联合应用细胞遗传学、巢式RT-PCR和FISH技术检测慢性髓系白血病治疗过程中的肿瘤负荷

Detection of Tumor Load in Chronic Myeloid Leukemia during Treatment with Transplantation by Conventional Cytogenetics,Nested-RT-PCR and FISH

本研究探讨常规细胞遗传学(conventional cytogenetics,CC)、巢式逆转录-聚合酶链反应(nested-reverse transcriptase polyme rase chain reaction,nested-RT-PCR)及双色双融合荧光原位杂交(dual-color and dual-fusion fluo-rescence in situ hybridization,D-FISH)三种技术监测慢性髓系白血病(chronic myeloid leukemia,CML)患者造血干细胞移植治疗过程中肿瘤负荷的灵敏度和特异性。联合应用CC、巢式RT-PCR和D-FISH三种技术对7例CML患者非清髓性异基因干细胞移植治疗前后的肿瘤负荷水平进行检测。检测结果显示:7例CML患者治疗前后的40份骨髓标本中,有29份标本检出不同比率的Ph染色体;3份因细胞数少CC分析失败;36份标本RT-PCR检测结果为阳性。病例1移植后12、18、26及38个月的4份标本Ph染色体及RT-PCR结果均为阴性。病例1移植后9和10个月、病例2移植后15个月、病例3移植后12个月的4份Ph(-)bcr/abl(+)标本经FISH检测,分别检出5.4%、0%、16.5%及1.5%的bcr/abl(+)细胞。病例5移植后20和60天、病例7移植后40天的3份标本因细胞数少而CC核型分析失败,对其行FISH检测,结果bcr/abl(+)细胞检出率分别为55.0%、27.5%和73.5%。病例1移植后12个月Ph(-)bcr/abl(-)的标本行FISH检测,结果bcr/abl(+)细胞检出率为0%。结论:CC可作为监测CML患者治疗过程中肿瘤负荷水平的基本手段。在移植后早期细胞数太少而无法进行CC检测,以及治疗病人体内肿瘤负荷降低到CC不能检出而RT-PCR仍为阳性时,借助FISH准确检测体内肿瘤负荷,以监测其动态变化。FISH检测bcr/abl转阴的病人需靠灵敏度更高的RT-PCR监测核定。

This study was purposed to investigate the sensitivity and specificity of conventional cytogenetics （ CC）, nested-reverse transcriptase polymerase chain reaction （nested-RT-PCR） and dual-color/dual-fusion fluorescence in situ hybridization （D-FISH） technique in monitoring the tumor load of chronic myeloid leukemia （CML） during treatment with transplantation. CC, nested-RT-PCR and interphase D-FISH were simultaneously carried out to detect the tumor load of 7 CML patients during treatment with non-myeloablative allogeneic stem cell transplantation （ allo-NSCT ）. 40 specimens from 7 CML patients before and after allo-NSCT were analyzed. The results showed that 29 specimens were Ph（ ＋ ） with different positive ratio and 3 specimens with lower cells were not analyzed by CC. 36 specimens were bcr/ abl mRNA （ ＋ ） by RT-PCR. 4 specimens from case 1 at 12,18,26 and 38 months after allo-NSCT were Ph（ - ） and bcr/abl mRNA （ - ） ,4 Ph（ - ） bcr/abl（ ＋ ） specimens containing 2 from case 1 at 9 and l0 months after allo-NSCT, 1 from case 2 at 15 months after allo-NSCT, 1 from case 3 at 12 months after allo-NSCT showed 5.4%, 0%, 16.5% and 1.5% bcr/abl（ ＋ ） cells by FISH. 3 specimens with lower cells containing 2 from case 5 at 20 and 60 days after allo- NSCT and 1 from case 7 at 40 days after allo-NSCT were analyzed by FISH and showed 55.0%, 27.5% and 73.5% bcr/abl（ ＋ ） cells. The Ph （ - ） bcr/abl（ - ） specimen from case 1 at 12 monthss post-allo-NSCT showed 0% bcr/abl （ ＋ ） cells by FISH. It is concluded that CC can be used as a basic tool to monitor the change of tumor load in CML during treatment. When specimen with lower cells can not be analyzed by CC in early period after allo-NSCT, or result of CC can not evaluate precisely dynamic change of tumor load and when tumor load in treated patient are lower to Ph （ - ） by CC while bcr/abl mRNA （ ＋ ） by RT-PCR, FISH must be used to detect precisely tumor load and monitor dynamic change of it. More sensitive RT-PCR is used to monitor tumor load when it is lower to bcr/abl（ - ） by FISH during treatment.