Multiplex quantitative PCR for the assessment of treatment response and outcome prediction in chronic myelogenous leukemia patients receiving Glivec treatment: a prospective study

慢性骨髓性白血病（CML）為造血幹細胞疾病，帶有染色體9 和22 移位異常，造成bcr 和abl 前致癌基因接合，產生之雜合蛋白帶有異常tyrosine kinase 活性，使用特殊的kinase 抑 制劑（STI571，Glivec）治療新診斷CML 有極高療效:96 ﹪血液緩解率及68 ﹪染色體緩解 率，少部分病人更得到分子緩解，即時定量RT-PCR 可以靈敏地定量白血病細胞，作為療效 評估和偵測微量殘存疾病。本實驗室過去已發展出單一步驟即時定量RT-PCR 法，在同一試 管內進行RT 及PCR 反應，靈敏度達到10 -5 與文獻報告使用二階段法相當。然而內在對照基因 GAPDH 仍需另外進行定量反應，可能成為定量誤差的一個潛在原因，使用多重基因定量 PCR，應可減少實驗操作及測量誤差，但文獻上尚未有運用於CML 報告。 本前瞻性研究收集102 位在2001 年6 月至2004 年10 月間接受Glivec 治療的CML 病人，包 括49 名慢性期、30 名加速期及23 名急性期病人。Glivec 治療時間平均為19.4 月(1~45 月)， 診斷至Glivec 治療時間平均為16 月(0~120 月)。2004 年7 月起大部分病人以Glivec 做為第一 線治療選擇。 新診斷時之骨髓和血液檢體分別作染色體和RT-PCR 分析，開始Glivec 治療前後每3 個 月收集檢體做MRD 分析定量。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BCR-ABL 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。單一步驟即時定量PCR：使用ABI Prism 7700 儀器及Taqman EZ RT-PCR Kit，Taqman 螢光性探針以Primer Express 軟體設計合成，每次反應均使用K562 細胞株作為陽性對照基 因以建立標準曲線，每個檢體至少做2 次反應。 研究結果發現血液學完全緩解率分別為慢性期98%、加速期70%、急性期35%，染色體 完全加部分緩解率分別為慢性期70%、加速期58%、急性期75%，基因緩解率(MRD 小於-3.0log) 分別為慢性期19%、加速期11%、急性期17%。三年白血病無惡化存活率分別為慢性期93%、 加速期51%、急性期4%，白血病無惡化存活率與染色體及基因緩解率均成相關。慢性期或 加速期病人治療後達到MRD 小於-2.0log 者，追蹤三年尚無人出現白血病惡化者。急性期病 人即使達到染色體或基因緩解，仍然無法有效避免復發惡化，研究發現其中多人出現 BCR-ABL 基因突變。以上結果與最近國外研究報告類似。 本研究成果證實Glivec(Imatinib)對於治療慢性期效果顯著，但是加速期或急性期則 需要合併其他藥物或改用新藥，以提升治療效果。多重基因即時定量PCR 分析法可以準確 定量殘存微量白血病，有效評估治療療效，為協助臨床決策判斷之最佳工具。(以上結果 已在2005 年台灣血液病學會年會發表，論文撰寫投稿中)Chronic myelogenous leukemia (CML) is a clonal disorder of hematopoietic stem cell characterized by t(9;22), which results in fusion of bcr and abl proto-oncogenes and expression of BCR-ABL chimeric protein with abnormal tyrosine-kinase activity. Inhibition of this kinase activity by STI571 (Glivec) resulted in 96% of hematological remission and 68% cytogenetic remission in newly diagnosed CML. Molecular remission is possible in a few patients. Quantification of t(9;22) carrying cells can be achieved by real-time quantitative RT-PCR (RQ-PCR) assay that is highly sensitive for evaluating treatment response and minimal residual disease (MRD). We had successfully developed one-step RQ-RT-PCR method that integrated RT and PCR reaction in single tube. The assay can detect one abnormal CML cells among 10 5 normal cells (5-log sensitivity). Amplification of GAPDH control gene was done in separate reaction. Since each PCR reaction has its own kinetics, separate PCR assay for target and control genes can potentially result in inaccurate quantification. Multiplex PCR can further minimize the laboratory procedure and increase the assay accuracy. Whether multiplex RQ-RT-PCR can be applied in detecting MRD in CML has not yet been reported in the literature. Between June 2001 and October 2004, a total of 102 CML patients had been treated with Imatinib in NTUH, including 49 patients in chronic phase (CP), 30 patients in accelerated phase (AP), and 23 patients in blastic phase (BC). Fifty-two were male and 50 female and the median age was 40 years old (ranges 3-80). The median interval from diagnosis to Imatinib treatment was 16 months (0-120) and median Imatinib duration 19.4 months (1-45). Since July 2004, most patients received Imatinib as first-line drug in early chronic phase. Results: the complete hematological rate (CHR) was 98% in CP, 70% in AP and 35% in BC patients (p < 0.001). Major cytogenetic remission (MCyR) was achieved in 70% of CP, 58% in AP and 75% in BC patients (p=0.08). However, only a few patients could achieved molecular remission as defined as MRD < -3.0log reduction: 19% in CP, 11% in AP and 17% in BC. (p=0.341). The probability of leukemic progression-free survival (PFS) at 3 years was 93% for CP, 51% for AP and 4% for BC (p<0.001). PFS was also correlated with cytogenetic response and molecular response. For patients treated in CP or AP stagesand had achieved MRD < -2.0log reduction, none had leukemic progression. Cytogenetic or molecular remission in BC stage didn ’t prevent disease relapse and progression. Sequencing analysis showed that most of them had acquired mutation at BCR-ABL kinase domain. In conclusion, Imatinib was highly effective in CML-CP. For advanced stages, higher doses of imatinib or combination with other drugs are needed to improve response and prevent leukemic progression to improve survival. Sequential monitoring of cytogenetic and molecular analysis is helpful in clinical decision-making. (The result was presented at the annual meeting of the Taiwan Society of Hematology, March 26, 2005 and manuscript in preparation for submission