兒童急性淋巴性白血病的預後因子探討:台灣的多中心研究

Abstract

背景：兒童急性淋巴性白血病是兒童最常見的惡性疾病，其成因是B-cell或是T-cell在分化過程中自我更新的能力失去調控，加上其他的基因缺失或是染色體的異常，進而引發不正常的細胞增生而導致白血病的形成。過去的四十年，兒童急性淋巴性白血病在臨床醫師不斷改進化學治療以及了解疾病的預後因子下，從不到10%的存活率，一直到現在先進國家的長期存活率已經可以到90%。這樣的進步主要是來自所謂的危險因子導向的治療，而不是來自於新藥的發明，亦即不斷去尋找預後因子修正化學治療的療程，才有如此的成績。而因為對預後因子的了解以及不斷的運用新的醫學檢驗的技術，如廣泛的使用基因晶片去分析急性淋巴性白血病的基因表達或基因變化，並和臨床的資料如疾病的復發、染色體的變化、化學治療的反應，藥物的副作用做結合分析，得到更多疾病發生的機轉的基因變異以及預後因子。 過去的二十年，在台灣小兒血液腫瘤科醫生的合作之下， 成立台灣兒童癌症研究小組(Taiwan Pediatric Oncology Group，簡稱為TPOG) ，對大部份的兒童惡性腫瘤使用相同的療程(protocols) ，這當然包括兒童急性淋巴性白血病，其治療成績的進展也已經發表。而在療程的制定上，根據復發的風險分不同的危險因子族群而給予不同強度的治療。台灣治療成果的進步，主要是來自化學治療強度的增加，並不是來自預後因子測量的改善，這在標準危險群以及高危險群的病人最為明顯。最高危險群的病人治療的進步有限，而大部份復發的病人其治療失敗的原因不明。加上部份藥物的使用，如mercatopurine 的劑量與西方國家差異太大，這讓我想去探討這些預後的背後有什麼差異性。因此，我們從癌細胞的染色體轉位，基因表達，病人本身的藥物基因路徑的多形性，一直到基因缺損去探討其在台灣兒童急性淋巴性白血病病人的預後意義。 研究主題：我們的研究主題如下(1)染色體的轉位以及對治療結果的影響 (2) BCL2L13，Livin和CASP8AP2在lymphoblasts的表達量和預後的關係 (3)十七個藥物代謝路徑基因多行性和預後的關係 (4)發展PCR加上毛細管電泳去分析IKZF1 deletions在B-cell progenitor急性淋巴性白血病的預後關係(5)探討 absence of biallelic TCRγ deletions (ABD) 在T-cell急性淋巴性白血病的預後意義。 方法與結果：總共369位兒童急性淋巴性白血病病人，分別來自台大醫院、中國醫藥大學、台中榮民總醫院、彰化基督教醫院、成大醫院以及高雄長庚醫院。在取得同意書的情況下，將其診斷，緩解或是復發時的骨髓檢體凍存。其中307位是B-cell progenitor急性淋巴性白血病，62位為T-cell急性淋巴性白血病。 染色體的轉位以及數目的變化是兒童急性淋巴性白血病的主要特徵，本論文使用了multiplex RT-PCR和nested PCR檢測148位病人發病時的檢體的四種常見的染色體轉位包括t(9;22)，t(1;19)，t(4;11)以及t(12;21)，並且加上傳統的染色體檢查做分析，這些染色體的轉位具備預後的意義。其中病人帶有t(12;21)，t(1;19)以及染色體的對數大於53的預後較好，反之病人帶有t(9;22)，t(4;11)以及其他MLL基因重組預後則較差。 此外，本論文使用定量PCR (Q-RT-PCR) 去定量90位病人三個與細胞凋亡(apoptosis)相關的基因( BCL2L13，Livin 和CASP8AP2) 在lymphoblasts的表達量以及和預後的關係做分析，在台灣的病人族群，lymphoblasts高表達BCL2L13是一個獨立的預後因子。 本論文總共針對病人本身的藥物代謝基因的多形性進行了預後的探討，使用的方法為PCR-based restriction fragment length polymorphism (RFLP)以及sequence-specific oligonucleotide (SSO) probe hybridization。本論文總共探好了十七個基因的多型性和預後的關係。其中105位病人接受TPOG-ALL-93 或TPOG-97-VHR 以及 91例接受TPOG-ALL-2002療程，結果發現MDR1基因，homozygotic MDR1 2677GG，3435CC，和2677G - 3435C基因型，與病人使用TPOG-ALL-93，在標準危險群病人的event-free survival (EFS)以及overall survival (OS)有顯著相關。但是這樣的預後意義並沒有在現行的療程TPOG-ALL-2002中發現，暗示化學治療的進步會讓一些不好的預後因子消失。 隨著更多的全基因組分析，愈來愈多的預後因子被發現。其中兩個比較重要的發現為在B-cell progenitor兒童急性淋巴性白血病，lymphoblasts如果有IKZF1 deletions，其EFS 和OS都不好。而在T-cell兒童急性淋巴性白血病如果lymphoblasts有absence of TCRγ deletions (ABD），非常容易發生引導期化學治療失敗，並且影響整體的EFS和OS。在本論文的病人族群中，B-cell progenitor急性淋巴性白血病病人如果帶有IKZF1 deletions，其EFS(p<0.001)，OS(p= 0.0016)都比沒有帶IKZF1 deletions的病人差。對於T-cell兒童急性淋巴性白血病，在多變數回歸分析之後，病人帶有ABD有較高的引導期化學治療失敗以及較差的OS(p值分別為 0.03和0.0196)。 結論：Lymphoblasts的染色體轉位具備預後的價值，而multiplex RT-PCR 可以輔助傳統染色體檢查的不足。藥物基因學的多形性在某些族群上有預後的意義，而這樣的意義與化學治療的強度有關。IKZF1 deletions 在B-cell progenitor急性淋巴性白血病具有很強的預後意義，在目前台灣臨床醫師所使用的療程，其治療成績仍不理想。而T-cell急性淋巴性白血病帶有ABD則容易引導失敗。某些預後因子，如lymphoblasts的基因變化，的確和白人的報告有所差異，或許部份解釋了台灣的病人治療成功率較低的原因。未來如何將這些預後因子結合風險導向治療，提供帶有不良預後因子的病人(如帶有IKZF1 deletions或ABD)其他的治療是未來努力的方向。

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatricmalignancy and accounts for 25% of childhood cancers. ALL is thought to originatefrom various important genetic lesions in blood-progenitor cells that are committed todifferentiate in the T-cell or B-cell pathway, including mutations that impart thecapacity for unlimited self-renewal and those that lead to stage-specific developmentalarrest. Cure rates have improved dramatically over the past 40 years, with 5-year overall survival rates increasing from <10% in the late 1960s to nearly 90% in 2000-2004 inadvanced countries. The success of treatment of childhood ALL depends upon theidentifications of risk factors. The advent of high-resolution genome-wide analyses ofgene expression, DNA copy number alterations and loss of heterozygosity, andwhole-gemone sequencing have led to the detection of many novel geneticabnormalities. These studies also provide new insights into the complex interactions ofmultiple genetic alterations in leukemogenesis and response to chemotherapy. Some ofthem also had strong prognostic implications. In 1988, the Taiwan Pediatric Oncology Group (TPOG) was formed with thecooperation of all leukemia treatment centers and has since initiated nationalcooperative group studies. The treatment results were published recently. Most of theadvance of treatment rely on the intensification of chemotherapy, not the improvementof identification of risk factors. For the purpose to improve the clinical outcomes by theidentifications of prognostic factors, this thesis focused upon the studies of geneticbackgrounds of the leukemic cells and the host. Thesis: The purpose of this thesis is to investigate the prognostic factors in childhoodALL in Taiwan. (1) Four common chromosomal translocations screening includingt(12;21), t(1;19), t(4,11) and t(9;22). (2) The prognostic value of three apoptoticBCL2L13, CASP8AP2, and Livin gene expression in the childhood ALL (3) seventeenpharmacogenetic polymorphisms for outcomes (4) the prognostic value of IKZF1deletions for B-lineage ALL (5) the prognostic impact of absence of biallelic TCRγdeletions (ABD) for patients with T-cell ALL. Methods and Results: Diagnostic bone marrow or peripheral blood and remissionsamples were obtained from 369 children diagnosed with ALL between July 1996 andJune 2010 at the National Taiwan University Hospital, National Cheng Kung UniversityHospital, Chang Gung Memorial Hospital-Kaohsiung Medical Center, China MedicalUniversity Hospital, Changhua Christian Hospital, and Veterans General Hospital-Taichung. Among them, 307 were newly diagnosed B-precursor ALL and 62 wereT-cell ALL. The hallmark of childhood ALL is chromosomal alterations. Multiplex RT-PCR and nested-PCR assays were used to detect the four common chromosomal translocations,including t(9;22), t(1;19), t(4;11), and 2 variants of t(12;21) in 148 leukemic samplesupon diagnosis. Patients with ETV6-RUNX1 fusion, hyper-diploidy, and t(1;19)/TCF-PBX1fusion had the most favorable outcomes whereas those with the t(9;22)/BCR-ABL1fusion or t(4;11) and other MLL gene re-arrangement had poor prognosis (p<0.001 forevent-free survival and overall survival). We investigated the expression of threeapoptosis related genes, BCL2L13, CASP8AP2, and Livin by the method of Q-RT-PCR, as well as their prognostic significance, in a retrospective study of 90 pediatric ALLpatients diagnosed between 1996 and 2007 in Taiwan. Multivariate analysis for EFSand OS demonstrated that high expression of BCL2L13 was an independent prognostic factor for childhood ALL in this ethnic group. Seventeen genetic polymorphisms in 13pharmacogenomic targets were analyzed by PCR-based RFLP and sequence-specificoligonucleotide (SSO) probe hybridization .The pharmacogenetic polymorphisms of the host were also evaluated. Three polymorphic alleles in the multi-drug resistant 1(MDR1) gene and homozygotic MDR1 2677GG, 3435CC, and 2677G-3435Cgenotypes were also highly associated with significant reduction in event-free survival(EFS) and overall survival (OS)in patients treated by the standard risk protocol(TPOG-ALL-93 SR). Recent genome-wide analyses have identified that an alteration of IKZF1 isassociated with very poor outcomes in B-cell progenitor ALL and the absence ofbi-allelic TCRγ deletions (ABD) reportedly predicts early treatment failure in childhoodT-cell ALL. In this cohort, patients with IKZF1 deletions had inferior event-freesurvival (p<0.001) and overall survival (p=0.0016). In T-cell ALL, patients with ABDhad higher incidences of induction failure and inferior OS than those without ABD(p=0.03 and 0.0196, respectively) after multivariate regression analysis. Conclusions: The multiplex RT-PCR can complement the cytogenetic study.Chromosomal alterations had prognostic implications in childhood ALL. Theexpression of BCL2L13 was an independent prognostic factor for childhood ALL in this ethnic group. Pharmacogenetic variations had some prognostic impacts in some subsets of patients with childhood ALL. IKZF1 deletions predict poor clinical outcomes forpatient with B-cell precursor ALL. ABD also predict induction failure in T-cell ALL.These prognostic genetic alterations may be included in risk-directed therapy in thefuture. Patients with poor outcomes (such as patients with IKZF1 deletions or T-cellALL with ABD) may benefit from alternative treatment regimes.