DataSheet_1_Peripheral blood marker of residual acute leukemia after hematopoietic cell transplantation using multi-plex digital droplet PCR.pdf

BackgroundRelapse remains the primary cause of death after hematopoietic cell transplantation (HCT) for acute leukemia. The ability to identify minimal/measurable residual disease (MRD) via the blood could identify patients earlier when immunologic interventions may be more successful. We evaluated a new test that could quantify blood tumor mRNA as leukemia MRD surveillance using droplet digital PCR (ddPCR).
MethodsThe multiplex ddPCR assay was developed using tumor cell lines positive for the tumor associated antigens (TAA: WT1, PRAME, BIRC5), with homeostatic ABL1. On IRB-approved protocols, RNA was isolated from mononuclear cells from acute leukemia patients after HCT (n = 31 subjects; n = 91 specimens) and healthy donors (n = 20). ddPCR simultaneously quantitated mRNA expression of WT1, PRAME, BIRC5, and ABL1 and the TAA/ABL1 blood ratio was measured in patients with and without active leukemia after HCT.
ResultsTumor cell lines confirmed quantitation of TAAs. In patients with active acute leukemia after HCT (MRD+ or relapse; n=19), the blood levels of WT1/ABL1, PRAME/ABL1, and BIRC5/ABL1 exceeded healthy donors (p<0.0001, p=0.0286, and p=0.0064 respectively). Active disease status was associated with TAA positivity (1+ TAA vs 0 TAA) with an odds ratio=10.67, (p=0.0070, 95% confidence interval 1.91 – 59.62). The area under the curve is 0.7544. Changes in ddPCR correlated with disease response captured on standard of care tests, accurately denoting positive or negative disease burden in 15/16 (95%). Of patients with MRD+ or relapsed leukemia after HCT, 84% were positive for at least one TAA/ABL1 in the peripheral blood. In summary, we have developed a new method for blood MRD monitoring of leukemia after HCT and present preliminary data that the TAA/ABL1 ratio may may serve as a novel surrogate biomarker for relapse of acute leukemia after HCT.

背景：造血细胞移植（hematopoietic cell transplantation, HCT）后，复发仍是急性白血病患者死亡的首要原因。通过血液样本检测微小残留病（minimal/measurable residual disease, MRD），有望在免疫干预效果更佳的早期阶段识别高危患者。本研究评估了一款新型检测手段，其可借助液滴数字聚合酶链式反应（droplet digital PCR, ddPCR）量化血液中肿瘤mRNA水平，用于白血病MRD监测。
方法：本多重ddPCR检测方法以表达肿瘤相关抗原（tumor associated antigens, TAA：WT1、PRAME、BIRC5）的肿瘤细胞系为开发基础，并以稳态基因ABL1作为内参。经伦理审查委员会（Institutional Review Board, IRB）批准的研究方案中，我们从31名造血细胞移植后急性白血病患者（共91份标本）以及20名健康志愿者的外周血单个核细胞中提取RNA。采用ddPCR技术同时定量检测WT1、PRAME、BIRC5及ABL1的mRNA表达水平，并计算造血细胞移植后伴或不伴活动性白血病患者的TAA/ABL1血液比值。
结果：肿瘤细胞系实验证实了该方法可对肿瘤相关抗原进行定量检测。造血细胞移植后伴活动性急性白血病（MRD阳性或复发；n=19）患者的WT1/ABL1、PRAME/ABL1及BIRC5/ABL1血液水平均显著高于健康志愿者（分别为p<0.0001、p=0.0286、p=0.0064）。活动性疾病状态与肿瘤相关抗原阳性（携带1种及以上TAA vs 未携带TAA）显著相关，比值比为10.67（p=0.0070，95%置信区间1.91–59.62）。受试者工作特征曲线下面积为0.7544。ddPCR检测结果的变化与临床标准检测手段捕获的疾病应答情况具有相关性，可准确判定15/16（95%）样本的疾病负荷正负状态。在造血细胞移植后MRD阳性或复发的白血病患者中，84%的患者外周血中至少有一种TAA/ABL1比值呈阳性。
综上，本研究开发了一种用于造血细胞移植后白血病血液MRD监测的新型方法，并提供了初步数据，证实TAA/ABL1比值可作为急性白血病造血细胞移植后复发的新型替代生物标志物。