Sufliarska S, Minarik G, Horakova J, Bodova I, Bojtarova E, Czako B, Mistrik M, Drgona L, Demitrovicova M, Lakota J, Krivosikova M, Kovacs L
Bone Marrow Transplantation Unit, Department of Pediatrics, Comenius University Medical School, Limbová 1, 833 40 Bratislava, Slovak Republic.
Neoplasma. 2007;54(5):424-30.
We describe the implementation, optimization, sensitivity determination and first clinical results of polymerase chain reaction (PCR) amplification of polymorphic short tandem repeat (STR) markers and Amelogenin locus coupled with fluorescent detection and capillary electrophoresis in chimerism monitoring of patients transplanted at three different transplant centers using a commercially available multiplex microsatellite assay. The chimerism analysis was performed with genomic DNA extracted from unselected peripheral blood leukocytes of one hundred pediatric and adult patients, who underwent allogeneic stem cell transplantation (SCT) from human leukocyte antigen (HLA) matched or one antigen mismatched related or unrelated donors for malignant (70 patients) and non-malignant (30 patients) diseases. Tested were 79 donor recipient pairs for 15 STR systems and identified an informative marker in all but one of them (98,7%), using 6 selected systems out of these fifteen, that appeared highly informative in our patients population. In 21 sex-mismatched donor recipient pairs we used the Amelogenin locus to distinguish the X and Y chromosome. In sixty-three out of these 100 patients chimerism was regularly analyzed from blood samples taken at various time points after SCT with the median follow up of 17 months. Complete chimerism (CC), maintained over the whole follow-up period, was detected in 24 (38, 1%), stable and decreasing mixed chimerism (MC) in 28 (44, 4%) and increasing MC in 11 patients (17, 5%). Patients with CC, stable and decreasing MC showed a significantly better (p 0,005) overall survival rate (0, 81), compared to those with increasing MC (0, 24). These results demonstrate that STR-based chimerism monitoring with sensitivity above 1% and high informativity (98, 7% of donor recipient pairs) is necessary in establishing the origin of engrafted cells after an allogeneic SCT, in detecting graft rejection and that it may contribute in identifying patients with imminent leukemia relapse.
我们描述了使用市售多重微卫星分析，对多态性短串联重复序列（STR）标记和牙釉蛋白基因座进行聚合酶链反应（PCR）扩增，并结合荧光检测和毛细管电泳，在三个不同移植中心对移植患者进行嵌合体监测的实施、优化、灵敏度测定及首次临床结果。嵌合体分析采用从100例儿科和成年患者未选择的外周血白细胞中提取的基因组DNA进行，这些患者接受了来自人类白细胞抗原（HLA）匹配或一个抗原不匹配的相关或无关供体的异基因干细胞移植（SCT），用于治疗恶性疾病（70例患者）和非恶性疾病（30例患者）。对79对供体-受体进行了15个STR系统检测，除其中1对（98.7%）外，其余均鉴定出信息性标记，从这15个系统中选择了6个，在我们的患者群体中显示出高度信息性。在21对性别不匹配的供体-受体中，我们使用牙釉蛋白基因座来区分X和Y染色体。在这100例患者中的63例中，在SCT后不同时间点采集的血样中定期分析嵌合体，中位随访时间为17个月。在24例（38.1%）患者中检测到在整个随访期内维持的完全嵌合体（CC），28例（44.4%）患者为稳定且下降的混合嵌合体（MC），11例患者（17.5%）为上升的MC。与上升的MC患者（0.24）相比，CC、稳定且下降的MC患者的总生存率显著更高（p<0.005）（0.81）。这些结果表明，基于STR的嵌合体监测灵敏度高于1%且信息性高（98.7%的供体-受体对），对于确定异基因SCT后植入细胞的来源、检测移植物排斥反应是必要的，并且可能有助于识别即将发生白血病复发的患者。
