【作者】 魏道林；

【导师】 王椿；

【作者基本信息】 复旦大学 ， 内科学， 2004， 博士

【摘要】 目的：观察改进培养方法对多发性骨髓瘤(multiple myeloma，MM)异常核型检出的影响，采用FISH技术回顾分析MM患者初诊时13号染色体的改变，分析13号染色体部分缺失在MM中的临床意义。 方法：对20例MM患者骨髓细胞进行常规细胞遗传学分析，观察MM的常见细胞遗传学改变以及改进培养方法(延长培养时间并添加生长因子)对MM异常核型检出的影响。以骨髓涂片档案片为标本，采用荧光原位杂交(fluorescence in situ hybridization，FISH)技术检测68例MM患者初诊时有无13号染色体上Rb-1基因缺失，并对44例患者中Rb-1、13q14.3两位点的缺失情况进行了比较。采用卡方检验分析13号染色体部分缺失与患者初诊时临床特征间关系；采用Log-rank检验进行单因素生存分析，用Cox回归作多因素生存分析，并用Kaplan-Meier法作图分析不同指标对生存率影响。 结果：通过R显带方法对16例复治MM患者的骨髓标本进行了染色体核型分析，发现培养方法对检测结果有影响：16例标本采取直接法、3天培养法均未发现异常核型，而培养6天并添加刺激剂有4例检出异常核型，其中3例为复杂异常核型，1例仅见del(13q14)；采用JAB Rb-1探针进行的FISH分析证实了显带分析的结果。FISH分析中，68例MM患者中有35例(51％)检出13号染色体部分缺失，有29例检出Rb-1基因缺失(43％)；其中44例患者有23例检出13q14.3位点缺失(52％)；两种探针检测结果相同的几率是66％(29／44)。卡方检验发现13号染色体部分缺失与血清肌酐水平、检出原浆细胞、血红蛋白水平、骨破坏数量、早期化疗反应、1年生存有关；单因素分析发现乳酸脱氢酶水平、骨髓象检出原浆细胞、轻链类型与预后有关相关；多因素分析中，骨髓象检出原浆细胞和血清肌酐水平与预后有密切联系；未发现13号染色体部分缺失与预后有关。 结论：延长培养时间、添加IL-6和GM-CSF等培养方法的改进有利于MM异常核型的检出，MM的细胞遗传学改变多为复杂核型异常。以骨髓涂片档案片为样本进行FISH检测方法简便、结果可靠，便于血液系统克隆性疾病的回顾性遗传学分析。Rb-1基因及13q14.3位点缺失是MM患者常见细胞遗传学改变，相当比例患者的13号染色体缺失涉及长臂较大片段。13号染色体部分缺失对MM的生物学行为有影响。骨髓象检出原浆细胞、高血清肌酐水平为MM的独立不良预后指标；受限于样本量，未能发现13号染色体部分缺失与预后有密切联系。更多还原

【Abstract】 OBJECTIVE: Evaluation of the effect of modified cell culture on detection of abnormal karyotype in MM patients. Retrospective analysis of the deletion chromosome 13 in MM patients at diagnosis were performed with FISH technique. Clinical significance of FISH-defined partial deletion chromosome 13 in MM patients were investigated.METHODS: RHG banding was used to study the common cytogenetic changes and to evaluate the effect of culture method modification(long term culture and addition of growth factors) on abnormal karyotype detection in 20 MM patients. Archival bone marrow smears from 68 MM patients at diagnosis were used to study and compare the deletion of Rb~l gene and locus 13ql4. 3 by FISH analysis. Chi-square test was used to study the relations between partial deletion of chromosome 13 and clinical featuress; Log-rank test, Cox’ s regression and Kaplan-Meier method were used in survival analysis. RESULTS: 16 cases analyzed by RHG-banding were evaluable, culture method had effect on the cytogenetic detection : abnormal karyotype were detected in 4 cases after 6 days culture with addi tion of IL-6 and GM-CSF; 3 cases with complex karyotype, 1 case with del(13ql4) only; these were proved by FISH using probes specific for Rb-1 gene. 35 out of 68(51%) cases were found with partial deletion of chromosome 13, 29 cases (43%) with deletion of Rb-1 gene; 23 out of 44 cases (52%) with deletion of 13ql4. 3; 66%(29/44) cases with the same result in above analysis. Chi-square test showed partial deletion of chromosome 13 was associated with serum creatine levels, detection of plasmablastic eel 1s, hemoglobin levels, numbers of bone lensions,early chemotherapy responses, 1 year survival; LDH levels, detection of plasmablastic cells and type of light chains were found associated with prognosis in Log-rank Lest; detection of plasmablastic cells and serum creatine levels were found to be significant single prognostic factors ; no prognostic implications were found for partial deletion of chromosome 13.CONCLUSIONS: Longer culture period with addition of IL-6 and GM-CSF improves the cytogenetic analysis of MM patients, complex karyotype arecommon changes in MM. Applicat ion of FISH on archival bone marrow smears is a simple, liable method, can be readly used to retrospective study of clonal hematopoieitc system diseases. Deletion of Rb~l gene and locus 13q 14.3 were common changes in MM, abnormalities of chromosome 13 may be larger deletion involving both Rb-1 and 13ql4.3 in some patients. Partial deletion of chromosome 13 affect the biological behavior of MM cases; detection of plasmablastic cells and serum creatine level were significantly and independly prognostic factors for MM patients, no prognostic implications were found for partial deletion of chromosome 13 due to the sample size.更多还原