2</em>), estrogen receptor 1 (<em>ESR1</em>), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (<em>PIK3CA</em>) mutations in a series of 32 plasma samples from patients with metastatic breast cancer. Forty-four mutations were detected, with an overall concordance of 95% (90/95) and a high degree of correlation (<em>R</em><sup>2</sup> = 0.9786). Interestingly, two <em>ESR1</em> mutations detected in multiplex drop-off digital PCR were also detected by targeted next-generation sequencing with comparable mutant allele frequencies, enabling the identification of these specific variants (p.D538N and p.536LYD>P). Moreover, an additional <em>PIK3CA</em> mutation (p.P539R) was first detected using targeted next-generation sequencing and later confirmed with a newly designed digital PCR assay. Although more expensive than multiplex digital PCR, these new types of small targeted next-generation sequencing gene panels could provide a rapid answer to a specific clinical question with a ready-to-use solution, which could benefit patients.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1202-1212"},"PeriodicalIF":3.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276489","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 : 2025-10-04DOI: 10.1016/j.jmoldx.2025.09.002
Yanca G. Oliveira , Marilia M. Montenegro , Vanessa T. Almeida , Eder A. Moura , Amom M. Nascimento , Gleyson Francisco da Silva Carvalho , Evelin A. Zanardo , Samar N. Chehimi , Beatriz M. Wolff , Lucas L. Vieira , Mariana R. Costa Siemann , Rafaela da Silva Mendes , Lissandro de Sousa Rolim , Karina M. Nascimento , Chong Ae Kim , Leslie D. Kulikowski
Genomic mosaicism is underdiagnosed owing to its variable tissue distribution and the limitations of single-tissue testing. Cytogenomic techniques applied across multiple tissues can uncover clinically actionable variants and clarify genotype-phenotype relationships. DNA from 21 patients (14 females and 7 males) with pigmentary mosaicism and global developmental delay was analyzed. Each patient underwent G-band karyotyping and array (Infinium CytoSNP-850K) on peripheral blood, skin fibroblasts, and buccal mucosa samples. Pathogenic or likely pathogenic variants were found in 13 of 21 patients (62%). Of these 13 patients, 10 (77%) exhibited mosaicism, with variant allele fractions as low as 15%. On the basis of tissue distribution, 3 cases were classified as germline events and 10 as somatic mosaicism. Patients with positive findings were subdivided into: i) mosaic numerical chromosomal alterations (n = 6), ii) pathogenic copy number variations (n = 5), and iii) structural rearrangements (n = 2). Notably, several mosaic variants—particularly aneuploidies—were detected exclusively in fibroblast DNA, underlining the added diagnostic yield of multitissue sampling. In this cohort, a multitissue cytogenomic approach achieved a 62% overall diagnostic rate and identified mosaicism in 77% of positive cases. These results support the routine incorporation of genomic arrays with multisample analysis into diagnostic workflows for rare developmental disorders, enhancing detection sensitivity and enabling precise genotype-phenotype correlations.
{"title":"Clinical Utility of Multitissue Genomic Arrays in Diagnosing Pigmentary Mosaicism Associated with Neurodevelopmental Delay","authors":"Yanca G. Oliveira , Marilia M. Montenegro , Vanessa T. Almeida , Eder A. Moura , Amom M. Nascimento , Gleyson Francisco da Silva Carvalho , Evelin A. Zanardo , Samar N. Chehimi , Beatriz M. Wolff , Lucas L. Vieira , Mariana R. Costa Siemann , Rafaela da Silva Mendes , Lissandro de Sousa Rolim , Karina M. Nascimento , Chong Ae Kim , Leslie D. Kulikowski","doi":"10.1016/j.jmoldx.2025.09.002","DOIUrl":"10.1016/j.jmoldx.2025.09.002","url":null,"abstract":"<div><div>Genomic mosaicism is underdiagnosed owing to its variable tissue distribution and the limitations of single-tissue testing. Cytogenomic techniques applied across multiple tissues can uncover clinically actionable variants and clarify genotype-phenotype relationships. DNA from 21 patients (14 females and 7 males) with pigmentary mosaicism and global developmental delay was analyzed. Each patient underwent G-band karyotyping and array (Infinium CytoSNP-850K) on peripheral blood, skin fibroblasts, and buccal mucosa samples. Pathogenic or likely pathogenic variants were found in 13 of 21 patients (62%). Of these 13 patients, 10 (77%) exhibited mosaicism, with variant allele fractions as low as 15%. On the basis of tissue distribution, 3 cases were classified as germline events and 10 as somatic mosaicism. Patients with positive findings were subdivided into: i) mosaic numerical chromosomal alterations (<em>n</em> = 6), ii) pathogenic copy number variations (<em>n</em> = 5), and iii) structural rearrangements (<em>n</em> = 2). Notably, several mosaic variants—particularly aneuploidies—were detected exclusively in fibroblast DNA, underlining the added diagnostic yield of multitissue sampling. In this cohort, a multitissue cytogenomic approach achieved a 62% overall diagnostic rate and identified mosaicism in 77% of positive cases. These results support the routine incorporation of genomic arrays with multisample analysis into diagnostic workflows for rare developmental disorders, enhancing detection sensitivity and enabling precise genotype-phenotype correlations.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1232-1241"},"PeriodicalIF":3.4,"publicationDate":"2025-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240189","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 : 2025-09-26DOI: 10.1016/j.jmoldx.2025.08.011
Jane Gibson , Hanadi El Achi , Dana Altenburger , Noah Brown , Amy S. Clark , Joshua Coleman , Rajyasree Emmadi , Amber M. Fussell , Meera Hameed , Danielle Jordan , Jennifer Laudadio , Anthony Provenzano , Robyn L. Temple-Smolkin , Christopher G. Suciu
Despite the increasing availability of next-generation sequencing (NGS) gene panel analysis for cancers, published reports suggest underutilization of testing, citing the shortage of credentialed professionals available to assist with the interpretation of test results among the key barriers. Obtaining a multidisciplinary consensus regarding a shared best practice NGS molecular biomarker reporting section template may facilitate introduction and/or increased testing for institutions that adopt similar report structures by improving report effectiveness and efficiency for both health care providers and laboratory professionals, leading to improved patient care. To address this challenge, the Association for Molecular Pathology convened a multidisciplinary collaborative expert working group to identify and utilize best practices from current reporting guidelines and approaches to develop an NGS biomarker report template to optimally present complex molecular profiling information for efficient use by oncologists and other health care providers. Seventeen non–small-cell lung cancer NGS biomarker reports from public, private, and academic laboratories were reviewed, and specific components (eg, report length, color use, formatting, presentation order of information, specific information included or omitted, tables, and figures) were assessed for their ability to be considered provider friendly. Based on this review, public and stakeholder input, available literature, and cumulative professional experience of the working group members, a guideline-concordant reporting template was developed based on expert opinion consensus and made freely available online with planned implementation assessment.
{"title":"Developing Consensus for a More Provider-Friendly Next-Generation Sequencing Molecular Biomarker Report","authors":"Jane Gibson , Hanadi El Achi , Dana Altenburger , Noah Brown , Amy S. Clark , Joshua Coleman , Rajyasree Emmadi , Amber M. Fussell , Meera Hameed , Danielle Jordan , Jennifer Laudadio , Anthony Provenzano , Robyn L. Temple-Smolkin , Christopher G. Suciu","doi":"10.1016/j.jmoldx.2025.08.011","DOIUrl":"10.1016/j.jmoldx.2025.08.011","url":null,"abstract":"<div><div>Despite the increasing availability of next-generation sequencing (NGS) gene panel analysis for cancers, published reports suggest underutilization of testing, citing the shortage of credentialed professionals available to assist with the interpretation of test results among the key barriers. Obtaining a multidisciplinary consensus regarding a shared best practice NGS molecular biomarker reporting section template may facilitate introduction and/or increased testing for institutions that adopt similar report structures by improving report effectiveness and efficiency for both health care providers and laboratory professionals, leading to improved patient care. To address this challenge, the Association for Molecular Pathology convened a multidisciplinary collaborative expert working group to identify and utilize best practices from current reporting guidelines and approaches to develop an NGS biomarker report template to optimally present complex molecular profiling information for efficient use by oncologists and other health care providers. Seventeen non–small-cell lung cancer NGS biomarker reports from public, private, and academic laboratories were reviewed, and specific components (eg, report length, color use, formatting, presentation order of information, specific information included or omitted, tables, and figures) were assessed for their ability to be considered provider friendly. Based on this review, public and stakeholder input, available literature, and cumulative professional experience of the working group members, a guideline-concordant reporting template was developed based on expert opinion consensus and made freely available online with planned implementation assessment.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1123-1136"},"PeriodicalIF":3.4,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145187375","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 : 2025-09-26DOI: 10.1016/j.jmoldx.2025.08.010
Raquel Herranz , Julia Oto , Emma Plana , Javier Pérez-Ardavín , Patricia Verger , Manuel Martínez-Sarmiento , César D. Vera-Donoso , Pilar Medina
Bladder cancer (BC) is a lethal urological malignancy, with current diagnostic and follow-up methods being invasive and costly. Cell-free DNA (cfDNA) in liquid biopsies has shown promise in cancer diagnostics, but its fragmentation and integrity in urine remain underexplored in BC, becoming the aim of this study. cfDNA was isolated from the urine of 156 patients with BC of most stages and 79 matched controls without renal or bladder conditions. The amount of a large (>250-bp) and a nested small (<125-bp) fragment of ACTB, AR, MYC, BCAS1, and STOX1 was quantified by quantitative real-time PCR. Fragmentation and integrity (ratio of large/small) were analyzed with ordinal logistic regression. The increase in the ratio of large/small ACTB fragments and the small fragments of AR and MYC may represent a valuable tool to diagnose and stage BC when classified as both non–muscle-invasive and muscle-invasive BC or considering grades and stages separately. The small fragment of MYC, leading the effect observed, displayed a valuable diagnostic capacity [area under the receiver operating characteristic (ROC) curve = 0.7221; 95% CI, 0.6527–0.7915; P < 0.0001; sensitivity = 50%; specificity = 95%], particularly for muscle-invasive BC (area under the ROC curve = 0.8098; 95% CI, 0.6674–0.9523; P < 0.0001; sensitivity = 70%; specificity = 97%). Herein, the analysis of urine cfDNA fragmentation and integrity of these surrogate markers is proposed as noninvasive biomarkers to diagnose and stage BC. Once validated, the proposed biomarkers could improve patient management by reinforcing or substituting current invasive and expensive techniques.
{"title":"Analysis of the Fragmentation and Integrity of Urine Cell-Free DNA as a Diagnostic and Staging Biomarker for Bladder Cancer","authors":"Raquel Herranz , Julia Oto , Emma Plana , Javier Pérez-Ardavín , Patricia Verger , Manuel Martínez-Sarmiento , César D. Vera-Donoso , Pilar Medina","doi":"10.1016/j.jmoldx.2025.08.010","DOIUrl":"10.1016/j.jmoldx.2025.08.010","url":null,"abstract":"<div><div>Bladder cancer (BC) is a lethal urological malignancy, with current diagnostic and follow-up methods being invasive and costly. Cell-free DNA (cfDNA) in liquid biopsies has shown promise in cancer diagnostics, but its fragmentation and integrity in urine remain underexplored in BC, becoming the aim of this study. cfDNA was isolated from the urine of 156 patients with BC of most stages and 79 matched controls without renal or bladder conditions. The amount of a large (>250-bp) and a nested small (<125-bp) fragment of <em>ACTB</em>, <em>AR</em>, <em>MYC</em>, <em>BCAS1</em>, and <em>STOX1</em> was quantified by quantitative real-time PCR. Fragmentation and integrity (ratio of large/small) were analyzed with ordinal logistic regression. The increase in the ratio of large/small ACTB fragments and the small fragments of <em>AR</em> and <em>MYC</em> may represent a valuable tool to diagnose and stage BC when classified as both non–muscle-invasive and muscle-invasive BC or considering grades and stages separately. The small fragment of <em>MYC</em>, leading the effect observed, displayed a valuable diagnostic capacity [area under the receiver operating characteristic (ROC) curve = 0.7221; 95% CI, 0.6527–0.7915; <em>P</em> < 0.0001; sensitivity = 50%; specificity = 95%], particularly for muscle-invasive BC (area under the ROC curve = 0.8098; 95% CI, 0.6674–0.9523; <em>P</em> < 0.0001; sensitivity = 70%; specificity = 97%). Herein, the analysis of urine cfDNA fragmentation and integrity of these surrogate markers is proposed as noninvasive biomarkers to diagnose and stage BC. Once validated, the proposed biomarkers could improve patient management by reinforcing or substituting current invasive and expensive techniques.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 12","pages":"Pages 1189-1201"},"PeriodicalIF":3.4,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145187330","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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