建立一套多對引子聚合酶連鎖反應同時偵測CEBPA、NPM與FLT3/ITD基因突變的方法

Abstract

急性骨髓性白血病 (Acute myeloid leukemia, AML)是由於造血前驅細胞發生突變，使得這些細胞無法分化為正常的顆粒性白血球(granulocyte)，且會有不受控制地增生的現象。CEBPA突變、NPM突變及FLT3內源性串連複製突變(FLT3 internal tandem duplication, FLT3/ITD)是目前在AML最常被發現的三種基因變異，它們除了可以用於評估疾病的預後，也由於在發病與復發時的血癌細胞所偵測到的CEBPA突變或NPM突變類型是相同的，因此這兩種突變也適合用作監測微量腫瘤殘餘狀態(minimal residue disease, MRD)的生物標誌。 本研究發展出一套以多對引子聚合酶連鎖反應(Mutiplex PCR)搭配螢光片段毛細管電泳分析的方法，並對102個AML病人的CEBPA突變、NPM突變及FLT3/ITD進行檢測，再以序列分析的結果進行確認。我們發現除了兩種無意義的TAD2突變外，有13個人(12.7%)具有CEBPA突變，共有17種突變類型；20個人(19.6%)具有NPM突變，共有7種突變類型；20個人(20.2%)具有FLT3/ITD的突變；但在CEBPA突變與NPM突變的兩群病人之間並無交集的情形。此方法在CEBPA突變的偵測率為89.5%，在NPM突變與FLT3/ITD的偵測率則達100%。我們也對於此方法的偵測限制及疾病狀態監測用途進行評估，結果顯示此方法的敏感度達到5%，並可用於監測治療後早期復發的情形。因此我們所建構出的這套螢光片段分析方法，可望在將來能應用在臨床上快速篩檢AML病人是否帶有CEBPA突變、NPM突變及FLT3/ITD，並可對於帶有CEBPA突變或NPM突變的病人在治療後是否出現復發的情形進行監測。

Acute myeloid leukemias (AML) are clonal disorders that are characterized by acquired somatic mutations in hematopoietic progenitors. Mutations of CCAAT/enhancer binding protein α (CEBPA), nucleophosmin (NPM), and Fms-like tyrosine kinase-3 (FLT3) genes have been reported as the most frequent genetic variations in AML patients, and they have the important value in predicting prognosis, especially in those with normal karyotype. Due to the reappearances of the same CEBPA mutation and NPM mutation at relapse, these mutations are suitable as the biomarkers for monitoring minimal residue disease (MRD) in AML. In this study, we designed a novel, rapid and reproducible method with high sensitivity and specificity for simultaneous screening of the CEBPA mutation, NPM mutation, and FLT3/ITD by multiplex PCR coupled with capillary electrophoresis and fluorescence detection. To verify this novel method, 102 AML patients were studied, and the results were then confirmed by PCR-coupled direct sequencing. In addition to two insignificant mutations, 17 distinct mutations in the CEBPA gene and seven in the NPM gene were found in the thirteen (12.7%) and twenty (19.6%) patients respectively, but none had both. Twenty patients (20.2%) had the FLT3/ITD mutation. The overall sensitivity of multiplex PCR for NPM mutation and FLT3/ITD were up to 100%, and that for CEBPA mutation was 89.5%. This novel method can detect mutant allele percentages down to 5% of total DNA and offer the ability to detect early relapse post-therapy. This simple and reproducible method which shows high sensitivity and apparent accuracy may be used as a screening and disease-monitoring tool for AML patients with CEBPA mutation, NPM mutation, and FLT3/ITD in the future.