Journal of Molecular Diagnostics最新文献_第5页

Analysis of the Fragmentation and Integrity of Urine Cell-Free DNA as a Diagnostic and Staging Biomarker for Bladder Cancer 尿无细胞DNA片段和完整性作为膀胱癌诊断和分期生物标志物的分析。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-26 DOI: 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.

膀胱癌（BC）是一种致命的泌尿系统恶性肿瘤，目前的诊断和随访方法是侵入性的和昂贵的。液体活检中的无细胞DNA （cfDNA）在癌症诊断中显示出前景，但其在BC患者尿液中的碎片性和完整性仍未得到充分探索，这成为本研究的目的。cfDNA从156例大多数阶段的BC患者和79例没有肾脏或膀胱疾病的对照组的尿液中分离出来。一个大（>250bp）和一个嵌套的小(

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引用次数: 0

Evaluation of Long-Read Genome Sequencing for Genomic Profiling of Myeloid Cancers 髓系癌基因组分析的长读基因组测序评估。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-26 DOI: 10.1016/j.jmoldx.2025.09.001

Haley J. Abel , Mohamed Mahgoub , Nidhi Davarapalli , Rohan Kodgule , Christopher A. Miller , Robert S. Fulton , Catrina Fronick , Christopher Markovic , Sharon Heath , Jacqueline E. Payton , Meagan A. Jacoby , Daniel C. Link , Matthew J. Walter , Eric J. Duncavage , Timothy J. Ley , David H. Spencer

Whole-genome sequencing (WGS) is a comprehensive approach for the genomic evaluation of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We recently described a streamlined tumor-only WGS assay (ChromoSeq) that uses Illumina short-read sequencing with targeted analysis to detect the full range of clinically relevant somatic mutations. Here we sought to determine the performance of this targeted analysis approach using long-read sequencing data from Oxford Nanopore Technologies and Pacific Biosciences. Samples from 26 patients with AML and MDS were sequenced to a mean of 52× coverage. Head-to-head comparison of reportable somatic variants to standard WGS revealed more than 96% recall and 91% precision for single nucleotide variants for both long-read platforms. Performance was lower for insertion/deletions (66% recall and 42% precision), especially in regions with few phased reads that facilitate accurate variant detection. The long-read platforms were 95% accurate for copy number calls, and they detected all recurrent structural variants with no false-positive findings. In addition, long reads properly identified intronic insertions near repetitive elements that were incorrectly identified as interchromosomal structural rearrangements by standard WGS. These results indicate that targeted, tumor-only analysis of long-read sequence data is a feasible approach for the genomic evaluation of myeloid cancers, and they show the utility of incorporating variants discovered via long-read sequencing to improve variant interpretation in short-read WGS.

全基因组测序（WGS）是一种用于急性髓性白血病（AML）和骨髓增生异常综合征（MDS）基因组评估的综合方法。我们最近描述了一种精简的肿瘤WGS检测（ChromoSeq），该检测使用Illumina短读测序和靶向分析来检测临床相关的全范围体细胞突变。在这里，我们试图利用牛津纳米孔技术公司和太平洋生物科学公司的长读序列数据来确定这种针对性分析方法的性能。对26例AML和MDS患者的样本进行测序，平均覆盖率为52倍。可报告体细胞变异与标准WGS的头对头比较表明，在两个长读平台上，单核苷酸变异（SNV）的召回率均大于96%，精确度为91%。插入/删除的性能较低（召回率为66%，准确率为42%），特别是在阶段读取较少的区域，这有助于准确检测变异。长读平台对拷贝数调用的准确率为95%，检测到所有复发性结构变异（SV），无假阳性。此外，长读数正确地识别了重复元件附近的内含子插入，这些插入被标准WGS错误地识别为染色体间结构重排。这些结果表明，针对长读序列数据的肿瘤分析是髓系癌症基因组评估的一种可行方法，并证明了将通过长读序列发现的变异纳入短读WGS的实用性，以改善变异解释。

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引用次数: 0

Analytical Validation of a Pan-Cancer Next-Generation Sequencing Assay for In-House Liquid Biopsy Testing 用于内部液体活检检测的泛癌症NGS检测的分析验证：一项国际多中心研究。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-25 DOI: 10.1016/j.jmoldx.2025.08.008

Gaëlle Lescuyer , Alexandre Harlé , Hari Shankar Kumar , Pantelis Constantoulakis , Nicole Pfarr , Ellen Heitzer , Clémence Michon , Gianluca Russo , Ernst-Jan M. Speel , Marie Piecyk , Marie Husson , Georgia Christopoulou , Eva-Maria Mayr , Mai-Lan Koppermann , Christophe Passot , Ricarda Graf , Anes Hadjadj Aoul , Violaine Bourdon , Hendrikus J. Dubbink , Ronald van Marion , Léa Payen

Liquid biopsy assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumor genetic alterations in circulating free DNA, which typically requires detecting variants at low allele frequencies and, therefore, a high sensitivity and specificity. This international, multicenter analytical performance study evaluated the Hedera Profiling 2 circulating tumor DNA test panel (HP2), a hybrid capture–based next-generation sequencing assay for the detection of somatic alterations in circulating free DNA. Covering 32 genes, HP2 enables the detection of single-nucleotide variants (SNVs), insertions and deletions (Indels), fusions, copy number variations, and microsatellite instability status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples precharacterized by orthogonal methods. In reference standards with variants spiked in at 0.5% allele frequency, sensitivity and specificity were 96.92% and 99.67%, respectively, for SNVs/Indels and 100% for fusions. In clinical samples, SNV/Indel detection showed high concordance (94% for European Society for Medical Oncology Scale of Clinical Actionability for Molecular Targets level I variants) with orthogonal methods. Evidence for solid sensitivity was also found for copy number variation detection and microsatellite instability status determination. Overall, the HP2 assay showed significant potential as a sensitive and efficient pan-cancer test for liquid biopsy testing in a decentralized molecular pathology laboratory setting.

液体活检（LBx）通过提供一种侵入性较小的组织活检替代方法，正在改变精确肿瘤学。这些检测筛选循环游离DNA （cfDNA）中的肿瘤遗传改变，这通常需要检测低等位基因频率的变异，因此具有高灵敏度和特异性。这项国际多中心分析性能研究评估了Hedera Profiling 2 ctDNA测试面板（HP2）（Hedera Dx, epalings，瑞士），这是一种基于混合捕获的NGS检测方法，用于检测cfDNA的体细胞改变。HP2覆盖32个基因，能够在单一dna工作流程中检测snv、indel、Fusions、cnv和MSI状态。采用参考标准和采用正交法预表征的137个临床样本的不同队列来评估分析性能。在等位基因频率为0.5%的参考标准中，SNVs/Indels的敏感性和特异性分别为96.92%和99.67%，Fusions的敏感性和特异性分别为100%。在临床样本中，用正交方法检测snv /Indels显示出高一致性（ESCAT I级变异为94%）。在CNV检测和MSI状态测定中也发现了固体灵敏度的证据。总的来说，在分散的分子病理实验室环境中，HP2检测显示出作为一种敏感和有效的泛癌检测LBx的巨大潜力。

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引用次数: 0

Validation and Clinical Utility of a Pan-Cancer Circulating Tumor DNA Assay as a First-Approach Test 泛癌循环肿瘤DNA检测作为第一步检测的验证和临床应用。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-25 DOI: 10.1016/j.jmoldx.2025.08.009

Nisha Kanwar , Michael B. Campion , Amber R. Schneider , Dragana Milosevic , Carlos Sosa , Antonina A. Wojcik , Kevin C. Halling , Kandelaria M. Rumilla , Ying-Chun Lo , Zhiyv Niu , Katherine B. Geiersbach , Margaret A. DiGuardo , Benjamin R. Kipp , Gang Zheng

The feasibility of circulating tumor (ct)-DNA assays in first-approach pan-cancer genomic profiling is not well established. Furthermore, low ctDNA levels limit assay sensitivity, which challenges adaptation to clinical genomic profiling. In this study, a 33-gene next-generation sequencing–based ctDNA panel was validated, and these issues were investigated using real-world clinical data. The cohorts included 123 patients who underwent first-approach ctDNA testing, and 48 patients for whom matched tissue was tested at the same time-point. The overall ctDNA assay failure rate was 0%. Insufficient tumor tissue was the main reason for liquid biopsy (69%). The most common primary cancer profiled was lung (39.0%), followed by colon (13.8%), bile duct (8.9%), pancreas (8.1%), and breast and prostate (each 4.1%). Tier I variants were detected in 33.3% of patients, and Tier I or II variants were detected in 65.0% (including 54.5% cholangiocarcinomas, in which tissue biopsy may be challenging due to anatomic location). Compared with matched tissue, ctDNA showed 76% sensitivity for Tier I variants. Actionable variants were increased by 14.3% with ctDNA versus tissue testing alone. ctDNA results preceded tissue results by an average of 21 days. High feasibility, actionability, and sensitivity support ctDNA assays as a potential first-line genomic test, especially in specific tumor types for advanced tumors with insufficient or unavailable tissue.

循环肿瘤DNA （ctDNA）检测在泛癌症环境中的可行性尚不明确。此外，低ctDNA水平限制了测定的敏感性，这对适应临床基因组图谱提出了挑战。验证了33个基因的下一代测序（NGS） ctDNA面板，并使用实际临床数据调查了这些问题。临床队列包括123例ctDNA检测作为第一种方法的患者，以及48例在同一时间点进行匹配组织检测的患者。总体ctDNA检测失败率为0%。肿瘤组织不足是液体活检的主要原因（69%）。最常见的原发癌是肺癌（39.0%），其次是结肠癌（13.8%）、胆管癌（8.9%）、胰腺癌（8.1%）、乳腺癌（4.1%）和前列腺癌（4.1%）。使用AMP/ASCO/CAP指南，在33.3%的患者中检测到I级变异，在65.0%的患者中检测到I级或II级变异（包括54.5%的胆管癌患者，由于解剖位置的原因，组织活检具有挑战性）。与匹配组织相比，ctDNA对I级变异的敏感性为76%。与单独的组织检测相比，并发检测使可操作变异的数量增加了14.3%。ctDNA结果比组织结果平均早21天。总之，高可行性、可操作性和敏感性支持ctDNA检测作为潜在的一线基因组检测，特别是在无法获得组织的晚期肿瘤的特定肿瘤类型中。

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引用次数: 0

Next-Generation Sequencing–Based T-Cell Receptor Gene Rearrangement Analysis in Nodal T Follicular Helper Cell Lymphoma, a Comparison with the EuroClonality/BIOMED-2 Assay 淋巴结T滤泡辅助细胞淋巴瘤中基于下一代测序的T细胞受体基因重排分析，与EuroClonality/BIOMED-2检测的比较

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.08.007

Ryuko Nakayama , Leonie I. Kroeze , Jeroen Luijks , Avital Amir , Jos Rijntjes , Konnie M. Hebeda , Patricia J.T.A. Groenen

Nodal T follicular helper cell lymphoma (nTFHL) can be difficult to diagnose because it often shows features of immune dysregulation and can have a low number of neoplastic T cells in involved lymph nodes. The analysis of T-cell receptor (TR) gene rearrangements by next-generation sequencing (NGS) and the conventional EuroClonality/BIOMED-2 were performed to compare their performance on this challenging diagnosis. DNA was extracted from 32 formalin-fixed, paraffin-embedded nTFHL samples from two pathology archives. NGS amplicon-based analysis of TRBV-TRBD-TRBJ, TRBD-TRBJ, and TRGV-TRGJ rearrangements was performed using the two-step PCR protocols developed by EuroClonality. The nucleotide sequences were analyzed for abundance, clonotype, and functionality. Both the NGS-based and the conventional clonality assays resulted in a high detection of clonality (97% and 94%, respectively), including both monoclonal and biclonal cases. There was an overrepresentation of TRBV20-1 and TRBV19 gene use that was in line with the frequent use of these genes in T cells of reactive lymph nodes and tonsils. The NGS-based approach detected two or more clonal targets in all clonal samples, whereas the conventional assay detected a single (isolated) dominant rearrangement in three cases. Hence, NGS enables more reliable detection of even small clones, as is frequent in nTFHL. NGS-based TR rearrangement analysis provides the abundance, the sequences, clonotypes, and productivity of the clonal rearrangements and thus stresses the need for novel guidelines for interpretation.

淋巴结T滤泡辅助细胞淋巴瘤（nTFHL）很难诊断，因为它经常表现出免疫失调的特征，并且在受病灶淋巴结中可能有少量的肿瘤T细胞。通过下一代测序（NGS）和传统的EuroClonality/BIOMED-2对t细胞受体（TR）基因重排进行分析，比较它们在这一具有挑战性的诊断中的表现。从2份病理档案中提取32份经福尔马林固定石蜡包埋的nTFHL样本，提取DNA。采用EuroClonality开发的两步pcr技术，对TRBV-TRBD-TRBJ、TRBD-TRBJ、TRGV-TRGJ重排进行NGS扩增子分析。分析核苷酸序列的丰度、克隆型和功能。基于ngs的和传统的克隆性测定均可获得较高的克隆性检测（分别为97%和94%），包括单克隆和双克隆病例。TRBV20-1和TRBV19基因的使用比例过高，这与这些基因在反应性淋巴结和扁桃体的t细胞中的频繁使用一致。基于ngs的方法在所有克隆样品中检测到两个或多个克隆靶点，而传统分析在三个病例中检测到单个（“分离的”）显性重排。因此，NGS能够更可靠地检测小克隆，这在nTFHL中很常见。基于ngs的TR重排分析提供了克隆重排的丰度、序列、克隆型和生产力，因此强调了对新的解释指南的需求。

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引用次数: 0

Celebrating 25 Years of Innovation with a Tribute to Our Contributors and the Future of Molecular Diagnostics 庆祝25年的创新，致敬我们的贡献者和分子诊断的未来

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.08.001

Ronald M. Przygodzki (Editor-in-Chief)

引用次数: 0

Analytical Validation of Blood-Derived Tumor Mutation Burden (bTMB) Assays 血源性肿瘤突变负荷（bTMB）测定的分析验证

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.06.008

Jonathan Baden , Mark Sausen , Andrew T. Anfora , Kevin M. D'Auria , Jennifer Dickey , James H. Godsey , Li Guan , Jennifer S. Lococo , Elizabeth Mansfield , Kristen L. Meier , David Merriam , Traci Pawloski , Soni Shukla , Daniel Stetson , Mark D. Stewart , Paul Wenz , Lauren C. Leiman

Immunotherapies have changed the treatment paradigm for patients with advanced and metastatic solid tumors, with tumor mutation burden representing one approach to identify patients who may experience clinical benefit. Circulating tumor DNA–based approaches have been developed for comprehensive analyses of clinically actionable biomarkers; however, blood tumor mutation burden (bTMB) represents a novel, complex biomarker. Although the clinical utility of bTMB is an evolving area of active development and has not led to consistent conclusions across studies, robust analytical validation of the underlying test is important to ensure that technical and biological limitations do not confound clinical interpretation of these results. To this end, the BLOODPAC bTMB Analytical Validation Working Group sought to identify key technical and biological issues associated with analytical validation of bTMB tests, along with a conceptual framework to address these challenges. This publication provides guidance for device manufacturers to demonstrate analytical performance of their test with the understanding that these data would be accompanied by an appropriately designed clinical validation study to demonstrate performance within the intended use population. Therefore, the specific algorithm to determine the bTMB result, along with the associated cutoff, is out of scope of this Perspective. Device manufacturers should also ensure that appropriate pre-analytical variables are accounted for and methods are incorporated to differentiate tumor-specific alterations from those associated with germline polymorphisms or clonal hematopoiesis.

免疫疗法已经改变了晚期和转移性实体瘤患者的治疗模式，肿瘤突变负担代表了确定可能经历临床获益的患者的一种方法。基于循环肿瘤dna的方法已被开发用于临床可操作的生物标志物的综合分析；然而，血液肿瘤突变负荷（bTMB）是一种新的、复杂的生物标志物。尽管bTMB的临床应用是一个不断发展的积极发展领域，并没有在所有研究中得出一致的结论，但对基础试验进行强有力的分析验证对于确保技术和生物学限制不会混淆这些结果的临床解释非常重要。为此目的，血凝血酶分析验证工作组力求确定与血凝血酶测试分析验证相关的关键技术和生物学问题，并提出解决这些挑战的概念框架。本出版物为器械制造商提供指导，以证明其检测的分析性能，并理解这些数据将伴随适当设计的临床验证研究，以证明在预期使用人群中的性能。因此，确定bTMB结果的具体算法以及相关的截止，超出了本透视图的范围。器械制造商还应确保考虑到适当的分析前变量，并采用方法区分肿瘤特异性改变与生殖系多态性或克隆造血相关的改变。

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引用次数: 0

Performance Evaluation of the Aptima hrHPV Nucleic Acid Amplification and Papanicolaou Co-Testing in Cervical Cancer Screening Aptima hrHPV核酸扩增和Papanicolaou联合检测在宫颈癌筛查中的性能评估：来自不同人群的回顾性研究

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.08.006

Sagee Tal , Jordan A. Vowels , Daniel González , Efstathia Polychronopoulou , Ranjana S. Nawgiri , Ping Ren

Cervical cancer screening is evolving, with guidelines increasingly favoring high-risk human papillomavirus (hrHPV) nucleic acid amplification tests (NAATs) as a primary method. Although hrHPV NAATs offer high sensitivity for HPV-driven cervical cancers, Papanicolaou (Pap) cytology can detect additional gynecologic malignancies, including HPV-independent ones. This study evaluated the Hologic Aptima hrHPV NAAT and Pap co-testing using 61,089 samples from patients aged ≥21 years screened between 2018 and 2023. The cohort was predominantly White (78.4%), with 16.5% Black, 4.1% Asian, and 38.9% Hispanic subjects. Overall percentage agreement (PA) between hrHPV NAAT and Pap was 87.9%, with high negative PA (93.6%) and lower positive PA (52.3%). Excluding atypical squamous/glandular cell abnormalities improved overall and positive PA to 93.0% and 78.3%, respectively. The Aptima hrHPV NAAT showed high sensitivity for high-grade squamous intraepithelial lesions (98.3%) and squamous cell carcinoma (90.0%) but lower sensitivity for low-grade lesions (73.3%), adenocarcinoma (13.3%), and other malignancies (28.6%). Notably, 29 high-grade lesions or malignant cases had abnormal Pap but were hrHPV negative; 24 (82.8%) were histologically confirmed, including endometrial, cervical, ovarian, and fallopian tube carcinomas (mostly non–HPV-related). These findings emphasize cytology's role in detecting malignancies potentially missed by hrHPV testing alone and support co-testing in diverse populations. The Aptima hrHPV NAAT is not approved by the US Food and Drug Administration for primary HPV screening, reinforcing this need.

宫颈癌筛查正在发展，目前的指南越来越倾向于高危人乳头瘤病毒（hrHPV）核酸扩增检测（NAAT）作为主要方法。虽然hrHPV NAATs对HPV驱动的宫颈癌具有高灵敏度，但巴氏细胞学可以检测到其他妇科恶性肿瘤，包括与HPV无关的肿瘤。该研究评估了Hologic Aptima hrHPV NAAT和PAP联合检测的性能，使用了2018年至2023年筛查的年龄≥21岁患者的61,089份样本。该队列以白人为主（78.4%），黑人占16.5%，亚洲人占4.1%，西班牙人占38.9%。hrHPV NAAT和PAP检测的总体一致性百分比（PA）为87.9%，其中高阴性PA（93.6%）和低阳性PA（52.3%）。排除非典型鳞状/腺细胞异常（AS/AG）将总体PA和阳性PA分别提高到93.0%和78.3%。Aptima hrHPV NAAT对高级别鳞状上皮内病变（HSIL）（98.3%）和鳞状细胞癌（90.0%）具有较高的敏感性，但对低级别鳞状上皮内病变（LSIL）（73.3%）、腺癌（13.3%）和其他恶性肿瘤（28.6%）的敏感性较低。值得注意的是，29例HSIL或恶性病例PAP异常，但hrhpv阴性；24例（82.8%）经组织学证实，包括子宫内膜癌、宫颈癌、卵巢癌和输卵管癌，大多数与hpv无关。这些发现支持细胞学在识别单独hrHPV检测可能遗漏的恶性肿瘤方面的重要作用，并支持在不同人群中进行联合检测。Aptima hrHPV NAAT未获fda批准用于原发性HPV筛查，这加强了这一需求。

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引用次数: 0

TAG-CLL, a Novel Tagmentation-Based Approach to Somatic Hypermutation Testing of IGHV Reveals the Weak Points of Both Sanger and Next-Generation Sequencing Methods TAG-CLL是一种新的基于标记的IGHV体细胞超突变检测方法，它揭示了Sanger和NGS方法的弱点。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.08.005

Marzena Wojtaszewska , Monika Szelest , Marta Szarawarska , Jarosław Grzyb , Beata Blajer-Olszewska , Michał Gniot , Emilia Jaskuła , Jarosław Dybko , Michał Soin , Katarzyna Wasilewska , Sylwia Czekalska , Magdalena Zawada , Magdalena Wojtas , Iwona Solarska , Agnieszka Kwak , Piotr Wójcik , Ewelina Nowak-Ozimek , Artur Kowalik , Tomasz Stokłosa , Agnieszka Chudy , Mirosław Markiewicz

Somatic hypermutation (SHM) status of IGHV gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose between inefficient Sanger sequencing protocols and expensive, recently developed next-generation sequencing–based methods. The performance of both types of methods seemed incomparable, and concerted validation of different protocols between laboratories was inconsiderable. Here, a new tagmentation-based approach to sequencing of IGHV locus is presented, which is agnostic to the amplification protocol used and enables direct comparison of the amplicons and libraries dedicated to different platforms (Sanger, IonTorrent, and Illumina). To demonstrate its potential, the 12 associated molecular diagnostics laboratories were asked to amplify an artificially prepared oligoclonal DNA sample containing a near-equimolar mixture of IGHV clonotypes from six different classes. The PCR products collected from laboratories were then tagmented and sequenced according to the common TAG-CLL workflow. The productivity, degree of germline identity, and SHM status concordance between laboratories have been analyzed. Moreover, systematic biases toward uneven amplification of different clonotypes and the prevalence of accidental artifacts in vitro and in silico have been evaluated, providing a framework for future validation of IGHV SHM methods and next-generation sequencing immunoinformatic pipeline benchmarking.

IGHV基因的体细胞超突变状态（SHM）尽管是慢性淋巴细胞白血病（CLL）的成熟诊断生物标志物，但给分子实验室带来了严重的方法学问题。他们可能会在效率低下的Sanger测序方案和最近开发的昂贵的下一代基于测序的方法（NGS）之间做出选择。这两种方法的性能似乎是无与伦比的，而且实验室之间对不同方案的一致验证——微不足道。本文提出了一种新的基于标记的IGHV基因座测序方法，该方法与所用的扩增协议无关，可以直接比较专用于不同平台的扩增子和文库（Sanger, IonTorrent, Illumina）。为了证明其潜力，12个相关的分子诊断实验室被要求扩增人工制备的寡克隆DNA样本，该样本包含来自6个不同类别的IGHV克隆型的近等摩尔混合物。然后根据通用的TAG-CLL工作流程对从实验室收集的PCR产物进行标记和测序。分析了实验室间的生产力、种系同一性程度和SHM状态一致性。此外，对不同克隆型扩增不均匀的系统性偏差以及体外和计算机中意外伪像的普遍存在进行了评估，为未来验证IGHV SHM方法和NGS免疫信息学管道基准提供了框架。

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引用次数: 0

A Mass Spectrometry–Based Multiplexed Targeted Assay for Detection of Hemoglobinopathies from Dried Blood Spots 一种基于质谱的多路靶向检测干燥血斑血红蛋白病的方法

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-09-23 DOI: 10.1016/j.jmoldx.2025.07.003

Anikha Bellad , Kannan Rangiah , Sandip Chavan , Jayesh Warade , Barnali Das , Akhilesh Pandey

Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. The goal was to develop and optimize a sensitive and specific mass spectrometry–based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated, and their corresponding heavy synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the most common mass spectrometer used in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal patients and patients with β-thalassemia, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous, or compound heterozygous states. The intra-assay and interassay precision CV were both <20%. We envision that such mass spectrometry–based assays could be used as first-line screening assay for hemoglobin variants, including sickle cell disease as well as thalassemias.

血红蛋白病是世界上最常见的遗传性疾病。准确分析血红蛋白变异是诊断血红蛋白病的关键。虽然高效液相色谱和毛细管区带电泳被广泛用作筛选工具，但它们具有固有的模糊性，往往妨碍准确检测血红蛋白变异。目的是开发和优化一种基于质谱的敏感和特异性检测方法，用于筛查和诊断血红蛋白病。生成典型的珠蛋白链特异性肽和常见血红蛋白变体对应的突变肽目录，并将其对应的重合成肽版本作为定量和计算珠蛋白链比率的内部标准。靶向质谱分析是通过液相色谱耦合三重四极杆质谱仪进行的，这是临床诊断中最常用的质谱仪。对716例个体（包括211例血红蛋白病患者）的干血斑进行分析。α:β-珠蛋白比率在正常患者和β-地中海贫血患者之间显示出显著差异，特别是当疾病是纯合子或混合结构变异（复合杂合）时。本文提出的方法允许在纯合子、杂合子或复合杂合子状态下识别变异。测定内、间精密度CV均为20%。我们设想，这种基于质谱的检测方法可用于血红蛋白变异的一线筛查，包括镰状细胞病和地中海贫血。

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