Detection of minimal residual disease in acute lymphoblastic leukemia by next-generation sequencing of IG/TCR
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摘要: 目的 比较免疫球蛋白(IG)/T细胞受体(TCR)重排二代测序技术(next-generation sequencing，NGS)与其他急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)微小残留病(minimal residual disease，MRD)检测手段间的差异，评估IG/TCR重排NGS在ALL MRD监测中的价值。方法 回顾性分析2018年9月至2023年12月本院收治的25例ALL患者，同时采用NGS、流式细胞术(FCM)和实时定量聚合酶链反应技术(q-PCR)检测MRD。结果 48.0%的患者初诊时NGS检测存在3个或以上异常高频克隆。81.3%的患者治疗后的主要残留克隆与初诊时最高频克隆一致。在FCM判定MRD阴性的69例样本中24例(34.8%)经NGS检测仍阳性，MRD中位水平为4.13×10-5，显著低于FCM阳性患者的MRD水平(4.70×10-3)(P < 0.05)。Ph+ALL患者同时采用NGS、q-PCR、FCM三种方法进行MRD检测，阳性率依次为59.1%、50.0%、27.3%，NGS阳性率显著高于FCM(P=0.035)。FCM判定阴性的16例样本中7例(43.8%)经NGS检测为阳性，中位MRD水平为1.99×10-5，较NGS/FCM双阳性组的中位MRD水平有降低趋势，但差异无统计学意义(P>0.05)。q-PCR判定阴性的11例样本中3例(27.3%)经NGS检测为阳性，中位MRD水平为7.59×10-6，较NGS/q-PCR双阳性组的中位MRD水平有降低趋势，但差异亦无统计学意义(P=0.053)。结论 NGS检测MRD的灵敏度和深度均优于FCM；Ph+ALL患者中NGS检测的灵敏度及深度较q-PCR有改善趋势。IG/TCR重排NGS是能保证足够检测深度的有效MRD检测手段。Abstract: Objective To compare the immunoglobulin(IG)/T cell receptor(TCR) rearrangement next-generation sequencing(NGS) with other methods of minimal residual disease(MRD) detection in acute lymphoblastic leukemia(ALL), and evaluating the value of IG/TCR rearrangement NGS in monitoring MRD.Methods A retrospective analysis was conducted on 25 ALL patients admitted to our hospital from September 2018 to December 2023. The MRD was determined by using NGS, flow cytometry(FCM) and real-time quantitative PCR(q-PCR).Results Three or more abnormal clones were detected by NGS in 48.0% of patients at initial diagnosis. The main residual clone of 81.3% of patients after treatment was consistent with the highest frequency clone at initial diagnosis. Among the 69 FCM MRD-negative(MRDneg) samples, 24 cases(34.8%) were NGS MRD-positive(MRDpos), and the median MRD level was 4.13×10-5, which was significantly lower than that of FCM MRDpos patients(4.70×10-3)(P < 0.05). In Ph+ALL patients, the positive rates of MRD detected by NGS, q-PCR and FCM were 59.1%, 50.0% and 27.3%, respectively. The MRD positive rate of NGS was higher than that of FCM(P=0.035). Among the 16 FCM MRDneg samples, 7 cases(43.8%) were NGS MRDpos. The median MRD level was 1.99×10-5, which had a decrease compared with the NGS/FCM MRDpos group, but there was no significant difference(P>0.05). Among the 11 q-PCR MRDneg samples, 3 cases(27.3%) were NGS MRDpos, and the median MRD level was 7.59×10-6, which showed a decreasing trend compared with the NGS/q-PCR MRDpos group(P=0.053).Conclusion The MRD detection sensitivity and depth of NGS are better than that of FCM. NGS also showed advantages over q-PCR in the detection of sensitivity and depth in Ph+ALL patients. NGS of IG/TCR is an effective method to ensure sufficient MRD detection depth.
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表 1 NGS和FCM检测治疗后MRD(89例次)
中位MRD FCM(+)(20例次) FCM(-)(69例次) NGS(+)(17例次) NGS(-)(3例次) NGS(+)(24例次) NGS(-)(45例次) NGS 4.53×10-2△ (1.21×10-6~6.50×10-1) MRD为阴性 4.13×10-5 (2.74×10-7~4.67×10-2) MRD为阴性 FCM 4.70×10-3△ (4.00×10-4~9.15×10-1) 3.00×10-4 (5.00×10-5~1.00×10-4) MRD为阴性 MRD为阴性 3.25×10-3△(4.00×10-4~9.15×10-1) MRD为阴性 MRD为阴性 与NGS(+)/FCM(-)比较，△P < 0.05。 
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表 2 Ph+ALL治疗后样本MRD检测(22例次)
中位MRD q-PCR(+)(11例次) q-PCR(-)(11例次) NGS(+)(10例次) NGS(-)(1例次) NGS(+)(3例次) NGS(-)(8例次) NGS 5.32×10-3 (1.26×10-5~2.62×10-1) MRD为阴性 7.59×10-6 (6.00×10-6~8.40×10-4) MRD为阴性 q-PCR 2.49×10-2 (2.00×10-3~1.82×10-1) 1.30×10-2 MRD为阴性 MRD为阴性 2.40×10-2(2.00×10-3~1.82×10-1) MRD为阴性 MRD为阴性
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