Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
Cell Therapy and Cell Engineering Facility, Memorial Sloan Kettering Cancer Center, New York, New York.
J Mol Diagn. 2023 Sep;25(9):634-645. doi: 10.1016/j.jmoldx.2023.06.002. Epub 2023 Jun 16.
Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.
尽管嵌合抗原受体 (CAR) T 细胞在体内的植入、扩增和持续存在是治疗效果的关键组成部分，但在常规临床实践中并未进行定量监测。我们描述了一种数字 PCR 检测方法的开发和分析验证，用于在治疗后超灵敏地检测 CAR 构建体，规避了已知低分区平台的技术限制。用于检测 axicabtagene、brexucabtagene 和 Memorial Sloan Kettering CAR 构建体的引物和探针被用于验证在 Bio-Rad 数字 PCR 低分区平台上的测试；结果与 Raindrop（一种高分区系统）作为参考方法进行比较。修改了 Bio-Rad 方案，以实现高达 500 ng 的 DNA 输入测试。使用双输入反应（20 和 500 ng）和综合分析方法，该检测在与参考方法相比时，在约 1 × 10（0.001%）左右表现出一致的靶标检测，具有出色的特异性和重现性以及 100%的准确性。对验证/实施阶段收到的 53 个临床样本进行专门分析表明，该检测有效地能够在早期扩增（第 6 天至 28 天）和长期持续（长达 479 天）的多个时间点进行监测。CAR 载体的检测水平从 0.005%到 74%（载体与参照基因拷贝数）不等。我们队列中观察到的最高水平与 2 级和 3 级细胞因子释放综合征诊断的时间诊断密切相关（P < 0.005）。在采样时，只有 3 名无法检测到构建体的患者发生疾病进展。
