Nga Yeung Tang, PhD, DABCC, Technical Director, STAT lab and Point of Care Testing, Corewell Health William Beaumont University Hospital, Royal Oak, MI; Assistant Professor, Department of Pathology, Oakland University William Beaumont School of Medicine, Auburn Hill, MI
Anastasia Gant Kanegusuku, PhD, DABCC, Associate Director, Regional Core Laboratory and Point of Care Testing; Assistant Professor of Pathology & Laboratory Medicine, Loyola University Medical Center, Chicago, IL
Preemptive pharmacogenomics (PGx) testing potentially improves clinical outcomes by having drug response predictions in hand. To provide testing to a large cohort of healthy individuals with a broad coverage of variants, pre-selected panels from manufacturers with diverse coverages and focuses could be implemented. Alternatively, to support specific populations, clinicians, and laboratorians could also develop custom-designed panels.
The recently published study from Dr. Tang’s group described the clinical validation of a custom-designed OpenArray PGx (OA-PGx) panel targeting 478 single-nucleotide variants (SNVs), which aimed to support clinical studies at the Center for Personalized Therapeutics at the University of Chicago (1). The selection of variants was based on the targeted population, clinical evidence, and the potential for variants to be clinically actionable in the future. References for drug-gene phenotypes for the selection included the Clinical Pharmacogenetics Implementation Consortium (CPIC), the Dutch Pharmacogenetics Working Group (DPWG), the Pharmacogene Variation Consortium (PharmVar), and the Food and Drug Administration (FDA) drug labels. The OA-PGx panel, a laboratory-developed molecular genetics test, underwent validation studies that included accuracy, precision, and sensitivity.
The accuracy study used reference genotypes determined by next-generation sequencing and the Sequenom MassARRAY iPLEX platform. The precision was assessed using Coriell Institute cell line DNA samples in triplicate to interrogate the reproducibility. The results showed that for 437 variants (including 35 variants on the RYR1 gene), all samples assayed had concordant calls with at least one reference genotype, demonstrating good accuracy. The sensitivity study showed that the call rates for DNA concentrations of 10 ng/µL and 50 ng/µL were 99.2% and 99.6%, respectively. Therefore, the manufacturer-recommended concentration of 50 ng/µL is preferred for patient genotyping.
While the manufacturer provides guidelines for the required concentration of genetic material, the laboratory plays an important role in determining which specimen types are acceptable for PGx testing. The study validated the OA-PGx panel using patient whole blood samples. As the clinical trials progressed at the Center for Personalized Therapeutics, it became evident that switching to a less invasive specimen type—buccal cells collected by buccal swab—would improve patient enrollment in the studies. To support this specimen type, the laboratory performed additional validation studies to ensure concordance between results obtained from the buccal swabs and whole blood samples.
Although the OpenArray is an ideal methodology for large-scale PGx testing, it comes with limitations. Because the OpenArray technology is based on allelic discrimination, each triallelic variant on the panel requires two assays for genotyping. Note that the allelic discrimination platform lacks the capability to detect novel variants. However, it remains suitable for well-studied variants. Recognizing the limitation of OpenArray technology for genotyping highly polymorphic genes, the OA-PGx panel is complemented by an extensive CYP2D6 assay based on Invader and TaqMan copy number assays (2).
针对一个个性化药物基因组学测试的临床验证
预测性药物基因组学 (PGx)测试通过提供药物反应的预测， 有望提高临床医疗效果。为了能够为一般健康人群提供全面的测试， 多个制造商提供了覆盖不同基因的预选测试套餐. 另外，为了支持对特定人群的筛查， 医生和实验室工作人员也在致力于开发个性化的测试组套。
在唐博士的团队最近发表的研究中，他们针对 478 个单核苷酸变异 (SNV) 的个性化 OpenArray PGx 测试套餐（OA-PGx）进行了临床验证。这组测试是为了支持芝加哥大学个性化治疗中心的临床研究（ 1) 。变异基因是基于研究对象、临床证据以及变异基因未来在临床上能被使用的可能性来选择的。选择过程中使用的药物基因表型参考了包括Clinical Pharmacogenetics Implementation Consortium (CPIC) 和Dutch Pharmacogenetics Working Group (DPWG)、Pharmacogene Variation Consortium (PharmVar) 以及美国食品和药物管理局 (FDA) 药物标签。 由于OA-PGx 测试套餐是实验室开发的测试，临床验证实验包括了准确性、可重复性和灵敏度。
准确性研究使用了通过新一代测序以及和 Sequenom MassARRAY iPLEX 平台确定的参考基因型做比较。可重复性实验采用 Coriell Institute的细胞系 DNA 样本，对每DNA 样本进行了三次测试。结果显示，对于437 个变异（包括RYR1 基因上的35 个变异），所有检测的样品都与至少一种参考基因型具有一致的结果，证明了测试的准确性。灵敏度的实验表明，DNA 浓度为 10 ng/μL 和 50 ng/μL 时的检出率分别为 99.2% 和 99.6%。因此，制造商推荐的浓度 50 ng/μL 是患者基因分型的首选浓度。
虽然制造商提供了所需遗传物质样本浓度的指南，但实验室在确定 PGx 测试可接受的样本类型方面发挥着重要作用。该研究验证了全血样本适合在OA-PGx测试上使用。随着个性化治疗中心临床试验的发展，中心需要改用侵入性较小的样本类型（比如通过口腔拭子收集的口腔细胞）去提高患者参与研究的人数。为支持这种样本类型，实验室进行了额外的验证研究，以确保从口腔拭子获得的结果与全血样本结果的一致性。
尽管 OpenArray 是大规模 PGx 测试的理想方法，它也有局限性。由于 OpenArray 技术基于等位基因的区分，因此测试中的每个三代型等位基因都需要两次基因分型测定。其他的局限性包括等位基因辨别平台缺乏检测新变异的能力。然而，它仍然适用于经过充分研究的基因变异。另一方面，由于 OpenArray 技术不是高度多态性基因分型的最佳平台，因此OA-PGx panel 需要Invader 和 TaqMan 拷贝数检测的广泛 CYP2D6 检测的补充及支持 (2)。
References:
1. Tang NY, Pei X, George D, House L, Danahey K, Lipschultz E, et al. Validation of a Large Custom-Designed Pharmacogenomics Panel on an Array Genotyping Platform. J Appl Lab Med. 2021;6:1505–16. https://doi.org/10.1093/jalm/jfab056
2. Leung EKY, Agolini E, Pei X, Melis R, McMillin GA, Friedman PN, et al. Validation of an Extensive CYP2D6 Assay Panel Based on Invader and TaqMan Copy Number Assays. The Journal of Applied Laboratory Medicine. 2017;jalm.2016.021923. https://doi.org/10.1373/jalm.2016.021923