Journal of Molecular Diagnostics最新文献_第4页

Clinical Utility and Performance of Methylation-Specific Triplet-Primed PCR for Fragile X Syndrome Diagnosis 甲基化特异性三胞胎引物PCR在脆性X综合征诊断中的临床应用和性能

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-28 DOI: 10.1016/j.jmoldx.2025.10.001

Areerat Hnoonual , Wipawan Arunthong , Oradawan Plong-on , Pornsiri Sangmanee , Pornprot Limprasert

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.

脆性X染色体综合征（FXS）是导致智力残疾的最常见原因。它通常是由FMR1中CGG三核苷酸重复（bbbb200个重复）扩增引起的，导致DNA超甲基化和基因沉默。传统的FXS诊断是基于聚合酶链反应（PCR）和Southern blot分析（SB）的结合，这在技术上具有挑战性且劳动密集型。甲基化特异性三联体重复引物PCR （msTP-PCR）已被提出，以克服检测FMR1扩增和甲基化状态的这些局限性。然而，其在临床实验室环境中FXS诊断中的表现仍有待广泛研究。我们验证并实施了msTP-PCR检测，并将其与临床样本中的常规诊断方案进行了比较。共420份临床样本（339名男性和81名女性）先前使用参考方法（基于pcr的方法和/或SB）进行FMR1 CGG重复扩增基因分型，使用msTP-PCR进行研究。结果在等位基因分类、重复数相关性和甲基化状态方面与参考结果高度一致。然而，在罕见的具有复杂嵌合正常、预突变和全突变等位基因的女性中观察到不一致的结果，这需要通过SB进一步证实。尽管如此，我们的结果证实了msTP-PCR方法是FXS诊断和产前检测的一种有用的替代技术，因为它快速、可靠、经济，并且可以潜在地减少SB分析的需求。

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引用次数: 0

Bridge Capture Permits Cost-Efficient, Rapid, and Sensitive Molecular Precision Diagnostics 桥捕获允许成本效益，快速和敏感的分子精密诊断。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-17 DOI: 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.

液体活检定量循环肿瘤DNA （ctDNA）突变的靶向下一代测序已经越来越流行。它们由各种库制备方法完成，每种方法都有其独特的优点和局限性。这项工作介绍了桥捕捉，一种超越市场领先的液体活检技术优势的新技术，消除了在可扩展性、成本效益、灵敏度或面板尺寸之间妥协的需要。采用Bridge Capture、Archer LIQUIDPlex和AmpliSeq CHPv2 for Illumina软件对24份模拟ctDNA的人工结直肠生物标本进行分析，比较变异等位基因频率（VAF）检测结果。Bridge Capture在测序深度要求、实验室间重复性、自动化和面板可扩展性方面进行了评估。在所有方法中，Bridge Capture检测到最低的VAF，所有VAF与Archer LIQUIDPlex （R2 = 0.995）和AmpliSeq CHPv2 Illumina （R2 = 0.988）密切相关。由于其独特的设计，Bridge Capture与常用的下一代测序平台兼容，并有效地利用测序能力，允许经济实惠和敏感的变异检测。该方法在独立实验室和自动化和手动工作流程之间具有高重复性。面板尺寸增加了300%，对探针的性能和交叉反应性的影响可以忽略不计，这意味着高复用能力。综上所述，Bridge Capture是一种具有成本效益、简单、快速和敏感的癌症诊断工具，具有显著改善突变检测的潜力。

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引用次数: 0

Enhanced Comprehension of the Pathogenicity of Splicing Variants 增强对剪接变异体致病性的理解：来自一系列体外和体内功能分析的证据。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-30 DOI: 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.

尽管有各种各样的计算机预测工具，但准确评估剪接区变异的致病性仍然有限。在本研究中，通过体外（minigene）或体内（逆转录聚合酶链反应）实验，对18个不确定意义剪接区变异（VUSs）进行了功能表征。通过建立三维蛋白质结构模型，进一步分析帧内突变对所指示蛋白质的影响。还比较了不同的计算机预测工具（SpliceAI、varSEAK和SPiP）的准确性。18个VUSs包括3个典型和15个非典型剪接变体。在16个变异（88.9%）中观察到剪接异常，外显子跳变（33.3%,6/18）是最常见的事件。其中，11个变异被预测产生过早终止密码子，3个导致帧内改变，2个导致两种结果。结构建模显示，在5个帧内改变中，有4个蛋白质二级结构明显破坏。综合功能和结构证据，15例VUSs（83.3%）被重新分类为致病性/可能致病性，从而为受影响家庭提供明确的诊断和知情的生殖决策。在人工智能工具中，SpliceAI在预测拼接异常方面表现出最高的准确性；然而，所有工具在分析特定剪接模式时的准确性都有所下降。本研究建立了一个在临床诊断中评估剪接VUS致病性的综合工作流程，为克服这一关键的诊断挑战提供了一种实用的方法。

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引用次数: 0

Clinical Validation of Duoseq, a Novel Assay for Clinical DNA and RNA Sequencing Duoseq的临床验证，一种用于临床DNA和RNA测序的新方法。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-28 DOI: 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.

下一代测序（NGS）已成为血癌临床检查不可或缺的一部分。然而，实施临床NGS仍然存在障碍，包括dna和RNAseq所需的单独工作流程，复杂的生物信息学分析和较长的周转时间。Duoseq是针对这些障碍开发的一种全面的、配套的解决方案，并作为血癌基因组分析工具实施，可同时检测单核苷酸变异（snv）、小插入/缺失（indels）和结构变异（SVs，易位和融合）。此外，Duoseq可以评估许多其他血癌诊断标志物，包括基因表达、拷贝数改变、起源细胞、致癌病毒状态（如EBV）、B/T细胞受体克隆性、免疫球蛋白重链突变状态和弥漫性大B细胞淋巴瘤亚型。Duoseq的分析和临床验证在两个临床机构并行进行。检测限snv为5%的变异等位基因频率（VAF）， indels为10%的变异等位基因频率，SVs为≥20%的肿瘤纯度。重复性研究显示>组组内和组间阳性预测值为99%，>组组间阳性预测值为96%。对福尔马林固定的石蜡包埋活检标本（N=197）进行Duoseq，使用NGS和/或FISH进行正交比较。snv、指数和sv的准确率达到了95%。这些结果确立了Duoseq作为血癌基因组分析工具的地位，使实验室能够及时、经济地提供关键的诊断信息。

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引用次数: 0

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) 尼拉帕尼联合醋酸阿比特龙和强的松（尼拉帕尼+ AAP）治疗后转移性阉割抵抗性前列腺癌患者同源重组修复基因改变的组织和血浆诊断分析的临床表现

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-28 DOI: 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.

本回顾性研究旨在证明两种诊断测试的临床应用，FoundationOneCDx （F1CDx）和Exact Sciences Resolution同源重组缺陷（HRD）（Resolution HRD测定）作为临床试验入组分析，以识别携带同源重组修复（HRR）基因改变的mCRPC患者。在3期量级研究中，从mCRPC患者收集的肿瘤组织和血浆使用F1CDx组织试验和/或Resolution HRD血浆试验进行测试。HRR改变的患者随机（1:1）接受尼拉帕尼（NIRA）和醋酸阿比特龙(AA)+强的松（NIRA+AAP）或安慰剂+AAP （PBO+AAP; NCT03748641）。疗效基于放射学无进展生存期（rPFS，主要终点）、到症状进展时间（TSP）、到细胞毒性化疗时间（TCC）和总生存期（OS）作为次要终点（中期分析1）。423例HRR患者中，F1CDx检测HRR阳性（HRR+） 291例（68.8%）（BRCA+:162例[38.2%]），F1CDx检测HRR阴性（HRR-） 38例（8.9%），Resolution HRD检测HRR+。此外，Resolution HRD法HRR+ (BRCA+:150[35.5%])的患者277/423例（65.5%），Resolution HRD法HRR-但F1CDx法HRR+的患者124例（29.3%）。通过组织或血浆检测检测BRCA+和HRR+患者，所有终点的临床意义获益均相当。这些结果证明了组织和血浆检测在确定需要NIRA+AAP治疗的患者中的临床应用。

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引用次数: 0

Validation of a Modular Gene Expression Assay for Risk Stratification and Subtyping Lymphomas 一种用于淋巴瘤风险分层和分型的模块化基因表达试验的验证。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-10 DOI: 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.

尽管常规诊断工作主要使用免疫组织化学染色和荧光原位杂交，但基因表达特征对于淋巴细胞恶性肿瘤的分类是重要的。这些传统的方法是劳动密集型的，可能会错误地分类潜在的致癌特征，导致不准确的预测。为了解决这个问题，我们开发了一个RNA表达面板，即淋巴瘤表达分析（LExA120） 120基因表达面板，使用NanoString平台对各种淋巴瘤亚型进行快速模块化分析。LExA120小组针对95个基因和25个管理基因来评估侵袭性b细胞淋巴瘤，包括弥漫性大b细胞淋巴瘤起源细胞、暗区和原发性纵隔大b细胞淋巴瘤特征、eb病毒（EBV）状态和典型霍奇金淋巴瘤移植后的风险。54份经福尔马林固定石蜡包埋的组织样本诊断为已知，51份为已知EBV状态。根据签名分数的Pearson相关系数，面板显示与先前验证的方法高度一致。该分析在重复测试和不同临床实验室中也显示出高再现性。我们的研究证实了该小组对ebv阳性和ebv阴性淋巴瘤进行分层的能力，具有很高的诊断确定性。虽然大约12%的病例需要原位杂交确认，但与传统技术的协同可能有助于更快速和更具成本效益的诊断。LExA120面板提供了淋巴瘤分类的多路方法，提高了淋巴瘤分型的效率和准确性。

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引用次数: 0

Preanalytical Histology Review Improves POLE Mutation Detection in Endometrial Carcinomas 分析前组织学检查提高了子宫内膜癌中POLE突变的检测。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-17 DOI: 10.1016/j.jmoldx.2025.09.008

Hehua Huang, Sara Hartinger, Rachel L.P. Geisick, Chandler Ho, Fei Dong

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.

分析前组织评估是肿瘤分子检测的重要步骤；然而，对分子检测结果的影响尚未得到系统评价。本研究描述一分子诊断实验室实施常规组织学检查之品质改善计画。使用多重PCR法对1752例子宫内膜癌进行了POLE突变检测。在实施一项方案后，对干预前和干预后的队列进行比较，该方案包括病理学家对组织学切片的审查，肿瘤内容的记录，以及肿瘤细胞含量低于40%的样本的肿瘤富集。干预前和干预后POLE突变率分别为6.3%和5.0% （p = 0.25）。最常见的突变是POLE p.Pro286Arg（47%）和POLE p.Val411Leu（21%）。干预后，肿瘤细胞的记录从29%增加到100% （p < 0.0001）。肿瘤富集率从1.4%增加到31.5% （p < 0.0001）。突变与野生型峰值比值的突变信号强度中位数从0.32增加到0.58 （p = 0.004）。重复检测率由8.8%降至2.3% （p < 0.0001）。系统的分析前组织学回顾提高了靶向POLE分析的分析性能，同时也提高了肿瘤细胞记录的符合性，增加了肿瘤富集，减少了重复测试。这些发现支持分析前评估，以提高低肿瘤含量病理标本的体细胞突变检测。

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引用次数: 0

Enhanced Detection of Splice-Altering Variants in Hematologic Malignancies Using Targeted RNA-Sequencing Data 利用靶向RNA-seq数据增强血液恶性肿瘤剪接改变变异的检测。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-10 DOI: 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.

基于rna的靶向测序有助于检测血液病和其他恶性肿瘤中的几种变异，包括剪接改变的变异。然而，由于人类转录组固有的复杂性和深度RNA测序数据中假阳性剪接的高流行率，准确识别临床相关的错误剪接事件仍然具有挑战性。为了解决这些挑战，SpliceChaser和BreakChaser被开发出来，它们是生物信息学工具，旨在增强相关剪接改变事件的检测和表征。SpliceChaser通过分析剪接连接周围的映射reads的侧翼序列内的读长多样性，提高了临床相关的非典型剪接的识别。BreakChaser处理软剪切序列和比对异常，以增强对与染色体内基因缺失产生的非典型剪接异构体相关的靶向缺失断点的检测。这些工具是使用来自慢性髓性白血病（CML）患者的1400多个RNA-seq样本进行开发和验证的。总的来说，SpliceChaser和BreakChaser在检测临床相关的非典型剪接改变变异或靶区域基因缺失方面达到了98%的阳性百分比一致性和91%的阳性预测值。通过将剪接和断点检测与强大的过滤策略相结合，这些工具有助于精确识别临床相关变异，为改进CML和其他恶性肿瘤的诊断和治疗策略铺平道路。

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引用次数: 0

Characterization of Large Genomic Rearrangements in BRCA1 and BRCA2 Genes in a Chinese High-Risk Cohort 中国高危人群中BRCA1和BRCA2基因大基因组重排的特征

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-12-23 DOI: 10.1016/j.jmoldx.2025.09.005

Ava Kwong , Cecilia Y.S. Ho , Henry C.M. Leung , Amy W.S. Leung , Chun Hang Au , Edmond S.K. Ma

Large genomic rearrangements (LGRs) account for at least 10% of the mutations in BRCA1 and 5% of BRCA2 mutations in outbred families with hereditary breast and ovarian cancer. A total of 21 probands with breast cancer who carried BRCA1 or BRCA2 LGRs were identified from a cohort of 4678 Chinese patients. There was a total of 13 BRCA1 LGR carriers and 8 BRCA2 LGR carriers, including 12 large genomic deletions and 1 duplication. Ten and three specific breakpoints from BRCA1 and BRCA2, respectively, were identified by either whole-genome sequencing by nanopore sequencing or long-range PCR. Five of these LGRs were recurrent LGRs. Three LGRs were novel founder LGRs in the southeast Chinese population. Chinese LGR carriers exhibited clinical phenotypes that were generally similar to those of non-LGR mutation carriers. However, there was a notable tendency for triple-negative breast cancer to be more prevalent among Chinese LGR carriers (P = 0.007), largely because of the predominance of BRCA1 mutations. This suggests a potential association that warrants further investigation.

大基因组重排（lgr）至少占遗传性乳腺癌和卵巢癌远亲家族BRCA1突变的10%和BRCA2突变的5%。从4678名中国患者中共鉴定出21名携带BRCA1或BRCA2 lgr的乳腺癌先证者。BRCA1 LGR携带者13例，BRCA2 LGR携带者8例，其中大基因组缺失12例，重复1例。通过纳米孔测序或远程PCR的全基因组测序，分别鉴定了BRCA1和BRCA2的10个和3个特定断点。其中5例为复发性lgr。三个lgr是中国东南地区新颖的创始人lgr。中国LGR携带者的临床表型与非LGR突变携带者大体相似。然而，三阴性乳腺癌在中国LGR携带者中更为普遍（P = 0.007），这主要是因为BRCA1突变占主导地位。这表明一种潜在的关联值得进一步调查。

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引用次数: 0

Comparative Analysis of TP53-Mutated Tumor DNA in Saliva and Plasma of Patients with Head and Neck Squamous Cell Carcinoma 头颈部鳞状细胞癌患者唾液和血浆中tp53突变肿瘤DNA的比较分析。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2026-01-01 Epub Date: 2025-10-28 DOI: 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.

液体活检为实体癌组织取样提供了一种有前途的非侵入性替代方法。唾液是一种易于获取的生物液体，可携带肿瘤DNA，但其与血浆循环肿瘤DNA （ctDNA）在头颈部鳞状细胞癌（HNSCC）中的临床应用仍不确定。对HNSCC患者的肿瘤样本进行新一代测序以鉴定TP53突变。分析匹配的血浆和唾液细胞游离DNA （cfDNA）样本是否存在肿瘤特异性突变。病理特征和生存数据与生物体液中突变的可检测性进行了评估。总体而言，85例肿瘤中有64例（75%）检测到TP53突变。液体活检分析包括来自40例患者的36份血浆和21份唾液样本。68%的口腔鳞状细胞癌检测到血浆ctDNA，而喉癌的这一比例为29%，并且与淋巴结转移相关（p = 0.034）。ctDNA检测显示无进展生存期更差（37.4个月对68.5个月，p = .134）。57%的病例在唾液cfDNA中发现肿瘤突变，无论疾病分期或是否存在区域转移。血浆ctDNA成为HNSCC的潜在预后标志物，而唾液cfDNA突变虽然经常检测到，但缺乏预后价值或与不良病理特征的相关性。进一步的研究需要阐明肿瘤DNA脱落到唾液中的机制，并确定其临床应用。

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引用次数: 0