Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.006
Somak Roy , Martine W. Tremblay , Edward Lockhart , Swaroop Aradhya , Pinar Bayrak-Toydemir , Mark Bowser , Jeana DaRe , Kristin Gibson , Michael Kennemer , Christopher Krueger , Matt Lebo , Rong Mao , Robert Nussbaum , Brendan O'Fallon , Andrew Rosato , Lisa V. Kalman , Birgit Funke
Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant genes and variants, there is an urgent need for reference materials to optimize, validate, and quality control NGS tests. This pilot study documents the paucity of physical reference materials for widely tested genes and demonstrates the utility of in silico mutagenized reference materials to supplement physical samples when developing NGS tests. We examined published, expert curated lists of clinically relevant variants for these widely tested genes and found that publicly available reference materials were available for only 29.4%. We outline the steps for generating in silico resources and used 49 curated variants to conduct a blinded proof-of-concept study with three experienced NGS laboratories. One laboratory detected all added variants, and two detected all but one. This study revealed common scenarios that could lead to false-negative results when common pathogenic variants cannot be tested during analytical validation. This work highlights the need to establish centralized knowledge bases for common, pathogenic variants, demonstrates the utility of in silico reference materials, and provides guidance for generating in silico reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.
{"title":"From Expert Knowledge to Validation Resources","authors":"Somak Roy , Martine W. Tremblay , Edward Lockhart , Swaroop Aradhya , Pinar Bayrak-Toydemir , Mark Bowser , Jeana DaRe , Kristin Gibson , Michael Kennemer , Christopher Krueger , Matt Lebo , Rong Mao , Robert Nussbaum , Brendan O'Fallon , Andrew Rosato , Lisa V. Kalman , Birgit Funke","doi":"10.1016/j.jmoldx.2025.07.006","DOIUrl":"10.1016/j.jmoldx.2025.07.006","url":null,"abstract":"<div><div>Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant genes and variants, there is an urgent need for reference materials to optimize, validate, and quality control NGS tests. This pilot study documents the paucity of physical reference materials for widely tested genes and demonstrates the utility of <em>in silico</em> mutagenized reference materials to supplement physical samples when developing NGS tests. We examined published, expert curated lists of clinically relevant variants for these widely tested genes and found that publicly available reference materials were available for only 29.4%. We outline the steps for generating <em>in silico</em> resources and used 49 curated variants to conduct a blinded proof-of-concept study with three experienced NGS laboratories. One laboratory detected all added variants, and two detected all but one. This study revealed common scenarios that could lead to false-negative results when common pathogenic variants cannot be tested during analytical validation. This work highlights the need to establish centralized knowledge bases for common, pathogenic variants, demonstrates the utility of <em>in silico</em> reference materials, and provides guidance for generating <em>in silico</em> reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1074-1083"},"PeriodicalIF":3.4,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-20DOI: 10.1016/j.jmoldx.2025.07.005
He Fang , Stephen M. Eacker , Yu Wu , Cate Paschal , Mary Wood , Brad Nelson , Alexander Muratov , Yajuan J. Liu
Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence in situ hybridization, and chromosome microarray analysis. These methods often lack the resolution and sensitivity needed for comprehensive characterization of chromosomal aberrations. To address these limitations, genomic proximity mapping (GPM), a genome-wide chromosome conformation capture (Hi-C) technology, was implemented in a clinical setting. Here, GPM was applied to a cohort of 123 individuals with constitutional disorders, achieving a 100% concordance in detecting 110 copy number variants (>25 kb) and 27 structural rearrangements, in addition to novel findings. GPM demonstrated unique advantages, such as resolving chromosomal rearrangements with precise (<5 kb) breakpoint resolution, maintaining robust performance with challenging samples, including formalin-fixed, paraffin-embedded tissues, and detecting mosaicism with high sensitivity. Furthermore, GPM reliably provided copy number and loss-of-heterozygosity profiles, streamlining workflows. GPM also uncovered 12 novel structural variants missed by standard methods, highlighting its superior detection capability. This analysis revealed that cases with more than two chromosomal rearrangements identified by traditional cytogenetics often harbor additional, cryptic rearrangements that remain undetected by standard-of-care methods. GPM represents a transformative tool for genomic diagnostics, offering a high-resolution, comprehensive approach to detecting genomic alterations. Its ability to address limitations of conventional cytogenetics methods positions GPM as a needed advance in the diagnosis, prognosis, and therapeutic management of genetic disorders.
{"title":"Evaluation of Genomic Proximity Mapping for Detecting Genomic and Chromosomal Structural Variants in Constitutional Disorders","authors":"He Fang , Stephen M. Eacker , Yu Wu , Cate Paschal , Mary Wood , Brad Nelson , Alexander Muratov , Yajuan J. Liu","doi":"10.1016/j.jmoldx.2025.07.005","DOIUrl":"10.1016/j.jmoldx.2025.07.005","url":null,"abstract":"<div><div>Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence <em>in situ</em> hybridization, and chromosome microarray analysis. These methods often lack the resolution and sensitivity needed for comprehensive characterization of chromosomal aberrations. To address these limitations, genomic proximity mapping (GPM), a genome-wide chromosome conformation capture (Hi-C) technology, was implemented in a clinical setting. Here, GPM was applied to a cohort of 123 individuals with constitutional disorders, achieving a 100% concordance in detecting 110 copy number variants (>25 kb) and 27 structural rearrangements, in addition to novel findings. GPM demonstrated unique advantages, such as resolving chromosomal rearrangements with precise (<5 kb) breakpoint resolution, maintaining robust performance with challenging samples, including formalin-fixed, paraffin-embedded tissues, and detecting mosaicism with high sensitivity. Furthermore, GPM reliably provided copy number and loss-of-heterozygosity profiles, streamlining workflows. GPM also uncovered 12 novel structural variants missed by standard methods, highlighting its superior detection capability. This analysis revealed that cases with more than two chromosomal rearrangements identified by traditional cytogenetics often harbor additional, cryptic rearrangements that remain undetected by standard-of-care methods. GPM represents a transformative tool for genomic diagnostics, offering a high-resolution, comprehensive approach to detecting genomic alterations. Its ability to address limitations of conventional cytogenetics methods positions GPM as a needed advance in the diagnosis, prognosis, and therapeutic management of genetic disorders.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 11","pages":"Pages 1054-1069"},"PeriodicalIF":3.4,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144976902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-07DOI: 10.1016/j.jmoldx.2025.07.002
Cassandra Michel , Alizée Simon , Andréa Witz , Idrissia Hanriot , Julie Dardare , Margaux Betz , Maxime Brun , Pauline Gilson , Jean Louis Merlin , Alexandre Harlé
Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. BRCA1 and RAD51C promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.
{"title":"A Novel Droplet Digital PCR Assay for BRCA1 and RAD51C Methylation","authors":"Cassandra Michel , Alizée Simon , Andréa Witz , Idrissia Hanriot , Julie Dardare , Margaux Betz , Maxime Brun , Pauline Gilson , Jean Louis Merlin , Alexandre Harlé","doi":"10.1016/j.jmoldx.2025.07.002","DOIUrl":"10.1016/j.jmoldx.2025.07.002","url":null,"abstract":"<div><div>Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious <em>BRCA1/2</em> mutation, these mutations are not the sole cause of HRD. Promoter methylation of <em>BRCA1</em> and <em>RAD51C</em> contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. <em>BRCA1</em> and <em>RAD51C</em> promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious <em>BRCA1</em>/2 mutation, and 2 (2.6%) a <em>RAD51C</em> mutation. Methylation analysis revealed <em>BRCA1</em> promoter methylation in 32 patients (41.6%) and <em>RAD51C</em> promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond <em>BRCA1</em>/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 1026-1036"},"PeriodicalIF":3.4,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-07-25DOI: 10.1016/j.jmoldx.2025.04.011
Cassandra Litchfield , Ronny Nienhold , Andreas Wicki , Michael Schmid , Domingo Aguilera-Garcia , Viktor Hendrik Koelzer
Formalin-fixed, paraffin-embedded (FFPE) tumor tissue is the standard in pathology due to logistical and quality constraints of fresh-frozen samples. Although whole-genome sequencing (WGS) offers diagnostic promise, its validity and utility in FFPE samples remain underexplored. This study bridges the gap by comparing FFPE-derived tumor WGS with next-generation sequencing results from FoundationOneCDx (F1CDx) and a melanoma-specific panel (MelArray) in 78 metastatic melanoma samples from the Swiss Tumor Profiler Study. A diagnostic pipeline was developed for quality control, variant annotation, and clinical actionability using public and commercial databases. FFPE-derived WGS displayed robust analytical validity, detecting 95% of somatic single nucleotide variants, 98% of multinucleotide variants, 90% of insertions/deletions, 76% of amplifications, and 96% of homozygous deletions identified by F1CDx. Tumor mutational burden strongly correlated with F1CDx (R = 0.98), particularly at the clinical threshold of ≥10 mutations per megabase, crucial for treatment decisions. WGS detected complex biomarkers such as UV-associated mutational signatures and genome-wide copy number alterations, aiding melanoma subtype distinction. Clinically, WGS suggested treatments or trials for all cases, identifying additional markers in 38% and 71% compared with F1CDx and MelArray, respectively. Novel therapeutic opportunities were found in 18% and 56% of cases. FFPE-derived WGS closely matches targeted panels in performance while providing comprehensive insights, enhancing therapeutic options. With decreasing costs, WGS could become a powerful routine diagnostic tool.
{"title":"Integrating Formalin-Fixed, Paraffin-Embedded–Derived Whole-Genome Sequencing into Routine Molecular Pathology","authors":"Cassandra Litchfield , Ronny Nienhold , Andreas Wicki , Michael Schmid , Domingo Aguilera-Garcia , Viktor Hendrik Koelzer","doi":"10.1016/j.jmoldx.2025.04.011","DOIUrl":"10.1016/j.jmoldx.2025.04.011","url":null,"abstract":"<div><div>Formalin-fixed, paraffin-embedded (FFPE) tumor tissue is the standard in pathology due to logistical and quality constraints of fresh-frozen samples. Although whole-genome sequencing (WGS) offers diagnostic promise, its validity and utility in FFPE samples remain underexplored. This study bridges the gap by comparing FFPE-derived tumor WGS with next-generation sequencing results from FoundationOneCDx (F1CDx) and a melanoma-specific panel (MelArray) in 78 metastatic melanoma samples from the Swiss Tumor Profiler Study. A diagnostic pipeline was developed for quality control, variant annotation, and clinical actionability using public and commercial databases. FFPE-derived WGS displayed robust analytical validity, detecting 95% of somatic single nucleotide variants, 98% of multinucleotide variants, 90% of insertions/deletions, 76% of amplifications, and 96% of homozygous deletions identified by F1CDx. Tumor mutational burden strongly correlated with F1CDx (<em>R</em> = 0.98), particularly at the clinical threshold of ≥10 mutations per megabase, crucial for treatment decisions. WGS detected complex biomarkers such as UV-associated mutational signatures and genome-wide copy number alterations, aiding melanoma subtype distinction. Clinically, WGS suggested treatments or trials for all cases, identifying additional markers in 38% and 71% compared with F1CDx and MelArray, respectively. Novel therapeutic opportunities were found in 18% and 56% of cases. FFPE-derived WGS closely matches targeted panels in performance while providing comprehensive insights, enhancing therapeutic options. With decreasing costs, WGS could become a powerful routine diagnostic tool.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 8","pages":"Pages 722-735"},"PeriodicalIF":3.4,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144703032","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 : 2025-07-25DOI: 10.1016/j.jmoldx.2025.03.005
Andrea Sboner , Jamal Benhamida , Julie W. Hirschhorn , Robyn L. Temple-Smolkin , Weiwei Zhang , Annette Leon , Association for Molecular Pathology Informatics Subdivision Leadership
{"title":"Informatics Powering Data to Shape the Future of Molecular Pathology","authors":"Andrea Sboner , Jamal Benhamida , Julie W. Hirschhorn , Robyn L. Temple-Smolkin , Weiwei Zhang , Annette Leon , Association for Molecular Pathology Informatics Subdivision Leadership","doi":"10.1016/j.jmoldx.2025.03.005","DOIUrl":"10.1016/j.jmoldx.2025.03.005","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 8","pages":"Pages 671-673"},"PeriodicalIF":3.4,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144703114","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 : 2025-07-23DOI: 10.1016/j.jmoldx.2025.06.002
Julius Honecker , Dirk Paffenholz , Julian Jungmann , Thomas Wieland , Angela A. Kou , Soo-Ryum Yang , Chad M. Vanderbilt , Vignesh Shanmugam , Meagan Montesion , Ryon P. Graf , Lee A. Albacker , Douglas I. Lin , Andreas Heilmann , Sotirios Lakis , Richard A. Hickman , Brennan Decker , Alex Robertson , Lauren L. Ritterhouse , Douglas A. Mata
Accurate detection of Epstein-Barr virus (EBV) in tumors is essential for refining diagnosis and guiding management of EBV-associated cancers. This validation study evaluated the performance of the FoundationOne CDx (F1CDx) and FoundationOne Heme (F1H) next-generation sequencing assays for detecting EBV compared with the reference standard EBV-encoded RNA (EBER) in situ hybridization (ISH). A total of 413 samples of gastric carcinoma, squamous cell carcinoma (predominantly nasopharyngeal carcinoma), and diffuse large B-cell lymphoma was assessed. The assays demonstrated a sensitivity of 94.0%, specificity of 99.1%, and accuracy of 98.3% with an area under the curve of 0.97 for EBV detection, showing high concordance with EBER ISH. Precision was 95.5%, and reproducibility across multiple runs was strong (CV = 4.7%). Dilution experiments demonstrated a linear relationship between EBV viral reads per million and the degree of cell culture dilution (Pearson's R > 0.99, P < 0.001), supporting assay linearity over a broad dynamic range. Characteristic mutational and cytogenetic profiles, including losses at 9p21 and gains at 11q13, were observed in EBV-positive samples, supporting the clinical utility of the method. These results indicate that F1CDx and F1H provide a reliable, in-parallel approach for detecting EBV along with somatic genomic alterations in a single assay. This method offers a promising alternative to conventional ISH, especially for identifying EBV in tumors without initial clinical suspicion, potentially streamlining diagnostic workflows.
在肿瘤中准确检测EBV对于改进EBV相关癌症的诊断和指导治疗至关重要。本验证性研究评估了FoundationOne CDx （F1CDx）和FoundationOne Heme （F1H）下一代测序（NGS）检测EBV的性能，并将其与参考标准EBV编码RNA （EBER）原位杂交（ISH）进行了比较。共有413例胃癌、鳞状细胞癌（主要是鼻咽癌）和弥漫性大b细胞淋巴瘤进行了评估。该方法检测EBV的灵敏度为94.0%，特异度为99.1%，准确度为98.3%，曲线下面积为0.97，与EBER - ISH具有较高的一致性。精密度为95.5%，重复性强（CV = 4.7%）。稀释实验表明，每百万eb病毒读数与细胞培养稀释度之间存在线性关系(Pearson’s R 0.99， P
{"title":"FoundationOne CDx and FoundationOne Heme Detect Epstein-Barr Virus with High Sensitivity and Specificity","authors":"Julius Honecker , Dirk Paffenholz , Julian Jungmann , Thomas Wieland , Angela A. Kou , Soo-Ryum Yang , Chad M. Vanderbilt , Vignesh Shanmugam , Meagan Montesion , Ryon P. Graf , Lee A. Albacker , Douglas I. Lin , Andreas Heilmann , Sotirios Lakis , Richard A. Hickman , Brennan Decker , Alex Robertson , Lauren L. Ritterhouse , Douglas A. Mata","doi":"10.1016/j.jmoldx.2025.06.002","DOIUrl":"10.1016/j.jmoldx.2025.06.002","url":null,"abstract":"<div><div>Accurate detection of Epstein-Barr virus (EBV) in tumors is essential for refining diagnosis and guiding management of EBV-associated cancers. This validation study evaluated the performance of the FoundationOne CDx (F1CDx) and FoundationOne Heme (F1H) next-generation sequencing assays for detecting EBV compared with the reference standard EBV-encoded RNA (EBER) <em>in situ</em> hybridization (ISH). A total of 413 samples of gastric carcinoma, squamous cell carcinoma (predominantly nasopharyngeal carcinoma), and diffuse large B-cell lymphoma was assessed. The assays demonstrated a sensitivity of 94.0%, specificity of 99.1%, and accuracy of 98.3% with an area under the curve of 0.97 for EBV detection, showing high concordance with EBER ISH. Precision was 95.5%, and reproducibility across multiple runs was strong (CV = 4.7%). Dilution experiments demonstrated a linear relationship between EBV viral reads per million and the degree of cell culture dilution (Pearson's R > 0.99, <em>P</em> < 0.001), supporting assay linearity over a broad dynamic range. Characteristic mutational and cytogenetic profiles, including losses at 9p21 and gains at 11q13, were observed in EBV-positive samples, supporting the clinical utility of the method. These results indicate that F1CDx and F1H provide a reliable, in-parallel approach for detecting EBV along with somatic genomic alterations in a single assay. This method offers a promising alternative to conventional ISH, especially for identifying EBV in tumors without initial clinical suspicion, potentially streamlining diagnostic workflows.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 920-935"},"PeriodicalIF":3.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144719035","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 : 2025-07-23DOI: 10.1016/j.jmoldx.2025.06.003
Julie Amiot , Corentin Levacher , Louise May Thibaut , Gwendoline Lienard , Stéphanie Vasseur , Olivier Quenez , Sophie Coutant , Steeve Fourneaux , Françoise Charbonnier , Hugo Thorn , Angélina Legros , Camille Aucouturier , Laurent Castéra , Raphaël Leman , Edwige Kasper , Stéphanie Baert-Desurmont , Sophie Krieger , Philippe Ruminy , Claude Houdayer
Diagnostic analysis of mRNA is essential because altered splicing is a frequent cause of genetic diseases. High-throughput splicing studies remain difficult to implement in routine diagnostics. This is why SEALigHTS (splice and expression analyses by ligation and high throughput sequencing), a cost-effective and easy-to-implement technique designed for simultaneous analysis of RNA from multiple patients on a panel of genes, was developed using probes designed at exon extremities. After reverse transcription and probing on cDNA, neighboring probes are ligated and the number of ligations quantified by using unique molecular identifiers and sequencing. A panel covering 42 genes (ie, 2195 probes) involved in breast/ovarian and colorectal cancer predispositions was designed. After a training phase on 40 samples, SEALigHTS was validated in another laboratory on 56 samples carrying various splicing variations previously characterized by RNA sequencing, with a sensitivity of 96% and specificity of 94%. Subsequently, in a series of 37 selected patients and 114 consecutive patients from the genetics clinic with a concomitant DNA panel, five new diagnoses were made, revealing the impact on splicing of a cryptic genomic variant (deep intronic, retrotransposon insertion), and the unexpected impact on splicing of six genomic variations was unmasked. Beyond increased diagnostic yield and classification of variants of uncertain significance, this comprehensive DNA and RNA combined approach highlights unexpected splicing defects and addresses genotype–phenotype correlation issues.
{"title":"Bridging the Diagnostic Gap in Hereditary Cancers with Simple, Cost-Effective, High-Throughput RNA Splicing Analysis","authors":"Julie Amiot , Corentin Levacher , Louise May Thibaut , Gwendoline Lienard , Stéphanie Vasseur , Olivier Quenez , Sophie Coutant , Steeve Fourneaux , Françoise Charbonnier , Hugo Thorn , Angélina Legros , Camille Aucouturier , Laurent Castéra , Raphaël Leman , Edwige Kasper , Stéphanie Baert-Desurmont , Sophie Krieger , Philippe Ruminy , Claude Houdayer","doi":"10.1016/j.jmoldx.2025.06.003","DOIUrl":"10.1016/j.jmoldx.2025.06.003","url":null,"abstract":"<div><div>Diagnostic analysis of mRNA is essential because altered splicing is a frequent cause of genetic diseases. High-throughput splicing studies remain difficult to implement in routine diagnostics. This is why SEALigHTS (splice and expression analyses by ligation and high throughput sequencing), a cost-effective and easy-to-implement technique designed for simultaneous analysis of RNA from multiple patients on a panel of genes, was developed using probes designed at exon extremities. After reverse transcription and probing on cDNA, neighboring probes are ligated and the number of ligations quantified by using unique molecular identifiers and sequencing. A panel covering 42 genes (ie, 2195 probes) involved in breast/ovarian and colorectal cancer predispositions was designed. After a training phase on 40 samples, SEALigHTS was validated in another laboratory on 56 samples carrying various splicing variations previously characterized by RNA sequencing, with a sensitivity of 96% and specificity of 94%. Subsequently, in a series of 37 selected patients and 114 consecutive patients from the genetics clinic with a concomitant DNA panel, five new diagnoses were made, revealing the impact on splicing of a cryptic genomic variant (deep intronic, retrotransposon insertion), and the unexpected impact on splicing of six genomic variations was unmasked. Beyond increased diagnostic yield and classification of variants of uncertain significance, this comprehensive DNA and RNA combined approach highlights unexpected splicing defects and addresses genotype–phenotype correlation issues.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 954-968"},"PeriodicalIF":3.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144719034","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 : 2025-07-23DOI: 10.1016/j.jmoldx.2025.07.001
Shiyi Xu , Xiaomei Luo , Bing Xiao , Huili Liu , Ting Xu , Linlin Chen , Tingting Yang , Na Xu , Yanjie Fan , Wenjuan Qiu , Ruifang Wang , Huiwen Zhang , Yanru Chen , Yongguo Yu , Yu Sun
Short tandem repeats (STRs) are associated with 70 genetic diseases. Because of the short read length of exome sequencing (ES), STR analysis is not routinely analyzed in clinical ES. So far, there has been limited systematic evaluation using large-scale clinical ES data to assess the diagnostic yield of pathogenic STR expansion. This study retrospectively analyzed 9580 exomes referred to our genetic laboratory between July 2019 and June 2024. The samples were divided into two groups: a genetically undiagnosed cohort (n = 4692) and a reference cohort with a low probability of carrying pathogenic STR expansions (n = 4888). An analysis pipeline was developed on the basis of the combination of multiple algorithms to analyze STRs detected in 30 known disease-related loci, achieving a precision of 54.9% and a sensitivity of 100%. STR verification by capillary electrophoresis analysis of STR confirmed 28 cases (0.6%) with pathogenic STR expansions in known disease-related loci. Fourteen of these cases (0.3%) could be explained by the STR findings, including seven neonates with DMPK expansions. The pipeline showed the potential to identity abnormal STR expansions at novel sites. In conclusion, this study demonstrates the clinical utility of ES-based STR analysis and advocates for its incorporation into the clinical ES workflow in genetic laboratories.
{"title":"Additional Diagnostic Yield through the Analysis of Short Tandem Repeats Based on Exome Sequencing Data","authors":"Shiyi Xu , Xiaomei Luo , Bing Xiao , Huili Liu , Ting Xu , Linlin Chen , Tingting Yang , Na Xu , Yanjie Fan , Wenjuan Qiu , Ruifang Wang , Huiwen Zhang , Yanru Chen , Yongguo Yu , Yu Sun","doi":"10.1016/j.jmoldx.2025.07.001","DOIUrl":"10.1016/j.jmoldx.2025.07.001","url":null,"abstract":"<div><div>Short tandem repeats (STRs) are associated with 70 genetic diseases. Because of the short read length of exome sequencing (ES), STR analysis is not routinely analyzed in clinical ES. So far, there has been limited systematic evaluation using large-scale clinical ES data to assess the diagnostic yield of pathogenic STR expansion. This study retrospectively analyzed 9580 exomes referred to our genetic laboratory between July 2019 and June 2024. The samples were divided into two groups: a genetically undiagnosed cohort (<em>n</em> = 4692) and a reference cohort with a low probability of carrying pathogenic STR expansions (<em>n</em> = 4888). An analysis pipeline was developed on the basis of the combination of multiple algorithms to analyze STRs detected in 30 known disease-related loci, achieving a precision of 54.9% and a sensitivity of 100%. STR verification by capillary electrophoresis analysis of STR confirmed 28 cases (0.6%) with pathogenic STR expansions in known disease-related loci. Fourteen of these cases (0.3%) could be explained by the STR findings, including seven neonates with <em>DMPK</em> expansions. The pipeline showed the potential to identity abnormal STR expansions at novel sites. In conclusion, this study demonstrates the clinical utility of ES-based STR analysis and advocates for its incorporation into the clinical ES workflow in genetic laboratories.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 1017-1025"},"PeriodicalIF":3.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144719033","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 : 2025-07-23DOI: 10.1016/j.jmoldx.2025.06.006
Pia S. Henkel , Eline Aas , Hege G. Russnes , Ingrid Dyvik , G. Live Fagereng , Åslaug Helland , Hanna Røgenes , Tonje G. Lien , Kine Pedersen
Detailed cost analyses of genomic profiling for precision cancer medicine can inform strategic planning and cost-effectiveness analysis. A flexible costing framework was developed in this study to conduct microcosting of genomic profiling in precision cancer medicine using the broad gene panel TruSight Oncology 500 and accounting for its integration into the molecular tumor board within the national Infrastructure for Precision Diagnostics in Norway. The framework enables calculation of costs per sample, by workflow steps and cost categories. Site visits and discussions with staff at Oslo University Hospital informed the diagnostic workflow, validation of the framework, and resource use inputs. Sensitivity analysis addressed alternative resource use estimates, higher batch sizes, and investment costs for automation of the library preparation step. Total costs per sample were $2944 USD, ranging from $2366 to $4307 when considering uncertainties in estimates. Consumables and personnel were the most resource-intensive cost categories across analyses. Automating the resource-intensive library preparation step enabled a higher weekly batch size with slightly lower costs per sample ($2881) despite the additional equipment costs. The dynamic costing framework highlights how the choice of equipment and batch sizes affects sample costs and personnel needs for genomic profiling. Consumables and personnel offer the largest potential for costs savings, but potential personnel bottlenecks need to be considered when further upscaling capacity.
{"title":"Microcosting Study of Genomic Profiling for Precision Cancer Medicine","authors":"Pia S. Henkel , Eline Aas , Hege G. Russnes , Ingrid Dyvik , G. Live Fagereng , Åslaug Helland , Hanna Røgenes , Tonje G. Lien , Kine Pedersen","doi":"10.1016/j.jmoldx.2025.06.006","DOIUrl":"10.1016/j.jmoldx.2025.06.006","url":null,"abstract":"<div><div>Detailed cost analyses of genomic profiling for precision cancer medicine can inform strategic planning and cost-effectiveness analysis. A flexible costing framework was developed in this study to conduct microcosting of genomic profiling in precision cancer medicine using the broad gene panel TruSight Oncology 500 and accounting for its integration into the molecular tumor board within the national Infrastructure for Precision Diagnostics in Norway. The framework enables calculation of costs per sample, by workflow steps and cost categories. Site visits and discussions with staff at Oslo University Hospital informed the diagnostic workflow, validation of the framework, and resource use inputs. Sensitivity analysis addressed alternative resource use estimates, higher batch sizes, and investment costs for automation of the library preparation step. Total costs per sample were $2944 USD, ranging from $2366 to $4307 when considering uncertainties in estimates. Consumables and personnel were the most resource-intensive cost categories across analyses. Automating the resource-intensive library preparation step enabled a higher weekly batch size with slightly lower costs per sample ($2881) despite the additional equipment costs. The dynamic costing framework highlights how the choice of equipment and batch sizes affects sample costs and personnel needs for genomic profiling. Consumables and personnel offer the largest potential for costs savings, but potential personnel bottlenecks need to be considered when further upscaling capacity.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 945-953"},"PeriodicalIF":3.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144719036","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 : 2025-07-22DOI: 10.1016/j.jmoldx.2025.06.005
Danielle K. Manning , Guruprasad Ananda , Cheryl Eifert , Vanesa Rojas-Rudilla , William Swanton , Shawn Keefe , Elizabeth P. Garcia , Michael D'Eletto , Erica Holdmore , Satyakam Mishra , Kirill Borziak , Pieter Lukasse , Phani Davineni , Monica D. Manam , Priyanka Shivdasani , Arezou A. Ghazani , Ai Ling Wang , Murat Bastepe , Himisha Beltran , Katherine A. Janeway , Laura E. MacConaill
Genomic profiling of cancers informs diagnostic and prognostic classification and aids in selection of targeted therapeutics. Targeted, next-generation sequencing of cancer-specific genes is clinically feasible and enables comprehensive somatic reporting; without a matched germline specimen, germline alterations can confound analyses of the somatic profile and generate uncertainty in interpretation. This work reports the validation and implementation of optional matched tumor/germline sequencing in a precision cancer medicine program. DNA from 63 patient samples was analyzed using OncoPanel, a hybrid capture-based sequencing assay of 461 genes. Three analytical pipelines were implemented: tumor only, matched tumor/germline, and germline only. For matched tumor/germline, germline alterations in 19 genes with actionable/therapeutic implications were rescued. Retrospective analysis of the first 1600 matched cases was done to determine the potential clinical utility of this approach. Limit of detection for point mutations/insertions and deletions was 3% allele fraction; reproducibility was >98%. Matched tumor/germline concordance across 938 somatic calls was 100%. The average tumor mutational burden (TMB) was approximately 4 mutations/Mb lower than tumor-only sequencing. TMB-high patients were accurately reclassified as TMB-low in 14% of cases. Twenty-five percent of validation cases (14% after launch) had a pathogenic or likely pathogenic germline variant conferring cancer susceptibility; 14% of validation cases (7% after launch) harbored a germline variant of therapeutic significance. Matched tumor/germline sequencing is more accurate than tumor-only sequencing, while still encompassing all genomic findings that inform targeted therapy selection.
{"title":"Clinical Implementation of Matched Tumor/Germline Sequencing Improves Accuracy of Tumor Genomic Profiling and Therapeutic Recommendations","authors":"Danielle K. Manning , Guruprasad Ananda , Cheryl Eifert , Vanesa Rojas-Rudilla , William Swanton , Shawn Keefe , Elizabeth P. Garcia , Michael D'Eletto , Erica Holdmore , Satyakam Mishra , Kirill Borziak , Pieter Lukasse , Phani Davineni , Monica D. Manam , Priyanka Shivdasani , Arezou A. Ghazani , Ai Ling Wang , Murat Bastepe , Himisha Beltran , Katherine A. Janeway , Laura E. MacConaill","doi":"10.1016/j.jmoldx.2025.06.005","DOIUrl":"10.1016/j.jmoldx.2025.06.005","url":null,"abstract":"<div><div>Genomic profiling of cancers informs diagnostic and prognostic classification and aids in selection of targeted therapeutics. Targeted, next-generation sequencing of cancer-specific genes is clinically feasible and enables comprehensive somatic reporting; without a matched germline specimen, germline alterations can confound analyses of the somatic profile and generate uncertainty in interpretation. This work reports the validation and implementation of optional matched tumor/germline sequencing in a precision cancer medicine program. DNA from 63 patient samples was analyzed using OncoPanel, a hybrid capture-based sequencing assay of 461 genes. Three analytical pipelines were implemented: tumor only, matched tumor/germline, and germline only. For matched tumor/germline, germline alterations in 19 genes with actionable/therapeutic implications were rescued. Retrospective analysis of the first 1600 matched cases was done to determine the potential clinical utility of this approach. Limit of detection for point mutations/insertions and deletions was 3% allele fraction; reproducibility was >98%. Matched tumor/germline concordance across 938 somatic calls was 100%. The average tumor mutational burden (TMB) was approximately 4 mutations/Mb lower than tumor-only sequencing. TMB-high patients were accurately reclassified as TMB-low in 14% of cases. Twenty-five percent of validation cases (14% after launch) had a pathogenic or likely pathogenic germline variant conferring cancer susceptibility; 14% of validation cases (7% after launch) harbored a germline variant of therapeutic significance. Matched tumor/germline sequencing is more accurate than tumor-only sequencing, while still encompassing all genomic findings that inform targeted therapy selection.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 10","pages":"Pages 969-988"},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144709677","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号