Schilhabel Anke, Szczepanowski Monika, van Gastel-Mol Ellen J, Schillalies Janina, Ray Jill, Kim Doris, Nováková Michaela, Dombrink Isabel, van der Velden Vincent H J, Boettcher Sebastian, Brüggemann Monika, Kneba Michael, van Dongen Jacques J M, Langerak Anton W, Ritgen Matthias
Hämatologie Labor Kiel, Medical Department II, Hematology and Oncology, University Medical Center Schleswig-Holstein, Kiel, Germany.
Laboratory Medical Immunology, Department of Immunology, Erasmus Medical Center (MC), University Medical Center Rotterdam, Rotterdam, Netherlands.
Front Oncol. 2023 Jan 16;12:1111209. doi: 10.3389/fonc.2022.1111209. eCollection 2022.
Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10 and 10 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.
使用实时定量（RQ）-PCR检测患者特异性和肿瘤特异性克隆重排免疫受体基因是血液系统恶性肿瘤精准医学领域公认的方法。由于每个患者和白血病克隆都需要单独的引物，因此为该方法建立性能规范面临独特挑战。对慢性淋巴细胞白血病（CLL）和急性淋巴细胞白血病（ALL）患者的一系列诊断检测结果表明，该方法的分析性能不依赖于患者的疾病特征。在所有检测中，90%的检测校准范围在10到10之间呈线性。当前标准化方法的检测限在100,000个白细胞中为1.8至4. .8个细胞。作为正交方法，RQ-PCR与流式细胞术和下一代测序的总体一致性约为90%。不同实验室之间以及在临床应用的微小/可测量残留疾病（MRD）临界值上下的准确性和精密度证明了该技术的稳健性。本文报道的全面的、体外诊断（IVD）指导的分析验证提供了证据，表明当使用标准化的数据生成和评估方案时，个性化诊断方法可生成可靠、可重复和特异的MRD数据。我们的方法也可以作为一个指导示例，说明如何根据欧洲IVD法规完成个性化内部诊断的分析验证。
