Journal of Molecular Diagnostics最新文献_第3页

Improving Specificity for Ovarian Cancer Screening Using a Novel Extracellular Vesicle–Based Blood Test 使用基于细胞外囊泡的新型血液检验提高卵巢癌筛查的特异性--训练和验证队列中的表现。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-24 DOI: 10.1016/j.jmoldx.2024.09.001

Emily S. Winn-Deen , Laura T. Bortolin , Daniel Gusenleitner , Kelly M. Biette , Karen Copeland , Aleksandra Gentry-Maharaj , Sophia Apostolidou , Anthony D. Couvillon , Daniel P. Salem , Sanchari Banerjee , Jonian Grosha , Ibukunoluwapo O. Zabroski , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Peter A. Duff , Brendan J. Manning , Troy B. Hawkins , Usha Menon

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity, and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%–99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%–99.2%), and an area under the curve of 0.97 (95% CI, 0.93–0.99) and detected 73.5% (61/83; 95% CI, 62.7%–82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests that it may have potential in OC screening.

卵巢癌（OC）发病率低，因此任何筛查策略都必须具有高灵敏度和高特异性。本研究探索了从血液中检测单个肿瘤相关细胞外囊泡 (EV) 上多个共定位蛋白或糖基化表位的实用性。新型 OC 检测试剂盒采用免疫亲和法捕获肿瘤相关的 EVs，然后进行邻近连接 qPCR，检测多达三种生物标记物的组合，以最大限度地提高特异性，并测量多种组合，以最大限度地提高灵敏度。利用来自 397 位女性的 EDTA 血浆样本组成的高级别浆液性癌（HGSC）病例对照训练集来锁定测试设计、数据解读算法以及癌症与非癌症的分界点。在 390 名女性（132 名对照组、66 名 HGSC、83 名非 HGSC OC、109 名良性）血清样本组成的独立盲法病例对照组中，对其性能进行了验证，并与 CA125 进行了比较。在验证研究中，OC 检验的特异性为 97.0%（128/132；95% CI：92.4%-99.6%），HGSC 敏感性为 97.0%（64/66；95% CI：87.8%-99.2%），AUC 为 0.97（95% CI 0.93-0.99），还检测出 73.5%（61/83；95% CI：62.7%-82.6%）的非 HGSC OC 病例。与 CA125 相比，该检测方法在良性卵巢肿瘤、非卵巢癌和炎症患者中的假阳性率较低。这一新检测方法的灵敏度和特异性表明，它在卵巢癌筛查中具有潜力。

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引用次数: 0

Colocalization of Cancer-Associated Biomarkers on Single Extracellular Vesicles for Early Detection of Cancer 癌症相关生物标记物在单个细胞外囊泡上的共定位，用于癌症的早期检测。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-24 DOI: 10.1016/j.jmoldx.2024.08.006

Daniel P. Salem , Laura T. Bortolin , Dan Gusenleitner , Jonian Grosha , Ibukunoluwapo O. Zabroski , Kelly M. Biette , Sanchari Banerjee , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Timothy Santos-Heiman , Gabrielle N. Barcaskey , Katherine S. Yang , Peter A. Duff , Emily S. Winn-Deen , Toumy Guettouche , Dawn R. Mattoon , Eric K. Huang , Joseph C. Sedlak

Detection of cancer early, when it is most treatable, remains a significant challenge because of the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. The detection of early-stage tumors is challenging given the lack of tumor-specific indicators (ie, protein biomarkers, circulating tumor DNA) to enable detection using a noninvasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line–derived EVs, where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer–associated biomarkers bone marrow stromal antigen-2, folate receptor-α, and mucin-1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.

由于缺乏足够灵敏的诊断方法来检测新生肿瘤，因此在癌症最容易治疗的早期发现癌症仍然是一项重大挑战。早期肿瘤相对于其原发组织而言较小，具有异质性，并且很少表现出临床症状。由于缺乏丰富的肿瘤特异性指标（如蛋白质生物标志物、循环肿瘤 DNA 等），因此使用非侵入性诊断方法检测肿瘤的存在变得更加困难。为了克服这些障碍，我们开发了一种液体活检检测方法，该方法可检测循环细胞外囊泡 (EV)，从而检测单个 EV 表面共定位的肿瘤特异性生物标记物。我们在人类癌细胞系衍生的EV中证明了这种方法的技术可行性，我们发现检测信号与卵巢癌相关生物标记物BST2、FOLR1和MUC1的细胞系基因/蛋白表达之间存在很强的相关性。此外，我们还证明，与单一生物标记物测量相比，检测 EV 表面不同的共定位生物标记物能显著提高判别性能。利用这种方法，我们在一项概念验证临床研究中观察到了区分高级别浆液性卵巢癌与良性卵巢肿块和健康妇女的良好效果。

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引用次数: 0

A Versatile and Upgraded Version of the LundTax Classification Algorithm Applied to Independent Cohorts 适用于独立队列的多功能升级版 LundTax 分类算法。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-24 DOI: 10.1016/j.jmoldx.2024.08.005

Elena Aramendía Cotillas , Carina Bernardo , Srinivas Veerla , Fredrik Liedberg , Gottfrid Sjödahl , Pontus Eriksson

Stratification of cancer into biologically and molecularly similar subgroups is a cornerstone of precision medicine. The Lund Taxonomy classification system for urothelial carcinoma aims to be applicable across the whole disease spectrum including both non–muscle-invasive and invasive bladder cancer. A successful classification system is one that can be robustly and reproducibly applied to new samples. However, transcriptomic methods used for subtype classification are affected by analytic platform, data preprocessing, cohort composition, and tumor purity. Furthermore, only limited data have been published evaluating the transferability of existing classification algorithms to external data sets. In this study, a single sample classifier was developed based on in-house microarray and RNA-sequencing data, intended to be broadly applicable across studies and platforms. The new classification algorithm was applied to 10 published external bladder cancer cohorts (n = 2560 cases) to evaluate its ability to capture characteristic subtype-associated gene expression signatures and complementary data such as mutations, clinical outcomes, treatment response, or histologic subtypes. The effect of sample purity on the classification results was evaluated by generating low-purity versions of samples in silico. The classifier was robustly applicable across different gene expression profiling platforms and preprocessing methods and was less sensitive to variations in sample purity.

将癌症分为生物和分子相似的亚组是精准医疗的基石。隆德分类学尿路上皮癌（UC）分类系统旨在适用于整个疾病谱，包括非肌层浸润性和浸润性膀胱癌。要使分类系统发挥作用，最重要的是该系统能稳健、可重复地应用于新样本。然而，用于亚型分类的转录组学方法会受到分析平台、数据预处理、队列组成和肿瘤纯度的影响。此外，评估现有分类算法在外部数据集上可移植性的数据也很有限。本研究基于内部微阵列和 RNA 序列数据开发了单样本分类器，旨在广泛适用于各种研究和平台。新的分类算法应用于 10 个已发表的外部膀胱癌队列（n=2560 例），以评估其捕获特征性亚型相关基因表达特征以及突变、临床结果、治疗反应或组织学亚型等补充数据的能力。通过在硅学中生成低纯度样本，评估了样本纯度对分类结果的影响。该分类器可在不同的基因表达谱平台和预处理方法中稳健应用，对样本纯度的变化不太敏感。

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引用次数: 0

Haplotype-Aware Detection of SERPINA1 Variants by Nanopore Sequencing 利用纳米孔测序技术检测 SERPINA1 变异的单倍型。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-12 DOI: 10.1016/j.jmoldx.2024.08.002

Mario A. González-Carracedo , Esther Herrera-Luis , María Marco-Simancas , Ainhoa Escuela-Escobar , Elena Martín-González , Olaia Sardón-Prado , Paula Corcuera , Jose M. Hernández-Pérez , Fabián Lorenzo-Díaz , José A. Pérez-Pérez

α-1 Antitrypsin (AAT) is an acute-phase reactant with immunomodulatory properties that mainly inhibits neutrophil elastase. Low serum levels cause AAT deficiency (AATD), an underdiagnosed condition that predisposes to pulmonary and hepatic diseases. The SERPINA1 gene, which encodes AAT, contains >500 variants. PI∗Z and PI∗S alleles are the most diagnosed causes of AATD, but the role of the SERPINA1 haplotypes in AAT function remains unknown. SERPINA1 gene was PCR amplified from 94 patients with asthma, using primers with tails for indexing. Sequencing libraries were loaded into a MinION-Mk1C, and MinKNOW was used for basecalling and demultiplexing. Nanofilt and Minimap2 were used for filtering and mapping/alignment. Variant calling/phasing were performed with PEPPER-Margin-DeepVariant. SERPINA1 gene was 100% covered for all samples, with a minimum sequencing depth of 500×. A total of 75 single-nucleotide variants (SNVs) and 4 insertions/deletions were detected, with 45 and 2 of them highly polymorphic (minor allele frequency >0.1), respectively. Nine of the SNVs showed differences in allele frequencies when compared with the overall Spanish population. More than 90% of heterozygous SNVs were phased, yielding 91 and 58 different haplotypes for each SERPINA1 amplified region. Haplotype-based linkage disequilibrium analysis suggests that a recombination hotspot could generate variation in the SERPINA1 gene. The proposed workflow enables haplotype-aware genotyping of the SERPINA1 gene by nanopore sequencing, which will allow the development of novel AATD diagnostic strategies.

α-1抗胰蛋白酶（AAT）是一种急性期反应物，具有免疫调节特性，主要抑制中性粒细胞弹性蛋白酶。血清中 AAT 含量低会导致 AAT 缺乏症（AATD），这种疾病诊断不足，容易引发肺病和肝病。编码 AAT 的 SERPINA1 基因含有超过 500 个变体。PI∗Z和PI∗S等位基因是导致AATD的最主要原因，但SERPINA1单倍型在AAT功能中的作用仍不清楚。使用带尾引物对 94 名哮喘患者的 SERPINA1 基因进行 PCR 扩增。测序文库被载入 MinION-Mk1C，MinKNOW 被用于基线分选和解复用。Nanofilt 和 Minimap2 用于过滤和映射/配准。使用 PEPPER-Margin-DeepVariant 进行变异调用/分期。所有样本的 SERPINA1 基因覆盖率均为 100%，最小测序深度为 500×。共检测到 75 个单核苷酸变异（SNV）和 4 个插入/缺失，其中 45 个和 2 个分别具有高度多态性（小等位基因频率大于 0.1）。其中 9 个 SNV 的等位基因频率与西班牙总体人群相比存在差异。超过90%的杂合SNV是分阶段的，每个SERPINA1扩增区域产生了91个和58个不同的单倍型。基于单倍型的连锁不平衡分析表明，重组热点可能导致 SERPINA1 基因的变异。所提出的工作流程可通过纳米孔测序对 SERPINA1 基因进行单倍型感知基因分型，从而开发出新型的 AATD 诊断策略。

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引用次数: 0

Comparison of Results from Two Commercially Available In-House Tissue-Based Comprehensive Genomic Profiling Solutions 两种市售内部组织综合基因组图谱分析解决方案的结果比较：仅供研究使用的 AVENIO 肿瘤组织综合基因组图谱分析试剂盒和 TruSight Oncology 500 分析法

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-11 DOI: 10.1016/j.jmoldx.2024.08.001

Hans-Peter Adams , Matthew C. Hiemenz , Kay Hertel , Frederike Fuhlbrück , Mara Thomas , James Oughton , Helle Sorensen , Ulrich Schlecht , Justin M. Allen , Martina Cantone , Sophie Osswald , David Gonzalez , Eli Pikarsky , Muriel De Vos , Ed Schuuring , Thomas Wieland

Increased adoption of personalized medicine has brought comprehensive genomic profiling (CGP) to the forefront. However, differences in assay, bioinformatics, and reporting systems and lack of understanding of their complex interplay are a challenge for implementation and achieving uniformity in CGP testing. Two commercially available, tissue-based, in-house CGP assays were compared, in combination with a tertiary analysis solution in a research use only (RUO) context: the AVENIO Tumor Tissue CGP RUO Kit paired with navify Mutation Profiler (RUO) software and the TruSight Oncology 500 RUO assay paired with PierianDx Clinical Genomics Workspace software. Agreements and differences between the assays were assessed for short variants, copy number alterations, rearrangements, tumor mutational burden, and microsatellite instability, including variant categorization and clinical trial-matching (CTM) recommendations. Results showed good overall agreement for short variant, known gene fusion, and microsatellite instability detection. Important differences were obtained in tumor mutational burden scoring, copy number alteration detection, and CTM. Differences in variant and biomarker detection could be explained by bioinformatic approaches to variant calling, filtering, tiering, and normalization; differences in CTM, by underlying reported variants and conceptual differences in system parameters. Thus, distinctions between different approaches may lead to inconsistent results. Complexities in calling, filtering, and interpreting variants illustrate key considerations for implementation of any high-quality CGP in the laboratory and bringing uniformity to genomic insight results.

越来越多的人开始采用个性化医疗，这使全面基因组分析（CGP）成为人们关注的焦点。然而，检测方法、生物信息学和报告系统的差异，以及对它们之间复杂的相互作用缺乏了解，是实施 CGP 检测并实现统一性所面临的挑战。我们比较了两种市场上销售的、基于组织的内部 CGP 检测方法，并将其与仅供研究使用（RUO）的三级分析解决方案相结合：AVENIO 肿瘤组织 CGP RUO 检测试剂盒与 navify Mutation Profiler (RUO) 软件配对，以及 TruSight Oncology 500 RUO 检测方法与 PierianDx Clinical Genomics Workspace 软件配对。评估了两种检测方法在短变异、拷贝数改变、重排、肿瘤突变负荷和微卫星不稳定性方面的一致性和差异，包括变异分类和临床试验匹配 (CTM) 建议。结果显示，短变异、已知基因融合和微卫星不稳定性检测的总体一致性良好。在肿瘤突变负荷评分、拷贝数变异检测和 CTM 方面存在重要差异。变异和生物标记物检测方面的差异可以用生物信息学方法中的变异调用、过滤、分层和归一化来解释；CTM 方面的差异则可以用报告的潜在变异和系统参数的概念差异来解释。因此，不同方法之间的区别可能会导致不一致的结果。调用、过滤和解释变异的复杂性说明了在实验室中实施任何高质量 CGP 的关键考虑因素，并使基因组洞察结果具有统一性。

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引用次数: 0

Analytical Validation of an Early Detection Pancreatic Cancer Test Using 5-Hydroxymethylation Signatures 利用 5-羟甲基化特征对胰腺癌早期检测试剂盒进行分析验证

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-09-03 DOI: 10.1016/j.jmoldx.2024.06.007

Shimul Chowdhury , Michael Kesling , Micah Collins , Vanessa Lopez , Yuan Xue , Glenn Oliveira , Verena Friedl , Anna Bergamaschi , David Haan , Wayne Volkmuth , Samuel Levy

Early detection of pancreatic cancer has been shown to improve patient survival rates. However, effective early detection tools to detect pancreatic cancer do not currently exist. The Avantect Pancreatic Cancer Test, leveraging the 5-hydroxymethylation [5-hydroxymethylcytosine (5hmC)] signatures in cell-free DNA, was developed and analytically validated to address this unmet need. We report a comprehensive analytical validation study encompassing precision, sample stability, limit of detection, interfering substance studies, and a comparison with an alternative method. The assay performance on an independent case-control patient cohort was previously reported with a sensitivity for early-stage (stage I/II) pancreatic cancer of 68.3% (95% CI, 51.9%–81.9%) and an overall specificity of 96.9% (95% CI, 96.1%–97.7%). Precision studies showed a cancer classification of 100% concordance in biological replicates. The sample stability studies revealed stable assay performance for up to 7 days after blood collection. The limit of detection studies revealed equal results between early- and late-stage cancer samples, emphasizing strong early-stage performance characteristics. Comparisons of concordance of the Avantect assay with the enzymatic methyl sequencing (EM-Seq) method, which measures both methylation (5-methylcytosine) and 5hmC, were >95% for all samples tested. The Avantect Pancreatic Cancer Test showed strong analytical validation in multiple validation studies required for laboratory-developed test accreditation. The comparison of 5hmC versus EM-Seq further validated the 5hmC approach as a robust and reproducible assay.

事实证明，早期发现胰腺癌可以提高患者的生存率。然而，目前还没有有效的胰腺癌早期检测工具。Avantect胰腺癌检测试剂盒利用无细胞DNA中的5-羟甲基化[5-羟甲基胞嘧啶（5hmC）]特征进行开发和分析验证，以满足这一尚未满足的需求。我们报告了一项全面的分析验证研究，包括精确度、样品稳定性、检测限、干扰物质研究以及与替代方法的比较。此前曾有报告称，该检测方法在独立病例对照患者队列中的性能表现为：对早期（I/II 期）胰腺癌的灵敏度为 68.3%（95% CI，51.9%-81.9%），总体特异性为 96.9%（95% CI，96.1%-97.7%）。精密度研究显示，生物重复样本中癌症分类的一致性为 100%。样本稳定性研究表明，采血后 7 天内检测性能稳定。检测限研究显示，早期和晚期癌症样本的检测结果相同，突出了早期癌症样本的强大性能特征。Avantect 检测法与同时检测甲基化（5-甲基胞嘧啶）和 5hmC 的酶法甲基测序（EM-Seq）法的一致性比较结果显示，所有检测样本的一致性均大于 95%。Avantect 胰腺癌检测试剂盒在实验室开发的检测认证所需的多项验证研究中显示出强大的分析验证能力。5hmC 与 EM-Seq 的比较进一步验证了 5hmC 方法是一种稳健、可重复的检测方法。

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引用次数: 0

Tailored Digital PCR Follow-Up of Rare Fusion Transcripts after Initial Detection through RNA Sequencing in Hematological Malignancies 在血液恶性肿瘤中通过 RNA 测序初步检测到罕见融合转录本后，对其进行定制的数字 PCR 追踪。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-08-31 DOI: 10.1016/j.jmoldx.2024.07.004

Marie-Laure Boulland , Amyra Aliouat , Elie Jalaber , Anne Desmares , Saloua Toujani , Damien Luque Paz , Margaux Wiber , Emeline Voirin , Sébastien Lachot , Audrey Basinko , Wayne-Corentin Lambert , Sylvain Carras , Elie Cousin , Tony Marchand , Marie de Tayrac , Thierry Fest , Roch Houot , Cédric Pastoret

Minimal residual disease (MRD) monitoring plays a pivotal role in the management of hematologic malignancies. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-sequencing (RNA-seq) approach (FusionPlex) with a whole-transcriptomic strategy (Advanta RNA-Seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r = 0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection of <0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets.

血液恶性肿瘤的治疗已进入一个新时代，其中最小残留病（MRD）监测发挥着关键作用。PML::RARA、CBFB::MYH11 或 RUNX1::RUNX1T1等公认的分子靶标通常通过定量反转录 PCR 追踪。最近，通过转录组分析，人们发现了更广泛的融合转录本。这些新发现的融合转录本可能成为 MRD 定量的新型分子标记。在本研究中，我们比较了靶向 RNA-seq 方法（FusionPlex）和全转录组策略（Advanta RNA-seq XT）在 21 个样本训练集中的融合检测。在已知融合的检测中，我们发现两者的一致性达到了 100%，而且两种技术在基因表达定量方面显示出良好的相关性（Spearman r=0.77）。此外，我们还在 126 例血液恶性肿瘤患者中对通过靶向 RNA-seq 鉴定融合进行了前瞻性评估。60名患者（48%）至少检测到一个融合转录本。我们设计了针对 11 种罕见融合的定制数字 PCR 检测方法，并验证了该技术在 MRD 定量中的检测限低于 0.01%。RNA-seq 和定制数字 PCR 的结合可能成为缺乏常规分子靶点的患者进行 MRD 评估的新标准。

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引用次数: 0

Genotype and Phenotype Correlation of the TPMT∗8 Allele in Thiopurine Metabolism TPMT*8：硫嘌呤代谢中一个重要的功能降低等位基因。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-08-31 DOI: 10.1016/j.jmoldx.2024.07.005

Rosalie M. Sterner, Patricia L. Hall, Dietrich Matern, John L. Black, Ann M. Moyer

Thiopurine 6-mercaptopurine (6-MP) is metabolized by thiopurine methyl transferase (TPMT). TPMT genetic variation results in some individuals having reduced or absent TPMT enzyme activity. If these individuals take a full thiopurine dose, life-threatening adverse events can occur. Testing identifies patients with reduced or absent TPMT activity and is recommended before initiation of therapy. The TPMT∗8 allele, defined by c.644G>A (p.Arg215His), is common among individuals of African ancestry (approximately 2.3% minor allele frequency) but is not included in genotyping recommendations due to its uncertain function. Here, a clinical TPMT enzyme activity assay was used to assess TPMT activity in red blood cells from 982 patients, including those with ∗1/∗8 (n = 22), ∗3A/∗8 (n = 1), and ∗3C/∗8 (n = 1) TPMT diplotypes. The average production of 6-methylmercaptopurine (primary TPMT product measured clinically) was 3.08 ± 0.16 nmol/mL per hour for ∗1/∗8 individuals, compared with 3.77 ± 0.03 nmol/mL per hour for normal metabolizers (P = 0.0001) and 2.39 ± 0.06 nmol 6-methylmercaptopurine/mL per hour for intermediate metabolizers (P < 0.0001). Individuals with a TPMT∗1/∗8 diplotype displayed reduced 6-MP metabolism between that of normal metabolizers and intermediate metabolizers, suggesting that TPMT∗8 is a reduced function allele.

硫嘌呤-6-巯基嘌呤（6-MP）由硫嘌呤甲基转移酶（TPMT）代谢。TPMT 基因变异会导致某些人的 TPMT 酶活性降低或消失。如果这些人服用足量的硫嘌呤，就会出现危及生命的不良反应。检测可识别 TPMT 活性降低或缺乏的患者，建议在开始治疗前进行检测。由 c.644G>A（p.Arg215His）定义的 TPMT*8 等位基因在非洲血统的个体中很常见（小等位基因频率为 2.3%），但由于其功能不确定，未被纳入基因分型建议中。本文采用临床 TPMT 酶活性测定法评估了 982 名患者红细胞中的 TPMT 活性，其中包括具有 *1/*8 （n=22）、*3A/*8 （n=1）和 *3C/*8 （n=1）TPMT 二联型的患者。*1/*8 患者的 6-MMP 平均产量（临床测量的主要 TPMT 产物）为 3.08 ± 0.16 毫摩尔/毫升/小时，而正常代谢者为 3.77 ± 0.03 毫摩尔/毫升/小时（p=0.0001），中间代谢者为 2.39 ± 0.06 毫摩尔 6-MMP/毫升/小时（p=0.0001）。

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引用次数: 0

Purity Independent Subtyping of Tumors (PurIST) Pancreatic Cancer Classifier PurIST 胰腺癌分类器：区分基础亚型和典型亚型的 16 种 RNA 表达特征的分析验证。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-08-22 DOI: 10.1016/j.jmoldx.2024.07.002

Yan Li , Jason D. Merker , Rachana Kshatriya , Dimitri G. Trembath , Ashley B. Morrison , Peyton C. Kuhlers , Naim U. Rashid , Jen Jen Yeh , Margaret L. Gulley

The two major molecular subtypes of pancreatic adenocarcinoma reportedly have differential response to FOLFIRINOX-based therapy. To promote rapid assignment of basal versus classical subtypes, an array-based single-sample classifier assay was developed and applied to 74 formalin-fixed, paraffin-embedded biopsy or resection specimens of known subtype based on transcriptomics. The Purity Independent Subtyping of Tumors (PurIST) algorithm assigns subtype based on relative expression of 16 RNAs counted by RNA sequencing (RNAseq) versus more practical array-based NanoString nCounter Elements XT technology. Subtype calls were largely concordant between RNAseq and array methods (72/74, 97% agreement). Compared with the lengthy RNAseq protocol, the array-based assay takes just 3 working days to analyze, permitting rapid reporting of tumor subtype. In conclusion, the PurIST pancreatic cancer classifier has robust performance to classify pancreatic adenocarcinoma into basal versus classical subtypes. Clinical validation studies are underway to evaluate outcome in patients whose standard-of-care chemotherapy regimen is selected on the basis of rapid subtype assignment (NCT04683315).

据报道，胰腺腺癌的两大分子亚型对基于FOLFIRINOX疗法的反应不同。为了促进基础亚型与经典亚型的快速分型，我们开发了一种基于阵列的单样本分类测定法，并将其应用于 74 份基于转录组学的已知亚型的 FFPE 活检或切除标本。肿瘤纯度独立亚型（PurIST）算法根据 RNAseq 计算的 16 种 RNA 的相对表达量与更实用的基于阵列的 NanoString nCounter Elements XT 技术进行亚型分配。RNAseq 和阵列方法对亚型的判定基本一致（72/74，一致率 97%）。与冗长的 RNAseq 程序相比，基于阵列的检测只需 3 个工作日即可完成分析，从而可以快速报告肿瘤亚型。总之，PurIST 胰腺癌分类器在将胰腺腺癌分为基底亚型和经典亚型方面表现出色。目前正在进行临床验证研究，以评估根据快速亚型分型选择标准化疗方案的患者的疗效（NCT04683315）。

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引用次数: 0

Performance Characteristics of Next-Generation Sequencing–Based Engraftment Monitoring and Microchimerism Detection in Allogeneic Hematopoietic Cell Transplantation 异基因造血细胞移植中基于 NGS 的移植监测和微嵌合体检测的性能特征：临床检测验证的实用方法》。

IF 3.4 3区医学 Q1 PATHOLOGY

Journal of Molecular Diagnostics

Pub Date : 2024-08-22 DOI: 10.1016/j.jmoldx.2024.07.003

Amanda G. Blouin , Wyatt Nelson , Daniel Geraghty , Medhat Askar , Fei Ye

Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showed high concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay and analysis software.

通过下一代测序（NGS）进行嵌合体分析是异基因造血细胞移植后移植监测的一种新兴方法。检测微嵌合体（嵌合体小于 1%）需要一种高灵敏度的方法，这种方法可能在早期复发检测、器官移植中的异体移植物监测和其他异体细胞疗法（如微移植）中具有临床实用性。随着越来越多的临床实验室采用这种方法，需要对其性能进行全面评估。本研究评估了一种基于 NGS 的检测方法，该方法同时使用 SNP 和 InDels 作为遗传标记。对准确性、线性度、灵敏度和重现性进行了评估。单供体的分析灵敏度为 0.2%，双供体的分析灵敏度为 0.5%。在整个嵌合率范围内，该检测方法显示出高度的可重复性。与 STR-PCR 的比较显示出很高的一致性；但 NGS 始终能检测到低于 5% 的嵌合率，而 STR-PCR 则检测不到。与 qPCR 的比较显示出很高的一致性，但在中间范围（40-60% 的嵌合率）相关性较低。总之，与 STR-PCR 和 qPCR 相比，该检测方法在单供体和多供体移植的整个嵌合率范围内表现出一致的高灵敏度和准确性。此外，我们还为测序性能和数据分析制定了质量指标标准。最后，我们讨论了临床实验室验证基于 NGS 的嵌合体检测和分析软件的注意事项。

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引用次数: 0