数字PCR技术在慢性髓系白血病微小残留病检测中的临床研究

目的：

比较数字 PCR（dPCR）与实时荧光定量 PCR（qPCR）检测慢性髓系白血病（CML）患者BCR::ABLl p210 国际标准（% IS）水平的差异，探讨 dPCR 在分子学反应（MR）动态监测中检测极低转录本水平的临床应用价值及其指导无治疗缓解（TFR）的潜在作用。

方法：

对 dPCR 体系进行性能验证；使用 qPCR 和 dPCR 检测 82 例 CML 治疗后样本的 BCR::ABLl p210 % IS 水平，对其中 47 例患者进行随访监测；对比分析 dPCR 与 qPCR 检测结果差异，并评估其在微小残留病（MRD）检测方面的差异。

结果：

dPCR 检测体系性能良好，对低 BCR::ABLl p210 % IS 水平具有良好的检出能力。在 82 例患者共 197 对样本中，dPCR 的阳性检出率显著高于 qPCR（P＜0.001），即便在深层分子学反应（DMR）阶段，dPCR 的阳性检出率仍显著优于 qPCR（P＜0.001）。两种方法的检测结果呈高度正相关（r＝0.7806，P＜0.001），且总体一致性良好（平均差值 -0.0511，95％一致性界限：-0.7719 至 0.6697）。MR分级整体一致性尚可（Kappa＝0.4020），分层分析后一致性较好（加权 Kappa＝0.6160，95％ CI：0.5440-0.6880，P＜0.001），完全一致率为 51.78％，且一致性随分子学缓解深度加深而降低。47 例患者的动态监测结果显示，部分 qPCR 持续阴性的患者 dPCR 仍可捕捉到低水平 BCR::ABLl p210 % IS 波动；dPCR 较 qPCR 可提前数月检出 MRD 变化。

结论：

dPCR 体系性能稳定、具有较好的低水平 MRD 检出能力，可在部分患者中检出 qPCR 未能检测到的 MRD 变化。在 CML 患者达到 DMR 后，dPCR 检测较 qPCR 存在一定优势。

Objective: 

To compare the differences of BCR::ABLl p210 international scale (% IS) levels betweendigital PCR (dPCR) and real-time quantitative PCR (qPCR) in patients with chronic myeloid leukemia (CML), and toevaluate the application value of dPCR for detecting very low transcript levels during dynamic monitoring of molecularresponse (MR) and its potential role in guiding treatment-free remission (TFR).

Methods: 

The analytical performance of the established digital PCR (dPCR) assay was validated. BCR::ABLl p210 (%IS) levels in post-treatment samples from 82 patients with chronic myeloid leukemia (CML) were measured by both qPCR and dPCR, and 47 of these patients were followed up longitudinally. Statistical analyses were performed to compare the results of the two methods and to evaluate their differences in minimal residual disease (MRD) detection.

Results: 

The dPCR assay demonstrated good analytical performance and reliable detection of low BCR::ABLl p210 (%IS) levels. A total of 197 paired measurements from 82 patients were analyzed. The positive detection rate of dPCR was significantly higher than that of qPCR (P < 0.001), and this difference remained significant in patients with deep molecular response (DMR) (P < 0.001). The two methods showed a high positive correlation (r = 0.7806, P < 0.001) with good overall agreement (mean difference: −0.0511; 95% limits of agreement: −0.7719 to 0.6697). Agreement in molecular response (MR) grading was moderate (kappa = 0.4020) and improved after weighting (weighted kappa = 0.6160, 95% CI : 0.5440–0.6880, P < 0.001), with an overall concordance rate of 51.78%; however, agreement decreased as the depth of molecular response increased. Dynamic monitoring of the 47 patients revealed that dPCR detected low-level fluctuations of BCR::ABLl p210 (%IS) in some patients who remained persistently negative by qPCR, and identified MRD changes several months earlier than qPCR.

Conclusion: 

The dPCR assay exhibits stable performance and good detection capability for low-level MRD, and can identify MRD changes that remain undetectable by qPCR in some patients. Once CML patients achieve deep molecular response (DMR), dPCR may offer certain advantages over qPCR for MRD monitoring.