Journal of Molecular Diagnostics最新文献_第9页

Evaluating Discordant Somatic Calls Across Mutation Discovery Approaches to Minimize False-Negative Drug-Resistant Findings 评估突变发现方法中不一致的体细胞呼叫，以尽量减少假阴性耐药结果。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-06-13 DOI: 10.1016/j.jmoldx.2025.04.012

Hsin-Fu Lin , Pei-Miao Chien , Chinyi Cheng , Tzu-Hang Yuan , Yu-Bin Wang , Pei-Lung Chen , Chien-Yu Chen , Jia-Hsin Huang , Jacob Shujui Hsu

Evaluating robustness of somatic mutation detections is essential when using whole-exome sequencing (WES) for treatment decision-making. A comprehensive evaluation was conducted using tumor WES from the US Food and Drug Administration–led Sequencing Quality Control Phase 2 project, in which multiple library kits sequenced identical DNA materials across three laboratories to benchmark analytical validity. These workflows included various read aligner (BWA, Bowtie2, DRAGEN-Aligner, DRAGMAP, and HISAT2) and mutation caller (Mutect2, TNscope, DRAGEN-Caller, and DeepVariant) combinations. The results revealed that DRAGEN exhibited superior performance, achieving mean F1 scores of 0.966 and 0.791 for single-nucleotide variant and insertion/deletion detection, respectively. Among open-source software, BWA Mutect2 and HISAT2 Mutect2 combinations showed the highest mean F1 scores for single-nucleotide variant (0.949) and insertion/deletion (0.722), respectively. The analyses indicated that high-quality data can be analyzed as having worse results, and vice versa. Evaluations of Catalog of Somatic Mutations in Cancer reported mutations unveiled discrepancies across enrichment kits. Integrated DNA Technologies enrichment kits showed a higher false-negative rate, whereas Agilent WES kits tended to miss mutations in CBL and IDH1, and Roche library kits tended to miss the mutations in PIK3CB. Sentieon TNscope tended to underestimate tumor mutation burden and overlook FLT3:c.G1879A for cytarabine resistance in leukemia and MAP2K1:c.G199A for BRAF inhibitors in melanoma. The findings highlight the importance of robust bioinformatic analysis in guiding clinical decision-making.

当利用全外显子组测序（WES）进行治疗决策时，评估体细胞突变检测的稳健性是必不可少的。使用来自fda主导的测序质量控制第二阶段（SEQC2）项目的肿瘤WES进行了全面评估，其中多个文库试剂盒在三个实验室对相同的DNA材料进行了测序，以基准分析有效性。这些工作流程包括各种读取对齐器（BWA, Bowtie2, DRAGEN-Aligner， DRAGMAP和HISAT2）和突变调用器（Mutect2, TNscope， DRAGEN-Caller和DeepVariant）组合。结果表明，DRAGEN检测SNV和INDEL的平均f1得分分别为0.966和0.791，具有较好的性能。在开源软件中，BWA Mutect2和HISAT2 Mutect2组合SNV和INDEL的平均f1得分最高，分别为0.949和0.722。分析表明，高质量的数据可以被分析为具有较差的结果，反之亦然。对COSMIC报告的突变的评估揭示了富集试剂盒之间的差异。IDT富集试剂盒假阴性率较高，而安捷伦WES试剂盒容易漏检CBL和IDH1突变，罗氏文库试剂盒容易漏检PIK3CB突变。对于药物相关的生物标志物，Sentieon TNscope倾向于低估肿瘤突变负担，忽视关键的耐药突变，如FLT3 （c.G1879A: p.A627T）用于白血病的阿糖胞苷耐药，MAP2K1 （c.G199A:p.D67N）用于黑色素瘤的BRAF抑制剂。这些发现强调了强大的生物信息学分析在识别肿瘤突变和指导临床决策方面的重要性。

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引用次数: 0

Morphological Bone Score as a Predictive Tool for Molecular Profiling Success 形态学骨评分作为分子谱分析成功的预测工具。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-06-13 DOI: 10.1016/j.jmoldx.2025.04.013

Kirill Kriukov , Dmitry Ivchenkov , Anna Bejanyan , Aleksandr Sarachakov , Aleksandra Kviatkovskaia , Gleb Khegai , Dominique Knipper-Davis , Amber Berlinski , Tayla Soares , Jochen K. Lennerz , Vladimir Kushnarev

Decalcification of bone-containing tumor samples serves to soften tissues before histologic processing. However, it can lead to nucleic acid degradation, resulting in next-generation sequencing failures that impede diagnostic solutions for patients. The Morphological Bone Score (MBS) described herein optimizes the assessment of decalcified tissue samples, consequently improving both diagnostic accuracy and cost efficiency in molecular genetic laboratories. The MBS, constructed using five key morphologic features, assigns scores from 0 to 11, reflecting low to high tissue damage and direct proportionality with nucleic acid yields per cell. The MBS threshold can be adjusted depending on the aims of a specific analysis while balancing between sensitivity and accuracy. In our next-generation sequencing workflow, the exclusion of poor-quality samples from downstream processing using MBS led to a savings of $1500 per sample. The MBS provides a cost-effective approach for maximizing tissue utilization and optimizing downstream profiling in precision oncology because its objectivity and consistency in evaluating pathologic samples ensure reliable and reproducible outcomes. With additional verification, this tool could be implemented in computational models for converting morphologic features into measurable units.

含骨肿瘤标本的脱钙有助于在组织学处理前软化组织。然而，它会导致核酸降解，导致下一代测序（NGS）失败，阻碍患者的诊断解决方案。本文描述的形态学骨评分（MBS）优化了脱钙组织样本的评估，从而提高了分子遗传学实验室的诊断准确性和成本效率。MBS使用五个关键形态学特征构建，评分从0到11，反映组织损伤程度从低到高，并与每个细胞的核酸产量成正比。MBS阈值可以根据具体分析的目的进行调整，同时在灵敏度和准确性之间取得平衡。在我们的NGS工作流程中，使用MBS将低质量样品从下游处理中排除，每个样品节省了1,500美元。由于MBS在评估病理样本时的客观性和一致性确保了可靠和可重复的结果，因此它为精确肿瘤学中最大化组织利用和优化下游分析提供了一种具有成本效益的方法。通过额外的验证，该工具可以在将形态特征转换为可测量单位的计算模型中实现。

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引用次数: 0

A Comparative Study of Medium-Coverage Genome Sequencing and SNP Array Technology in Identifying Chromosomal Abnormalities to Advance Prenatal and Postnatal Diagnosis 中覆盖基因组测序和SNP阵列技术在染色体异常识别中的比较研究，以促进产前和产后诊断。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-06-06 DOI: 10.1016/j.jmoldx.2025.04.009

Jialun Pang , Lin Zhou , Jiancheng Hu , Hanzhe Kuang , Hui Xi , Na Ma , Shuting Yang , Wenxian Yu , Yanan Zhang , Qian Zhang , Victor Wei Zhang , Jing Chen , Ying Peng

This study compared the performance of 5-fold genome sequencing (GS) with single nucleotide polymorphism (SNP) array technology in detecting chromosomal abnormalities, particularly in the context of prenatal and postnatal diagnostics. A total of 42 samples, previously analyzed by SNP array, were re-examined using 5-fold GS to evaluate the detection of clinically significant copy number variations (CNVs), mosaicism, and absence of heterozygosity (AOH). The results revealed a 100% concordance between the two methods for the identification of clinically relevant CNVs, with both technologies detecting similar CNV size ranges. However, 5-fold GS demonstrated better precision in defining CNV breakpoints and exhibited a lower false-positive rate, as confirmed by quantitative PCR validation. Additionally, 5-fold GS detected mosaicism with comparable sensitivity to SNP array, capturing mosaic levels as low as 17%, whereas SNP array identified levels between 15% and 84%. For AOH detection, 5-fold GS identified all candidate AOH regions with a slightly better sensitivity, achieving a detection size limit of 4.8 Mb compared with SNP array's 5.08 Mb. Overall, 5-fold GS shows potential as a reliable method for chromosomal abnormality detection, offering high accuracy and clinical utility in both prenatal and postnatal genetic testing.

本研究旨在比较5倍基因组测序（GS）和单核苷酸多态性（SNP）阵列技术在检测染色体异常方面的性能，特别是在产前和产后诊断方面。先前通过SNP阵列分析的总共42个样本，使用5倍GS重新检查，以评估临床显著拷贝数变异（CNVs），镶嵌性和缺乏杂合性（AOH）的检测。结果显示，两种方法鉴定临床相关CNV的一致性为100%，两种技术检测的CNV大小范围相似。然而，qPCR验证证实，5倍GS在确定CNV断点方面表现出更好的精度，并表现出更低的假阳性率。此外，5倍GS检测马赛克的灵敏度与SNP阵列相当，捕获的马赛克水平低至17%，而SNP阵列识别的马赛克水平在15%至84%之间。对于AOH检测，5倍GS识别出所有候选AOH区域的灵敏度略高，与SNP阵列的5.08 Mb相比，检测大小限制为4.8 Mb。总体而言，5倍GS显示出作为染色体异常检测的可靠方法的潜力，在产前和产后基因检测中都具有较高的准确性和临床实用性。

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引用次数: 0

Validation of Human Papillomavirus Genotyping by Oxford Nanopore Sequencing in Formalin-Fixed, Paraffin-Embedded Tissues and ThinPrep Anal and Gynecologic Samples 牛津纳米孔测序在FFPE组织和ThinPrep肛门和妇科样本中的HPV基因分型验证。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-06-05 DOI: 10.1016/j.jmoldx.2025.04.010

Carolina Hernandez , Luz H. Patiño , Milena Camargo , Ching Yi Wang , Feng Chen , Bernadette Liggayu , Liyong Cao , Carlos Cordon-Cardo , Emilia M. Sordillo , Alberto Paniz-Mondolfi , Juan D. Ramírez

Human papillomavirus (HPV) is linked to various cancers, including cervical, anal, and head and neck cancers. Conventional methods for HPV genotyping and commercial platforms are limited to detecting high-risk HPV genotypes primarily in gynecologic samples. Because of changing trends in the epidemiology and pathogenesis, there is a growing need for HPV genotyping techniques applicable to emerging clinical contexts involving diverse sample types, such as head and neck or anal samples, particularly for formalin-fixed, paraffin-embedded (FFPE) tissues. This study aimed to validate amplicon-based sequencing with Oxford Nanopore Technologies (ONT) for the detection and genotyping of HPV in 181 samples, including FFPE head and neck samples, and ThinPrep liquid-based cytology samples from anal and gynecologic tissues. Sanger sequencing was used as a reference for genotyping accuracy. The ONT sequencing method demonstrated a limit of detection of 1 copy/μL for HPV16 and HPV18. Perfect agreement (κ coefficient = 1.0) was observed for HPV detection across all sample types. Genotyping accuracy exceeded 95%, and ONT identified additional genotypes in certain anal and gynecologic samples that were undetected by Sanger sequencing. The assay showed high reproducibility, with consistent results across intrarun and interrun analyses. This study is the first to validate ONT sequencing for HPV genotyping in FFPE head and neck samples. ONT provides a rapid, cost-effective method for comprehensive HPV genotyping in diverse sample types.

人类乳头瘤病毒（HPV）与各种癌症有关，包括宫颈癌、肛门癌、头颈癌。传统的HPV基因分型方法和商业平台仅限于检测主要在妇科样本中的高危HPV基因型。由于流行病学和发病机制的变化趋势，越来越需要适用于涉及不同样本类型的新临床情况的HPV基因分型技术，例如头颈部或肛门样本，特别是用于福尔马林固定石蜡包埋（FFPE）组织。本研究旨在验证基于扩增子测序的牛津纳米孔技术在181个样本中的HPV检测和基因分型，包括FFPE头颈部样本，以及肛门和妇科组织的ThinPrep液体细胞学样本。Sanger测序作为基因分型准确性的参考。我们的ONT测序方法显示HPV16和HPV18的检测限为1拷贝/μL。所有样本类型的HPV检测结果完全一致（kappa系数= 1.0）。基因分型准确率超过95%，ONT在某些肛门和妇科样本中发现了Sanger测序未检测到的额外基因型。该分析具有高重复性，在运行内和运行间分析结果一致。这项研究首次验证了FFPE头颈部样本中HPV基因分型的ONT测序。ONT提供了一种快速，经济有效的方法，全面的HPV基因分型在不同的样本类型。

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引用次数: 0

Comparison of the Mutational Profile between BCL2- and BCL6-Rearrangement Positive Follicular Lymphoma BCL2-和bcl6重排阳性滤泡性淋巴瘤突变谱的比较。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-06-05 DOI: 10.1016/j.jmoldx.2025.05.002

Haruka Ikoma , Joaquim Carreras , Yara Yukie Kikuti , Masashi Miyaoka , Shunsuke Nagase , Yusuke Kondo , Atsushi Ito , Makoto Orita , Sakura Tomita , Shinichiro Hiraiwa , Hiroshi Kawada , Juan F. Garcia , Giovanna Roncador , Elias Campo , Naoya Nakamura

It was recently reported that follicular lymphoma (FL) with BCL6 rearrangement (R) is associated with favorable progression-free survival, whereas BCL2-R and BCL2-6-R cases are associated with disease progression. However, the pathologic mechanism remained unexplored. This study analyzed the mutational landscape and immune microenvironment of 31 FL cases, including 16 BCL2-R, 11 BCL6-R, and 4 BCL2-6-R FL cases. The method included an in-house next-generation targeted sequencing panel of 168 genes associated with aggressive B-cell lymphoma and FL, whole genome copy number change microarray (OncoScan), and immunohistochemistry for the immune microenvironment focused on M2-like tumor-associated macrophages, regulatory T lymphocytes, and programmed cell death protein 1 (PDCD1; alias PD-1)–positive follicular T helper cells. The resulting mutational profile was compatible with a previously reported conventional FL series featuring frequent mutations in CREBBP, KMT2D, TNFRSF14, STAT6, and CD36. Moreover, BCL6-R cases had mutations in ARID1B, ARID5B, and RHOA; low frequency of mutations in other genes, such as OSBPL10, PTPRD, ATM, and HLA-B; 6q loss; and absence of disease progression. In comparison with BCL6-R cases, BCL2-R and BCL2-6-R cases had mutations in EZH2, chromosome 18 copy number gain, and disease progression in some cases. The immune microenvironment profile was heterogeneous; however, BCL6-R cases demonstrated higher infiltration of colony-stimulating factor 1 receptor– and leukocyte immunoglobulin like receptor B3 (LILRB3; alias CD85a)–positive cells. In conclusion, compared with BCL2-R, FL with BCL6-R exhibited some differences in mutational profiles and immune microenvironment.

我们最近报道了伴有bcl6重排(R)的滤泡性淋巴瘤（FL）与有利的无进展生存相关，而BCL2-R和BCL2-6-R病例与疾病进展相关。然而，病理机制尚不清楚。本研究分析了31例FL的突变景观和免疫微环境，其中16例为BCL2-R， 11例为BCL6-R， 4例为BCL2-6-R。该方法包括内部新一代靶向测序（NGS） 168个与侵袭性b细胞淋巴瘤和FL相关的基因，全基因组拷贝数改变微阵列（OncoScan），以及免疫微环境的免疫组织化学，重点是m2样肿瘤相关巨噬细胞，调节性T淋巴细胞和PD-1 （PDCD1）阳性滤泡T辅助细胞。由此产生的突变谱与先前报道的传统FL系列相一致，该系列在CREBBP、KMT2D、TNFRSF14、STAT6和CD36中具有频繁突变。此外，BCL6-R病例存在ARID1B、ARID5B和RHOA突变；其他基因（如OSBPL10、PTPRD、ATM和HLA-B）的低频率突变、6q缺失和无疾病进展。与BCL6-R病例相比，BCL2-R和BCL2-6-R病例发生EZH2突变，18号染色体拷贝数增加，部分病例出现疾病进展。免疫微环境具有异质性；然而，BCL6-R病例表现出更高的CSF1R-和CD85A （LILRB3）阳性细胞的浸润。综上所述，与BCL2-R相比，FL与BCL6-R在突变谱和免疫微环境方面存在一定差异。

{"title":"Comparison of the Mutational Profile between BCL2- and BCL6-Rearrangement Positive Follicular Lymphoma","authors":"Haruka Ikoma , Joaquim Carreras , Yara Yukie Kikuti , Masashi Miyaoka , Shunsuke Nagase , Yusuke Kondo , Atsushi Ito , Makoto Orita , Sakura Tomita , Shinichiro Hiraiwa , Hiroshi Kawada , Juan F. Garcia , Giovanna Roncador , Elias Campo , Naoya Nakamura","doi":"10.1016/j.jmoldx.2025.05.002","DOIUrl":"10.1016/j.jmoldx.2025.05.002","url":null,"abstract":"<div><div>It was recently reported that follicular lymphoma (FL) with <em>BCL6</em> rearrangement (R) is associated with favorable progression-free survival, whereas <em>BCL2</em>-R and <em>BCL2-6</em>-R cases are associated with disease progression. However, the pathologic mechanism remained unexplored. This study analyzed the mutational landscape and immune microenvironment of 31 FL cases, including 16 <em>BCL2</em>-R, 11 <em>BCL6</em>-R, and 4 <em>BCL2-6-</em>R FL cases. The method included an in-house next-generation targeted sequencing panel of 168 genes associated with aggressive B-cell lymphoma and FL, whole genome copy number change microarray (OncoScan), and immunohistochemistry for the immune microenvironment focused on M2-like tumor-associated macrophages, regulatory T lymphocytes, and programmed cell death protein 1 (PDCD1; alias PD-1)–positive follicular T helper cells. The resulting mutational profile was compatible with a previously reported conventional FL series featuring frequent mutations in <em>CREBBP</em>, <em>KMT2D</em>, <em>TNFRSF14</em>, <em>STAT6</em>, and <em>CD36</em>. Moreover, <em>BCL6</em>-R cases had mutations in <em>ARID1B</em>, <em>ARID5B</em>, and <em>RHOA</em>; low frequency of mutations in other genes, such as <em>OSBPL10</em>, <em>PTPRD</em>, <em>ATM</em>, and <em>HLA-B</em>; 6q loss; and absence of disease progression. In comparison with <em>BCL6</em>-R cases, <em>BCL2</em>-R and <em>BCL2-6-</em>R cases had mutations in <em>EZH2</em>, chromosome 18 copy number gain, and disease progression in some cases. The immune microenvironment profile was heterogeneous; however, <em>BCL6</em>-R cases demonstrated higher infiltration of colony-stimulating factor 1 receptor– and leukocyte immunoglobulin like receptor B3 (LILRB3; alias CD85a)–positive cells. In conclusion, compared with <em>BCL</em>2-R, FL with <em>BCL</em>6-R exhibited some differences in mutational profiles and immune microenvironment.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"27 8","pages":"Pages 796-807"},"PeriodicalIF":3.4,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144250606","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}

引用次数: 0

Clinical Bioinformatician Body of Knowledge—Bioinformatics and Software Core 临床生物信息学知识体系-生物信息学和软件核心：分子病理学协会的报告。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-05-19 DOI: 10.1016/j.jmoldx.2025.04.008

Sabah Kadri , Kelly E. Craven , Amber M. Fussell , Elaine P.S. Gee , Danielle Jordan , Eric W. Klee , Niklas Krumm , Robyn L. Temple-Smolkin , Ahmet Zehir , Weiwei Zhang , Andrea Sboner

With the evolution of next-generation sequencing–based testing in molecular diagnostics laboratories, the clinical role of bioinformaticians has also evolved. The Association for Molecular Pathology's Clinical Bioinformatician Body of Knowledge aims to define the various roles the clinical bioinformatician operates individually or within a clinical bioinformatics team, along with proficiencies and skill sets that may be required or desirable across these roles. One of the most common professional responsibilities of a clinical bioinformatician is to implement bioinformatics pipelines, either vendor supplied or custom built for the assays in the molecular diagnostics laboratory, along with analysis and quality control of clinical genomics data. This second article in the series describes the various stages in the life cycle of a clinical bioinformatics pipeline and the considerations, areas of expertise, and skill sets required in each stage. This information may help laboratory professionals to better work with clinical bioinformaticians and laboratory directors to hire the appropriate expertise based on the specific needs of the laboratory.

随着分子诊断实验室中基于测序的新一代检测技术的发展，生物信息学家的临床角色也在不断发展。分子病理学协会的临床生物信息学家知识体系旨在定义临床生物信息学家单独或在临床生物信息学团队中操作的各种角色，以及这些角色可能需要或期望的熟练程度和技能集。临床生物信息学家最常见的专业职责之一是实施生物信息学管道，无论是供应商提供的还是定制的，用于分子诊断实验室的分析，以及临床基因组学数据的分析和质量控制。该系列的第二份手稿描述了临床生物信息学管道生命周期的各个阶段，以及每个阶段所需的考虑因素、专业领域和技能集。这些信息可以帮助实验室专业人员更好地与临床生物信息学家和实验室主任合作，根据实验室的具体需求雇用适当的专业人员。

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引用次数: 0

Standardizing Laboratory Practices in Pharmacogenomics (STRIPE) Consensus Conference 标准化药物基因组学实验室实践（STRIPE）共识会议：实验室挑战工作组报告。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-05-17 DOI: 10.1016/j.jmoldx.2025.04.006

Victoria M. Pratt , Betsy Bove , Raymond A. Lorenz , Annette K. Taylor , Bronwyn Ramey

引用次数: 0

Path to Health Equity and Improved Outcomes through Inclusive Sex and Gender Data Collection in Genomic Testing 通过基因组检测中包容性的性别和社会性别数据收集实现健康公平和改善结果之路。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-05-15 DOI: 10.1016/j.jmoldx.2025.04.004

Marco L. Leung , Ina Amarillo , Danielle Jordan , Matthew S. Lebo , Rizwan C. Naeem , David I. Suster , Robyn L. Temple-Smolkin , Cigdem H. Ussakli , Honey V. Reddi

As the demand for health services among sexual and gender diverse (SGD) individuals rises, there is a growing need for comprehensive and equitable standards of care across the health care system. Despite progress in various research areas, there is a relative lag in genetics and genomics. In this Perspective, the Association for Molecular Pathology Working Group presents survey data on how the sex and gender identity of patients, including SGD individuals, is collected, interpreted, and reported within current genomic laboratory practices during the preanalytical, analytical, and postanalytical phases. Recommendations and guidelines related to the care of the SGD community are explored, identifying knowledge and practice gaps in each phase. On the basis of the survey results, review of existing available literature, and collective professional experience, the Working Group provides future considerations to enhance affirmative and inclusive processes, improve test quality, advance health equity, and enhance patient outcomes.

随着性和性别多样化（SGD）个体对卫生服务的需求上升，对整个医疗保健系统的全面和公平的护理标准的需求日益增长。尽管在各个研究领域取得了进展，但在遗传学和基因组学方面相对滞后。从这个角度来看，分子病理学协会工作组提出了关于在分析前、分析和分析后阶段，当前基因组实验室如何收集、解释和报告患者（包括SGD个体）的性别和性别认同的调查数据。研究了与SGD社区护理相关的建议和指导方针，确定了每个阶段的知识和实践差距。根据调查结果、对现有文献的审查以及集体专业经验，工作组提出了未来应考虑的事项，以加强肯定性和包容性进程、提高检测质量、促进卫生公平和改善患者预后。

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引用次数: 0

Smart Nonuniformity for Calibrating Sequencing Depth of a Targeted Gene Panel to Simultaneously Detect Somatic and Germline Variants 智能非均匀性：校准目标基因面板的测序深度，同时检测体细胞和种系变异。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-05-15 DOI: 10.1016/j.jmoldx.2025.04.007

Robert L. O'Reilly , Philip Harraka , Jared Burke , Daniele Belluoccio , Paul Yeh , Kerryn Howlett , Kiarash Behrouzfar , Amanda Rewse , Helen Tsimiklis , Graham G. Giles , John L. Hopper , Kristen J. Bubb , Stephen J. Nicholls , Roger L. Milne , Melissa C. Southey

Targeted gene panel sequencing that measures genomic variation at different depths has potential diagnostic application. A targeted gene panel, smart nonuniformity sequencing, was developed to detect somatic variants associated with clonal hematopoiesis of indeterminate potential (CHIP), which requires an optimal sequencing depth of >500×; and germline variants requiring a lower ≥50× depth (panel 1). This was achieved by adjusting probe ratios for genomic regions relevant to identifying CHIP in comparison to those relevant to germline variation analysis. An additional custom panel containing only the genomic regions relevant to the identification of CHIP (panel 2) was also manufactured to confirm that panel 1 did not miss variants because of the complex design. Both panels were used to sequence 150 blood-derived DNAs; 94 DNAs from research participants aged 64 to 75 years; 16 DNAs with known germline variants; 16 DNAs with known germline variants (titrated from 0% to 100%); 24 DNAs from individuals aged <40 years; and 3 commercial CHIP controls and 3 high-molecular-weight DNA controls. The sequencing median depth ratio between the CHIP and germline relevant genomic regions was 4.7:1. Fourteen CHIP-associated variants were called in both panel 1 (1382× median variant depth) and panel 2 (1665× median variant depth). All known germline variants were identified (251× median variant depth). Smart nonuniformity sequencing reliably detects variants with allele frequency in the range >0.01 to 1 in one workflow.

在不同深度测量基因组变异的靶向基因面板测序具有潜在的诊断应用。开发了一个靶向基因面板，用于检测1)与不确定潜力克隆造血（CHIP）相关的体细胞变异，这需要bb0 500X的最佳测序深度，以及2)需要更低的≥50X深度的种系变异[面板1]。这是通过调整与鉴定CHIP相关的基因组区域的探针比例与与种系变异分析相关的探针比例来实现的。还制作了一个额外的定制面板，仅包含与CHIP鉴定相关的基因组区域[面板2]，以确认面板1不会因复杂的设计而遗漏变异。两个面板都用于对150个血液来源的dna进行测序；来自64-75岁研究参与者的94个dna；16个已知种系变异的dna；16个已知种系变异的dna（从0-100%滴定）；在一个工作流程中，来自0.01-1岁个体的24个dna。

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引用次数: 0

Leveraging RNA from DNA Extraction Lysate to Rescue “Insufficient” Samples for More Comprehensive Genomic Profiling in Patients with Scant Tumor Specimens 利用DNA提取裂解液中的RNA来拯救“不足”的样本，以便在肿瘤标本稀少的患者中进行更全面的基因组分析。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2025-05-15 DOI: 10.1016/j.jmoldx.2025.04.005

Mark D. Ewalt , Sara E. DiNapoli , Kerry Mullaney , Alison Urvalek , Minji Kim Uh , Purvil Sukhadia , J. Keith Killian , Michael Zaidinski , Hun Jae Jung , Tiffany McFarlane , Kelly Rios-Papachristos , Alexander Drilon , Mark G. Kris , Khedoudja Nafa , Maria E. Arcila , Marc Ladanyi , Ahmet Zehir , Michael Offin , Ryma Benayed

Tissue availability is often a limiting factor in obtaining comprehensive genomic profiling to identify actionable oncogenic drivers in tumors from patients with cancer. The utility of performing complementary DNA and RNA sequencing to better identify targetable gene fusions was previously reported. Here, we report our experience using RNA recovered from lysate material, following DNA extraction, to perform targeted RNA sequencing and identify gene fusions and oncogenic transcript variants in a large cohort of patients with solid tumors. To validate this approach, RNA-sequencing results of lysate-extracted RNA and direct formalin-fixed, paraffin-embedded (FFPE) extracted RNA from the same tumors were compared. After finding equivalent identification of oncogenic gene fusions and transcript variants, efforts were expanded to a larger cohort across more diverse tumor types. Lysate-extracted RNA performed comparably to freshly FFPE extract RNA, with 97% and 96% success rates, respectively. Within the lysate-extracted group, it was documented that lysate was the only material available for RNA extraction (n = 1862, 42% of all tested samples) and, within this subgroup, 364 (20%) samples were positive for actionable fusions or oncogenic isoforms. Using RNA recovered from lysate can permit sequential or simultaneous comprehensive DNA/RNA sequencing from scant FFPE samples in laboratories where dual sample extraction is not logistically possible, allowing more complete profiling to enhance the identification of actionable oncogenic gene fusions to guide care.

组织可用性通常是获得全面基因组谱以确定癌症患者肿瘤中可操作的致癌驱动因素的限制因素。我们之前报道了进行互补DNA和RNA测序以更好地识别靶基因融合的效用。在这里，我们报告了我们的经验，使用从裂解物中回收的RNA，在DNA提取后，进行靶向RNA测序，并在大量实体肿瘤患者中鉴定基因融合和致癌转录物变异。为了验证我们的方法，我们比较了从同一肿瘤中裂解液提取的RNA和直接FFPE提取的RNA的RNA测序结果。在发现致癌基因融合和转录变体的等效鉴定后，我们将研究范围扩大到更大的队列，涵盖更多不同的肿瘤类型。裂解液提取的RNA与新鲜ffpe提取的RNA的成功率分别为97%和96%。在裂解液萃取组中，我们证明裂解液是唯一可用于RNA提取的材料（n = 1,862，占所有测试样品的42%），并且在该亚组中，364（20%）样品在可操作融合或致癌同种异构体中呈阳性。使用从裂解液中回收的RNA可以在实验室中对缺少的FFPE样品进行顺序或同时进行全面的DNA/RNA测序，因为双重样品提取在后勤上是不可能的，允许更完整的分析，以增强可操作的致癌基因融合的识别，以指导护理。

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