https://scholars.lib.ntu.edu.tw/handle/123456789/188193
標題: | 急性骨髓性白血病治療後偵測微量殘存白血病細胞之研究 | 其他標題: | Therapeutic Monitoring of Minimal Residual Disease in Acute Myelogenous Leukemia | 作者: | 唐季祿 | 關鍵字: | 急性骨髓性白血病;殘存微量白血病;即時定量PCR;WT-1 基因;acute myeloid leukemia;minimal residual leukemia;real-time RT-PCR;WT-1 gene | 公開日期: | 2003 | 出版社: | 臺北市:國立臺灣大學醫學院內科 | 摘要: | 復發是白血病化學治療最常見治療失敗原因,定量殘存微量白血病(minimal residual leukemia, MRL )可有效預測治療能否成功。最近二年我們利用即時定量RT-PCR 放大特異 性染色體移位產生之雜合RNA ,能靈敏偵測MRD 達到10 -4 ~10 -5 ,但只有20-30%病人適用, 無法廣泛使用。WT1 抑癌基因已知在急性骨髓性白血病(AML) 60-80%會出現WT1 過度表 現,和Bcl-2 基因維持白血病細胞活性,可能影響預後。本計畫評估以 Wilms’ tumor (WT1) 基因作為MRD 標的的可行性,篩選高危險群病人和預測移植後復發之關連性。 我們設計多重基因即時定量(multiplex RQ-PCR)法,可在單一試管中同時放大WT1 和 GAPD 基因(內在對照基因,以控制RNA 品質及總量)。利用不同螢光探針,同時偵測定量 PCR 產物,評估發現其靈敏度與準確度均不亞於分開作RQ-PCR 結果。 研究結果發現正常人在捐贈骨髓移植或周邊血液幹細胞移植時收集的正常幹細胞檢體 WT1 RNA 含量很低或完全偵測不到,反之89 例新診斷AML 病人中65 例(77%)骨髓過量 表達WT1-RNA ,可適用MRD 偵測(靈敏度為10 -3 ~10 -5 )。5 例同時帶AML-ETO 及WT1 異 常病人,分別以此2 種基因標的作MRD 定量,比較結果發現有良好一致性。再系列追蹤 29 位病人化學治療後WT1 含量變化,發現化療後MRD 持續>10 -3 者,白血病復發率高, 其他病人初期療效良好,進入分子緩解,復發機率則明顯減少。以上結果與最近國外研究 報告類似。 本研究成果證實Wilms’ tumor (WT1) 基因可作為AML 病人MRD 偵測之分子標的, 篩選高危險群AML 病人和預測復發。可以更有效評估化學治療療效,協助發展有效之腫 瘤根除技術以減少術後復發,提高移植成功率。 Intensive chemotherapy and bone marrow transplantation have achieve high complete remission rate (60~80%) and 30-70% long-term survivor in acute myeloid leukemia (AML). However, many patients still died of relapse eventually. In the past 2 years, we have developed a real-time quantitative RT-PCR (RQ-PCR) assay that can accurately detect one leukemic cell out of 10 4 to 10 5 normal cells (4-5 Log) with 100% specificity. This assay was highly correlated with leukemic status and was able to identify high-risk patients before clinical relapse occurred. However, this technique is applicable to 20-30% of AML patients with specific molecular fusion genes—AML1-ETO in t(8;21) and PML-RAR α in t(15;17). Recently, WT-1 gene, gene responsible for Wilms’ tumor, was found to be over-expressed in 60-80% of AML and was associated with poor survivor. Quantitation of WT-1 RNA level may be a candidate molecular target useful in monitoring the level of minimal residual leukemia (MRL) after treatment. The goal of this project is to extend our current technique in using RQ-PCR in therapeutic monitoring of more than 50% of AML patients which have important clinical utility in detecting residual leukemia, selecting high risk patients and predicting leukemic relapse. In a retrospective study between Jan. 2000 and Apr. 2003, WT1 expression level in 84 adult AML at diagnosis was performed by a novel one-step multiplex quantitative real time reverse transcriptase polymerase chain reaction (M-RQ-PCR) technique. WT1 overexpression was defined as ≧ 1% expression level of K562 cell line. Normal samples collected from 3 BMT and 11 PBSCT donors contained very low or undetectable levels of WT1, suggesting that normal stem cells expressed very low level of WT1. WT1 overexpression was detected in 65/84 (77%) AML and 5/7 (71%) ALL and were eligible for MRD monitoring. There was no association of WT1 expression level with sex, age, initial WBC counts, CD34%, blast%, and cytogenetics. WT1 overexpression occurred frequently in most FAB subtypes except for M5 subtype. In 5 AML patients with both t(8;21) and WT1 overexpression, MRD level was estimated by AML1-ETO RQ-PCR and WT1 RQ-PCR assays respectively and there was good linear correlation (r 2 =0.769, N=35). In some cases, WT1 level was useful in differential diagnosis of early relapse. False negative results may be encountered in poor quality samples and should be interpreted cautiously. Serial follow-up of WT1 kinetics in 52 patients showed that WT1-MRD level persistently > 10 -3 after chemotherapy was associated with leukemic relapse. By contrast, relapse occurred in some patients who initially achieved molecular remission. In conclusion, WT1 RQ-PCR assay is potentially useful molecular marker for MRD monitoring in AML patients. Whether WT1 level after induction and consolidation therapy is correlated with relapse risk and clinical outcome deserves further study. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/23592 | 其他識別: | 912314B002180 | Rights: | 國立臺灣大學醫學院內科 |
顯示於: | 醫學系 |
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912314B002180.pdf | 173.27 kB | Adobe PDF | 檢視/開啟 |
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