- 學位論文
TEL-AML1及BCR-ABL(p190)融合基因變異在南台灣小兒急性淋巴性白血病之盛行率以及應用在細微殘存疾病之偵測價值評估研究
The incidence of TEL-AML1 and BCR-ABL(p190) fusion transcripts in childhood acute lymphoblastic leukemia of Southern Taiwan and application of the fusion transcripts in the detection of minimal residual disease
摘要
在百分之八十至百分之九十的兒童急性白血病可找到染色體的異常，這些染色體的異常不只具有診斷及預測預後的價值，也可以提供我們進一步瞭解白血病細胞轉型(transformation)及增殖(proliferation)在分子生物學上的線索。 自從第一個融合基因BCR-ABL in t(9;22)被發現以後，許多與其他染色體有關的融合基因也被找出來：譬如在兒童急性淋巴性白血病中最常見的TEL-AML1 融合基因，是第十二對染色體的TEL gene與第二十一對染色體的AML1 gene因染色體轉位而融合在一起；特別出現在嬰兒白血病的MLL-AF4融合基因，則為第四對染色體的AF4 gene與第十一對染色體的MLL gene因染色體轉位而融合在一起。 隨著分子生物技術的發展，許多比細胞基因學更敏感的方法可用來偵測這些白血病專一性的染色體異常：如螢光原位雜交染色法(FISH)、南方漬點法(Southern blot analysis) 以及反轉錄聚合酶鍊鎖反應(RT-PCR)。除了可用來預知疾病病程，這些白血病專一性的染色體異常還可當作監控細微殘存疾病的分子標記。所謂細微殘存疾病就是當病人達到完全緩解後，體內仍殘存的癌細胞。許多研究已顯示殘存的癌細胞量與疾病復發有密切的關係，所以利用適合的分子標記設計精確的實驗室方法來測量細微殘存疾病可讓我們瞭解在抗癌藥物的治療下細微殘存疾病的變化，進一步根據每個病人不同的情形設計適合的療程。 在本研究中，我們以兒童急性淋巴性白血病為對象，針對兩種白血病專一性的染色體異常: t(9;22)-BCR/ABL p190 fusion genes 及 t(12;21)-TEL/AML1 fusion genes，利用反轉錄聚合酶鍊鎖反應來偵測融合基因的存在，並且在有這些基因異常者追蹤其治療後這些融合基因是否仍存在。本研究共收錄自西元2003年一月至西元2004年十月間被診斷出患有急性淋巴性白血病的個案25位（包括20位新診斷的個案及5位復發的個案），急性骨髓性白血病3位（2位新診斷及1位復發的個案），以及一位新診斷的幼年型骨髓單核球型白血病個案。 其中25個急性淋巴性白血病的個案中共有8位偵測到TEL-AML1 fusion transcript (32%)，其中20位新診斷的個案中有6位（30%） ，5位復發的個案中有2位偵測到 (40%)， 在 BCR-ABL p190 部分， 則是25個急性淋巴性白血病的個案中僅有2位偵測到（8%）。而這兩位皆為新個案，所以在新個案中偵測到BCR-ABL p190 的比例是 (10%)， 這些TEL-AML1 fusion transcript positive 個案的臨床特徵（包括免疫分型、白血球數、年齡），皆與其他文獻報告類似，但在本研究中發現有兩個年紀低於1歲的案例也出現TEL-AML1 fusion transcript，在這兩個案例中，TEL-AML1 fusion transcript的序列是否與其他個案一樣，以及是否與其白血病形成有直接相關需進一步探討。而是否 TEL-AML1 fusion transcript positive為一個好的預後因子，以及BCR-ABL p190 positive 為一個不好的預後因子，因本研究所收20個新診斷的個案中，除了一位沒有達到完全緩解（此位個案無TEL-AML1 fusion gene 或BCR-ABL p190 fusion gene）之外，其餘個案皆於引導療程結束時達到緩解，其中僅有一位呈現TEL-AML1 positive的病人於鞏固療程時出現復發，並於後來因疾病進行而死亡。其餘個案至目前為止追蹤期最長者為兩年，所以尚未能對此兩項融合基因與預後之關聯提出結論。 我們以漬點法進行TEL-AML1 fusion transcript positive 急性淋巴性白血病的微小殘存疾病的定量檢測，發現六位有收取到治療後檢體的個案中，有四位的TEL-AML1 fusion transcript隨著疾病緩解而下降，其中三位下降幅度較大。惟整體個案數不多，並且在唯一一位治療中途復發的個案中沒有明確顯示出TEL-AML1 fusion transcripts 的表現量與復發的相關性，因此後續仍須更多的個案來證實是否以TEL-AML1 fusion transcripts 為基因標的，使用我們的定量方法可以達到精確掌握病情發展的目的。
並列摘要
Chromosome abnormalities were found in 80% to 90% of childhood acute lymphoblastic leukemia (ALL) cases. These leukemia-specific chromosome aberrations not only have prognostic value but also provide important clues for further investigation of leukogenesis , leukemic cell transformation and proliferation. Since the first fusion gene , BCR-ABL in t(9;22) , was discovered , many other chromosome aberrations with fusion genes have been identified. For instance , t(12;21) with TEL-AML1 fusion gene have been noted as the most frequent rearrangement in child hood ALL, whereas t(4;11) with MLL-AF4 fusion gene has been identified particularly in infant ALL. Advancement in molecular biology leads to accurate detection of these leukemia-specific chromosome aberrations with sensitive techniques, such as fluorescence in situ hybridization (FISH) , Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Leukemia-specific fusion genes have been used as molecular markers for minimal residual disease (MRD) monitoring. The remaining leukemic cells below the threshold of cytomorphological techniques (sensitivity around 1%~5%) was referred to as MRD. Several studies showed that existence of MRD during the maintenance phase is a independent poor prognostic factor. Monitoring MRD at consecutive time points can give clinical relevant insight into the effectiveness of treatment. In the present study, we apply RT-PCR technique in the detection of two leukemia-specific chromosome fusion genes, TEL-AML1 fusion gene and BCR-ABL p190 fusion gene. We also monitor the expression level of these fusion genes at sequential time points of the treatment course. Twenty-nine patients were enrolled in the study including 20 newly-diagnosed ALL, 5 relapsed ALL, 2 newly-diagnosed AML , 1 relapsed AML and one JMML. Of total 25 ALL cases, TEL-AML1 fusion gene was detected in 8 patients (6 newly-diagnosed and 2 relapsed cases) and BCR-ABL p190 was detected in only 2 newly-diagnosed cases. The incidence of TEL-AML1 fusion gene and BCR-ABL p190 fusion gene in our cases were 32 % and 10 % respectively. The clinical features of our eight TML-AML1 positive ALL cases were similar to the other studies except two of them were younger than 12 months old. Also, no t(12;21) was detected by the conventional cytogenetic study in our cases as it was identified as a cryptic chromosome translocation. All cases but one (TML-AML1 negative and BCR-ABL p190 negative) achieved cytomorphological remission after induction therapy and one patient among the 8 TML-AML1 positive cases relapsed during the consolidation phase. The other 18 newly-diagnosed ALL patients remained in remission status with follow-up period ranged from 1 month to 24 months. Blotting analysis was used for minimal residual disease detection by TEL-AML1 fusion transcript in the eight TML-AML1 positive ALL patients. Among six patients whose bone marrow or peripheral blood samples were obtained after treatment , reduction of TEL-AML1 expression levels were found in four. For further investigation of the accuracy and clinical implication of our methods , more experience and a large study group will be needed.