Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China.
School of Public Health, Shanxi Medical University, Taiyuan, Shanxi, China.
Eur J Haematol. 2018 Sep;101(3):291-296. doi: 10.1111/ejh.13084. Epub 2018 Jul 12.
This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment.
Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods.
At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R ≥ 0.99, with conversion equation of Y = 33.148X (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR.
In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.
本研究旨在建立一种用于监测 BCR/ABL（P210）阳性慢性髓系白血病（CML）患者微小残留病（MRD）的液滴数字 PCR（dd-PCR），从而实现肿瘤负荷的深度监测，并确定指导临床个体化治疗的疗效。
采用 dd-PCR 和 RT-qPCR，通过梯度稀释从 K562 细胞和正常外周血单个核细胞中获得两种细胞悬液，并在细胞水平进行测量。在外周血（PB）水平，在酪氨酸激酶抑制剂（TKI）治疗和定期随访后，获得 61 例 CML-慢性期（CML-CP）病例。通过 RT-qPCR，在连续 3 次分析中，每 3 个月进行 1 次，PB 中均未检测到 BCR/ABL（P210）融合基因。同时，同时进行最后等量的 cDNA 的 dd-PCR。对 10 例 MR4.5 的 CML 患者同时采用两种方法进行随访。
在细胞水平上，dd-PCR 和 RT-qPCR 的结果一致性达到 R≥0.99，转换方程为 Y=33.148X（Y：dd-PCR 结果；X：RT-qPCR 结果）。在 dd-PCR 检测中，61 例 CML 患者中有 11 例（18.03%）检测阳性，差异有统计学意义（P<0.01）。在 10 例 MR4.5 的 CML 患者的随访中，所有患者均失去 MR4.0，其中 4 例通过 dd-PCR 比 RT-qPCR 早 3 个月检测阳性。
与 RT-qPCR 相比，dd-PCR 更敏感，因此通过转换方程可以将 dd-PCR 结果准确转换为国际标准的 RT-qPCR 结果，从而实现基于更深分子生物学的 BCR/ABL（P210）MRD 的更精细的分层。对临床监测疾病进展具有一定的参考价值。
