实时定量PCR监测B细胞恶性肿瘤患者造血干细胞移植后的IgH水平及意义

本研究评价实时定量PCR(RQ-PCR)技术检测IgH水平对B细胞恶性肿瘤患者造血干细胞移植(HSCT)后残留肿瘤细胞监测的意义.采用家族一致性TaqMan探针联合等位基因特异性寡核苷酸(ASO)上游引物技术检测22例B细胞恶性肿瘤患者HSCT前后骨髓单个核细胞的IgH水平动态变化.IgH水平以内参基因GAPDH进行归一化.结果表明,RQ-PCR实验可重复灵敏度为1个拷贝.9例IgH单克隆重排患者,在HSCT后1个月骨髓中IgH的拷贝数较初治时明显降低(6.67×103/106 GAPDH vs 29/106 GAPDH,p＜0.01).3例移植后15个月IgH的拷贝数持续小于102/106 GAPDH,18个月后IgH水平为0的患者获得了完全的临床和分子遗传学缓解(CCyR);5例移植后3个月以内IgH拷贝数持续小于102/106 GAPDH,随后IgH拷贝数持续小于103/106 GAPDH的患者临床获得完全缓解(CR).1例患者移植后近3个月时IgH拷贝数为4.5×103/106 GAPDH,4个月时临床复发.RQ-PCR检测8例干细胞采集物的肿瘤污染水平为3.68×102(0-1720)/106 GAPDH,外周血采集物中的肿瘤污染小于骨髓[75(0-890)/106 GAPDH vs 1.1×103(527-1720)/106 GAPDH,p＜0.05].采集物可用于RQ-PCR检测的8例患者无论肿瘤污染程度如何,临床均无复发.采集物中肿瘤污染的水平与初治及移植后1个月的IgH水平呈正相关(r值分别为0.810、0.708,p＜0.05).结论RQ PCR能够有效监测B细胞恶性肿瘤患者移植后IgH水平动态变化.移植后3个月内IgH拷贝数大于103/106 GAPDH可能是预测患者复发的标志.

Monitoring IgH Levels in Patients with B -Cell Malignancy by Real-time Quantitative PCR after Hematopoietic Stem Cell Transplantation and Its Significance

The study was purpose to evaluate the value of real time quantitative-PCR for monitoring IgH level in patients with B-cell malignancy after hematopoietic stem cell transplantation (HSCT). Quantification of IgH levels was performed on bone marrow mononuclear cells from 9 patients with B-cell malignancy before and after HSCT by PCR using the consensus JH TaqMan probe in combination with an allele-specific oligonucleotide (ASO) upstream primer. The IgH levels was normalized by control gene GAPDH. The results indicated that the reproducible sensitivity of RQ-PCR was 1 copy, the significant reduction of IgH copies was observed in bone marrow samples of 9 patients at one month post HSCT (6.67x10(3)/10(6) GAPDH vs 29/10(6) GAPDH, p<0.01). 3 out of 9 patients who achieved complete clinical and molecular cytogenetic remission (CCyR) contained persistently measurable low IgH level of 10(2)/10(6) GAPDH within 15 months and no detectable IgH at 18 months post HSCT. Whereas 5 out of 9 patients whose IgH copies were less than 10(2)/10(6) GAPDH within 3 months and less than 10(3)/10(6) GAPDH 3 months post HSCT achieved a sustained complete remission (CR). IgH copies in one patient were 4.5x10(3)/10(6) GAPDH at 3 months post HSCT, who relapsed at 4 months post HSCT. The median levels of tumor contamination in the stem cell harvests from 8 patients measured by RQ PCR were 3.68x10(2) (0-1720)/10(6) GAPDH. RQ PCR showed that PBPC harvests were less contaminated than BM harvests [75 (0-890)/10(6) GAPDH vs 1.1x10(3) (527-1720)/10(6) GAPDH, p<0.05]. 8 patients whose stem cell harvest were avaiable for RQ PCR were still in CR despite of the tumor contamination. The level of tumor contamination in stem cell harvest well correlated with IgH levels at diagnosis and one month after HSCT (r=0.810, r=0.708, p<0.05). It is concluded that RQ PCR can effectively monitor the IgH levels in patients with B-cell malignancy after auto-HSCT. 10(3)/10(6) GAPDH within 3 months post HSCT may be a cut-off level of IgH copies, which may be used to evaluate different prognoses of patients.