RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo

BCR-ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML-LBC) lineage and BCR-ABL1⁺ acute lymphoblastic leukemia (BCR-ABL1⁺ ALL). The recombination-activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML-LBC and BCR-ABL1⁺ ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild-type (WT) RAG and catalytically inactive RAG-expressing BCR-ABL1⁺ and BCR-ABL1⁻ cell lines, respectively, and demonstrate that BCR-ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG-expressing BCR-ABL1⁺ cell lines and primary CD34⁺ bone marrow cells from CML-LBC samples maintain more double-strand breaks (DSB) compared to catalytically inactive RAG-expressing BCR-ABL1⁺ cell lines and RAG-deficient CML-CP samples, which are measured by γ-H2AX. WT RAG-expressing BCR-ABL1⁺ cells are biased to repair RAG-mediated DSB by the alternative non–homologous end joining pathway (a-NHEJ), which could contribute genomic instability through increasing the expression of a-NHEJ-related MRE11 and RAD50 proteins. As a result, RAG-expressing BCR-ABL1⁺ cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR-ABL1 signaling but independent of the levels of BCR-ABL1 expression and mutations in the BCR-ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR-ABL1⁺ leukemia through its endonuclease activity.     

BCR-ABL融合蛋白多样性及其与白血病表型的关系

本文报告了BCR-ABL融合蛋白及其与白血病表型的关系。首先报告BCR-ABL融合基因的结构及其转录本,其中包括这些融合基因可分为M-bcr,m-bcr和u-bcr三种类型及其见于那些白血病;其次报告了BCR断点位置的变化,ABL的特殊断点,BCR-ABL信息RNA的拼接,BCR-ABL融合蛋白的结构与白血病表型的关系;此外,还介绍了BCR/ABL细胞有染色体的其它异常和Ph染色体的变种等;最后论述了BCR/ABL融合蛋白起源于何种细胞系,何种成熟阶段。少数文献报告此蛋白起始于多能干细胞。     

回输自体来源的树突状细胞治疗慢性粒细胞白血病的临床研究——附3例报告

目的:观察体外诱导生成的自体慢性粒细胞白血病(慢粒)来源树突状细胞(dendriticcell,DC)体内回输的疗效、安全性及对体内免疫功能的影响.方法:3例慢粒慢性期患者(Ph染色体、BCR-ABL融合基因均为阳性)接受体外诱导的自体慢粒来源DC的静脉回输,DC回输数量为(4.1～5.9)×106,每周1次,共回输4次.通过检测回输前后迟发型超敏反应,回输前后外周血CD3 、CD4 、CD8 T细胞百分率的变化,以了解机体产生的免疫反应.回输前后定期检查患者的白细胞计数、骨髓细胞形态学、外周血Ph染色体阳性的细胞数、BCR-ABL融合基因阳性细胞所占比率,以观察慢粒的缓解情况.结果:3例患者在DC回输前迟发型超敏反应均为阴性,在第2次DC回输后呈阳性.DC回输后CD3 T细胞百分率明显升高,其中2例患者的CD4 T细胞百分率明显升高;在DC回输的过程中及之后的3个月,白细胞基本在正常值范围内,骨髓象为慢粒缓解期.Ph染色体阳性的细胞数有下降趋势,BCR-ABL融合基因阳性细胞所占比率有所下降.结论:回输自体来源DC激活了机体抗肿瘤免疫反应,可能具有促进慢粒缓解的作用,为慢粒的治疗提供了新的方法.     

Molecular monitoring of chronic myeloid leukemia: present and future

Introduction: Fusion of BCR-ABL1 genes causes chronic myeloid leukemia (CML). As a reliable marker of disease burden, it also serves as the target of tyrosine kinase inhibitors (TKIs). New more sensitive molecular diagnostic tools for BCR-ABL1 can contribute to therapeutic decision-making, especially in considering drug discontinuation for patients enjoying prolonged deep molecular response.

Areas covered: Several novel platforms are transforming CML molecular diagnostics to enable faster point-of-care devices, better understanding of clonal diversity and resistance mutations. Here, we review these molecular platforms, knowing implementation in other hematological malignancies will ensue.

Expert commentary: Treatment with TKI in CML is the first example of a highly effective targeted therapy. Monitoring of BCR-ABL1 mRNA is standard in assessing disease burden being highly predictive of outcomes recommended by both European LeukemiaNet (ELN) and National Comprehensive Cancer Network (NCCN); however, studies has demonstrated poor adherence to these recommendations. In both clinical practice and assay performance, further optimizing of BCR-ABL1 monitoring can be envisioned including point-of-care methods for increased availability of rapid, standardized testing and increasingly sensitive molecular assays that allow for quantification of MRD and detecting resistance mutations.     

实时定量PCR技术检测BCR-ABL水平在异基因造血干细胞移植治疗慢性粒细胞白血病中的应用

目的探讨实时定量PCR（qRT-PCR）技术检测慢性粒细胞白血病（CML）患者异基因造血干细胞移植（allo．HSCT）治疗后BCR．ABL水平对改善患者预后的临床价值。方法研究纳入河北联合大学附属医院血液科2005年5月至2013年5月接受allo-HSCT治疗的CML患者15例,其中慢性期患者8例,加速期患者3例,急变期患者4例。分别在治疗前及治疗后1,2,3,6,12个月留取骨髓血标本,采用基于TaqMan探针的qRT-PCR技术检测BCR-ABL水平。根据BCR-ABL水平的动态变化,评估患者复发程度,对高复发风险的患者给予早期干预性治疗。两组问均数比较采用t检验。结果慢性期及进展期（加速期＋急变期）患者在治疗前平均BCR—ABL水平分别为（10．120±6．035）％及（43．750±14．173）％,差异具有统计学意义（t=-2．289,P〈0．05）。治疗后第1,2,3,6,12个月,慢性期和进展期患者平均BCRABL水平相比,差异均无统计学意义（t=-1．936,-2．003,-0．687,-1．613和-1．171,均P〉0．05）。治疗后第12个月,8例慢性期患者的BCR-ABL水平均为0,7例进展期患者中有5例为0。2例慢性期患者和5例进展期患者接受早期干预性治疗,除1例急变期患者在治疗后12个月复发,行再次移植无效而死亡外,其余6例患者均达完全分子生物学缓解,至随访截止时均无病存活。结论qRT-PCR是评估CML患者治疗后复发情况的良好方法,通过BCR-ABL水平的动态监测,可筛选出高复发风险的患者,配合早期干预性治疗措施可以提高患者无病生存率。     

15例成人Ph染色体和/或BCR-ABL阳性混合表型急性白血病的临床及实验室特征分析

本研究总结成人Ph染色体和/或BCR-ABL融合基因阳性的混合表型急性白血病（Ph＋ MPAL）患者的临床和实验室特征.回顾性分析2009年1月-2012年9月在我院诊治的15例Ph＋ MPAL的特征及细胞形态学、免疫学、细胞遗传学和分子生物学检测以及临床随访结果.Ph＋ MPAL的诊断采用WHO-2008分类标准.结果表明,ph＋在同时期总MPAL群体中的检出率为17.2％（15/87）.15例患者中男性7例、女性8例;中位年龄为51（16-81）岁;初诊时中位WBC计数为69（12.7-921）×109/L.形态学检查显示为急性髓系白血病（AML）者2例（13.3％）,急性淋巴细胞白血病（ALL）者6例（40.0％）、杂合型白血病（HAL）者7例（46.7％）.免疫学分析表明,此15例患者均为B淋系和髓系混合表达,93.3％患者的白血病细胞表面有CD34的表达,其中64.3％（9/14）的患者CD34表达率在60％以上.染色体R显带技术检测显示,正常核型者2例（13.3％）,单纯Ph＋者8例（53.3％）,Ph＋伴附加异常者5例（33.3％）.附加的染色体异常分别为-7（2例）、＋Ph（1例）、dic（7;9）（1例）、i（8q）和-16（1例）;有残余正常分裂相者4例.多重巢式PCR检测发现,e1a2型融合基因6例（40.0％）、b2a2或b3a2型共9例（60.0％）.7例Ph＋ MPAL患者中有4例检测到IKZF1基因缺失.本组中10例患者诱导治疗一疗程后完全缓解（CR）率为70.0％,其中联合酪氨酸激酶抑制剂（TKI）组的CR率（83.3％;5/6）,优于单纯化疗组（50.0％;2/4）,但差异无统计学意义（P＞0.05）.随访至今,6例患者由于疾病复发或进展而死亡（60.0％;6/10）.异基因造血干细胞移植（allo-HSCT）组患者总生存优于单纯化疗组（P =0.004）.结论：Ph＋ MPAL主要为B淋系和髓系混合表达,多数年龄较大,高表达CD34抗原.在BCR-ABL融合基因亚型和基因突变方面类似于Ph＋急性白血病.初诊时WBC计数是独立的预后因素,复发进展仍是此类患者死亡的主要原因.联合TKI和allo-HSCT或许能改善预后,有必要进行前瞻性、多中心的临床试验来验证.     

Myelodysplastic/myeloproliferative neoplasms

The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) include clonal myeloid neoplasms that overlap the MDS and MPN categories and at the time of initial diagnosis exhibit some clinical, laboratory, or morphologic features supporting the diagnosis of myelodysplastic syndrome (MDS) and at the same time show proliferative features in keeping with the diagnosis of a myeloproliferative neoplasm (MPN). Although the clinical, morphologic, and laboratory findings vary along a continuum from MDS to MPN, distinctive features are usually present that allow assignment of most of the cases to 1 of 3 distinct subtypes recognized by the 2008 World Health Organization (WHO) classification: chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR-ABL(-)(aCML, BCR-ABL1(-)), and juvenile myelomonocytic leukemia (JMML). The WHO classification also recognizes a provisional category of the MDS/MPN, unclassifiable (MDS/MPN, U), including the provisional entity of refractory anemia with ring sideroblasts and thrombocytosis (RARS-T). In the past 2 to 3 years since the publication of the WHO classification in 2008, dynamic progress in array technologies and next-generation amplicon deep sequencing has provided new insights into the molecular pathogenesis of MDS/MPN, especially CMML and JMML. In this review we will give an overview of these neoplasms and focus on adult MDS/MPN, especially CMML. We will give only brief updates for aCML and RARS-T; JMML will be discussed in a separate article.     

Nilotinib 300 mg BID as frontline treatment of CML: Prospective analysis of the Xpert BCR-ABL Monitor system and significance of 3-month molecular response

Sixty patients with early chronic phase CML (ECPCML) received Nilotinib on a phase II study which included a comparison of the Xpert BCR-ABL Monitor™ PCR system with standardized (IS) BCR-ABL1 real-time quantitative PCR (RQ-PCR). 88% patients achieved MMR with 45% achieving MR4.5. At 3 months BCR-ABL1/ABL1 IS >1% and <10% was associated with a lower likelihood of subsequent MR4.5 compared to patients with lower levels (p = 0.018). No significant difference was observed between methodologies in identifying MMR. Nilotinib induces high molecular response rates in ECPCML and the Xpert BCR-ABL Monitor™ system merits further investigation in this setting.