Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.     

Oncogenic lesions and molecular subtypes in adults with B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL), a genetically heterogeneous disease, is classified into different molecular subtypes that are defined by recurrent gene rearrangements, gross chromosomal abnormalities, or specific gene mutations. Cells with these genetic alterations acquire a leukemia-initiating ability and show unique expression profiles. The distribution of B-ALL molecular subtypes is greatly dependent on age, which also affects treatment responsiveness and long-term survival, partly accounting for the inferior outcome in adolescents and young adults (AYA) and (older) adults with B-ALL. Recent advances in sequencing technology, especially RNA sequencing and the application of these technologies in large B-ALL cohorts have uncovered B-ALL molecular subtypes prevalent in AYA and adults. These new insights supply more precise estimations of prognoses and targeted therapies informed by sequencing results, as well as a deeper understanding of the genetic basis of AYA/adult B-ALL. This article provides an account of these technological advances and an overview of the recent major findings of B-ALL molecular subtypes in adults.     

Low leukemia burden improves blinatumomab efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia

Background

A lower baseline bone marrow blast percentage (bBMB%) is associated with better outcomes in patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving blinatumomab. The objective of this analysis was to investigate the association between bBMB% and treatment outcomes in relapsed/refractory (R/R) B-ALL.

Methods

Data from five trials of blinatumomab for R/R B-ALL were pooled for analyses. Patients were placed in one of three groups: group 1, ≥50% bBMBs; group 2, ≥25% to <50% bBMBs; group 3, ≥5% to <25% bBMBs. Response and survival outcomes were compared between groups.

Results

Data from 683 patients (166 pediatric, 517 adult) were analyzed. Collectively, patients in groups 2 and 3 had significantly higher odds of achieving a complete remission (CR) (odds ratio [OR], 3.50 [95% confidence interval (CI), 2.23–5.48] and 3.93 [95% CI, 2.50–6.18], respectively; p < .001) and minimal/measurable residual disease response (OR, 2.61 and 3.37, respectively; p < .001) when compared with group 1 (reference). Groups 2 and 3 had a 37% and 46% reduction in the risk of death (hazard ratio [HR], 0.63 and 0.54, respectively; p < .001) and a 41% and 43% reduction in the risk of an event (relapse or death) (HR, 0.59 and 0.57, respectively; p < .001) compared with group 1. No significant differences in response or survival outcomes were observed between groups 2 and 3. Seven of nine patients whose bBMB% was lowered to <50% with dexamethasone achieved CR with blinatumomab.

Conclusion

Any bBMB% <50% was associated with improved efficacy following blinatumomab treatment for R/R B-ALL.     

Establishment and characterization of a B-cell line from a patient with acute lymphoblastic leukemia

A permanent lymphoblastoid cell line was established from the peripheral blood of a child with acute lymphoblastic leukemia. The cell line, designated SDK, grows in a stationary suspension culture, forming aggregates, in RPMI medium supplemented with 10% FCS, with a doubling time of 50-60 h. Immunologic markers and cytological features suggested that the SDK cells should be identified as being of B-cell origin. The cells failed to form rosettes with sheep erythrocytes, did not express T-cell antigens as defined by monoclonal antibodies, and exhibited surface and cytoplasmic immunoglobulin determinants. Chromosome analysis revealed the presence of three cell populations with (a) 46XY; (b) t(8q-; 14q+) or 2p-; 14q+) and (c) cells with unidentifiable markers. SDK demonstrated susceptibility to TPA-induced differentiation toward plasma cells.     

儿童与成人急性B淋巴细胞白血病免疫表型的差异分析

目的:对儿童和成人急性B淋巴细胞白血病(B-ALL)免疫表型存在的差异进行分析,探讨不同年龄段B-ALL分型特征和临床意义。方法:以260例儿童和127例成人B-ALL患者为研究对象,使用流式细胞术对患者初发时骨髓标本进行免疫表型检测,分析抗原表达情况。结果:在全部B-ALL患者中B系抗原阳性率高的抗原有CD19,CD22,CD79a,分别达到了100%,99.73%,99.19%;而成熟B系抗原CD20和cIgM阳性率为31.27%和21.29%,阳性率较低。 CD10的阳性率为94.88%,在成人组和儿童组中存在明显差异,儿童组明显高于成人组(P＜0.05)。造血干/祖细胞抗原CD34、HLA-DR、CD38、cTdT阳性率分别为76.82％、98.38％、98.92％和92.72%,其中儿童患者CD34明显低于成人患者(P＜0.05)。髓系相关抗原CD33、CD13和CD15的表达最常见,分别达20.49％、20.49％、7.01％,其中CD33和CD15在儿童组中阳性率明显低于成人组(P＜0.05)。结论:免疫分型对儿童与成人B-ALL的诊断和微小残留病检测方案选择具有重要意义。     

同胞间脐血移植治疗小儿急性淋巴细胞性白血病的时机选择与随访（附3例分析）

目的：同胞脐血具有组织相容性高,易获得HLA配型一致而不易被排斥的特点,本文报道同胞间脐血移植治疗3例小儿急性淋巴细胞性白血病的疗效并进行随访。方法：对3例急性淋巴细胞性白血病（2例高白细胞性高危CR1,1例标危并发中枢神经系统白血病CR2）,进行了同胞间脐血移植,HLA高分辨2例全相合,1例5个位点相合。预处理选用白消安/环磷酰胺为主方案。于0d回输脐血,MNC数量为2.65×107/kg,2.23×107/kg,2.18×107/kg;CD34＋细胞数量为1.92×107/kg,1.39×105/kg。预防移植物抗宿主病采用环孢霉素A、甲基泼尼松龙和甲氨蝶呤的方案。结果：中性粒细胞绝对值（ANC）≥0.5×109/L的天数分别是＋13d、＋24d、＋20d,PLT≥20×109/L的时间为＋19d、＋46d、＋21d,移植证据均为供者型。3例出现Ⅰ-Ⅱ度GVHD,均控制。随访中发生Ⅱ度慢性GVHD 1例,现长期存活2例,生活良好。结论：同胞间脐血移植是儿童高危白血病有效的治疗手段,急、慢性GVHD发生率较低,存在移植物抗白血病作用,更为安全可靠。     

同胞间脐血移植治疗小儿急性淋巴细胞性白血病的时机选择与随访(附3例分析)

目的:同胞脐血具有组织相容性高,易获得HLA配型一致而不易被排斥的特点,本文报道同胞间脐血移植治疗3例小儿急性淋巴细胞性白血病的疗效并进行随访。方法:对3例急性淋巴细胞性白血病(2例高白细胞性高危CR1,1例标危并发中枢神经系统白血病CR2),进行了同胞间脐血移植,HLA高分辨2例全相合,1例5个位点相合。预处理选用白消安/环磷酰胺为主方案。于0d回输脐血,MNC数量为2.65×107/kg,2.23×107/kg,2.18×107/kg;CD34+细胞数量为1.92×107/kg,1.39×105/kg。预防移植物抗宿主病采用环孢霉素A、甲基泼尼松龙和甲氨蝶呤的方案。结果:中性粒细胞绝对值(ANC)≥0.5×109/L的天数分别是+13d、+24d、+20d,PLT≥20×109/L的时间为+19d、+46d、+21d,移植证据均为供者型。3例出现Ⅰ-Ⅱ度GVHD,均控制。随访中发生Ⅱ度慢性GVHD 1例,现长期存活2例,生活良好。结论:同胞间脐血移植是儿童高危白血病有效的治疗手段,急、慢性GVHD发生率较低,存在移植物抗白血病作用,更为安全可靠。     

Methods of dendritic cell preparation for acute lymphoblastic leukemia immunotherapy in children

Cell immunotherapy through dendritic cells (DC) presents a hopeful strategy for the treatment of various tumors. The aim of our study was to find which progenitor cells are most suitable for the preparation of dendritic cells in acute lymphoblastic leukemia (ALL) in pediatric patients, whether blasts from bone marrow or dendritic cells generated from peripheral blood mononuclear cells taken at the time of remission after induction chemotherapy. DC generated from the BM blasts of patients with B-ALL and T-ALL (n=15) at the time of diagnosis expressed low levels of costimulatory molecules and CD markers typical for mature DC. In contrast, DC cultivated from peripheral mononuclear cells of patients (n=9) had comparable morphology and expression of costimulatory molecules to DC obtained from healthy individuals, which was even higher after tumor lysate pulsing. Autologous lymphocyte proliferation increased after DC blasts lysate pulsation and further after lymphocyte restimulation, showing evidence of induction of specific cytotoxic lymphocytes. When comparing both cell sources for the preparation of DC in patients with ALL, it appears that peripheral mononuclear cells obtained after chemotherapy are more suitable than bone marrow leukemic blasts due to similar morphology, phenotypic, and functional capacity to monocytes of healthy donors. Despite this, it is necessary to take into account individual variability when preparing DC-based vaccines. The final verification of the efficiency of immunotherapy against residual hematopoietic malignant cells in patients with ALL can only be obtained through a clinical study.     

New insights into the pathophysiology and therapy of adult acute lymphoblastic leukemia

Significant advances have been made in the last decade toward a better understanding of the disease pathogenesis and the development of novel therapies that target specific subsets of adult acute lymphoblastic leukemia (ALL). Risk‐adapted strategies are transforming the disease treatment and prognosis. With current treatment regimens, long‐term survival is achieved by approximately 50% of patients with B‐cell ALL, 50% to 60% of patients with Philadelphia chromosome–positive ALL, and approximately 80% of patients with Burkitt's leukemia. Genomic profiling in ALL has identified new prognostic markers, new therapeutic targets, and novel ALL subtypes. These may be amenable to future targeted therapies that can further improve outcomes. The early recognition of early precursor T‐cell ALL, a distinct pathobiological entity with a poor prognosis, is essential for the development of an effective clinical management strategy. The role of monoclonal antibodies and cytotoxic T‐cell therapies continues to be defined. Many of the approaches are currently being evaluated for ALL salvage. Their incorporation into frontline adult ALL therapy, in concomitant or sequential strategies, may increase the cure rates to levels achieved in pediatric ALL and may reduce the need for prolonged intensive and maintenance chemotherapy. Cancer 2015;121:2517–2528 . © 2015 American Cancer Society.     

Prophylaxis and treatment of neoplastic meningeosis in childhood acute lymphoblastic leukemia

The introduction of cranial radiotherapy (CRT) has provided efficient control of overt or subclinical meningeosis in acute leukemia. Especially due to the long-term toxicity of CRT, reduction or elimination of radiotherapy appeared mandatory after cure rates of more than 70% had been achieved in acute lymphoblastic leukemia (ALL). Several large clinical trials of the Berlin-Frankfurt-Münster (BFM) Study Group with more than 3500 patients since 1981 have demonstrated that intensive systemic and intrathecal chemotherapy without or with limited CRT can efficiently prevent central nervous system (CNS) relapses in a large percentage of patients. However, only in low-risk patients prophylactic radiotherapy can be completely and safely replaced by conventional doses of methotrexate. In addition, reduction of chemotherapy in low-risk ALL increased the rate of relapses with CNS involvement. Thus, only a combination of multidrug induction, high-dose methotrexate (HD-MTX) consolidation, and reintensification allowed safe elimination of CRT in low-risk ALL. This approach combined with CRT with 12Gy and 18 Gy in medium and high risk ALL, respectively, reduced the incidence of relapses with CNS involvement to less than 5% (trial ALL-BFM 86). Patients with inadequate response to therapy, or with T-cell ALL, or with overt CNS disease are at particularly high risk for relapse with CNS involvement, and require more systemic and intrathecal chemotherapy combined with cranial irradiation. In B-cell ALL, short intensive chemotherapy pulses including HD-MTX could completely replace radiotherapy. In AML, post-consolidation CRT appears to be advantageous with regard to control of extramedullary as well as systemic relapses.     

microRNA与急性淋巴细胞性白血病关系的研究进展

microRNA是一类长度为19～25个核苷酸的内源性非编码小分子RNA,通过抑制靶基因的表达参与包括细胞增殖、分化、凋亡、炎症调节、干细胞发育等几乎所有重要的生物学过程,许多microRNA在肿瘤细胞中异常表达,提示可能与肿瘤的发生发展有关。急性淋巴细胞性白血病（ALL）是最常见的儿童肿瘤,临床表现、形态学、免疫表型及遗传学特征极具异质性。现已发现若干microRNA在ALL 中异常表达,且与其生物学特性以及临床特征、预后和治疗相关。对microRNA的了解有助于帮助人们更深入地认识ALL 的发病机理,有助于在寻找合适的诊断、判定预后的分子标志物以及潜在的治疗靶点方面取得新突破。      

Inhibition of autophagy enhances the anticancer activity of bortezomib in B-cell acute lymphoblastic leukemia cells

B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging disease to treat in adults because of the high rates of relapse and refractory. Bortezomib, as a proteasome inhibitor, exerts obvious cytotoxicity against ALL cells and increases the sensitivity of ALL cells to conventional chemotherapeutic agents. We observed that bortezomib inhibited proliferation, induced apoptosis, arrested the cell cycle and induced autophagy in the Nalm-6 cell line and CD34⁺ primary cells. Additionally, we demonstrated that bortezomib promoted the disruption of the Bcl-2/Beclin-1 complex and increased the formation of the Beclin-1/PI3KC3 complex, leading to the initiation of autophagy. Autophagy inhibitors were employed in this study, and we found that autophagy inhibitors enhanced the anti-ALL activity of bortezomib. Taken together, these results revealed that autophagy protected B-ALL cells against the cytotoxicity of bortezomib and, in combination with autophagy inhibitors, can enhance the anticancer effects of bortezomib.     

成人早期前体T急性淋巴细胞白血病（ETP-ALL）与非ETP-ALL的临床特点对比

目的:对比分析成人早期前体T急性淋巴细胞白血病（ETP-ALL）与非早期前体T急性淋巴细胞白血病（non-ETP-ALL）的临床特点。方法:回顾性分析于我科系统诊治的成人T细胞急性淋巴细胞白血病（T-ALL）患者19例,其中ETP-ALL 6例,non-ETP-ALL 13例,对比两组患者临床基本状况、血液及骨髓检测结果、免疫分型结果及诱导治疗后缓解情况。结果:ETP-ALL组患者白细胞水平显著低于non-ETP-ALL组患者,血小板水平显著高于non-ETP-AL组患者,主要见于pro-T-ALL,首次诱导治疗后完全缓解或接近完全缓解（CR/CRi）率显著低于后者。结论:ETP-ALL患者具有较独特的临床特点,对常规诱导治疗反应差,有必要积极探索新的治疗方法和药物。     

Disulfiram联合铜对B淋巴细胞白血病细胞株增殖与凋亡影响机制探讨

目的 研究Disulfirum(DS)联合Cu(DS/Cu)对B淋巴细胞白血病(B-ALL)细胞株(nalm6细胞)增殖及凋亡的影响,并探讨其机制.方法 CCK8法检测不同浓度DS/Cu对Nalm6的增殖抑制作用,Annexin V/PI法检测不同浓度DS/Cu对Nalm6细胞的诱导凋亡作用,JC-1法检测不同浓度DS/Cu处理nalm6后线粒体膜电位的变化,蛋白质印迹法检测药物处理后线粒体通路相关蛋白(Bcl-2和Bcl-xl)的表达变化.结果 0.5 μmol/L Cu作用24h后对nalm6细胞的增殖抑制率为(6.39±4.93)％,与对照组比较差异无统计学意义,t=-2.244,P=0.154.而不同浓度的(0.05、0.1、0.2、0.4、0.8、1.6、3.2和6.4μmol/L)DS联合0.5 μmol/L Cu对nalm6细胞均具有显著的增殖抑制作用,抑制率分别为(22.29±6.69)％、(48.66±11.58)％、(50.83±12.61)％、(59.24±9.43)％、(62.74±9.17)％、(66.7±7.63)％、(72.13±7.94)％和(77.86±5.43)％,与对照组比较差异均有统计学意义,P值均＜0.05;24 h的IC50为(0.18±0.08) μmol/L.Annexin V/PI检测细胞凋亡结果显示,0.5 μmol/L Cu作用24 h后nalm6细胞的凋亡比例为(7.82±5.13)％,与对照组(8.34±6.23)％比较差异无统计学意义,t=0.112,P=0.916.而不同浓度的DS/Cu对nalm6细胞均具有显著的诱导凋亡作用,0.025、0.05、0.1、0.2和0.5 μmol/L的DS联合Cu(0.5 μmol/L)作用24h后的凋亡率分别为(17.84±7.68)％、(31.39±5.86)％、(60.41±13.87)％、(69.26±13.29)％和(81.37±12.72)％,与对照组比较差异有统计学意义,P值均＜0.05.JC-1法检测线粒体膜电位变化结果显示,单药Cu(0.5μmol/L)组的JC-1多聚体/单体比值为1.12±0.09,与对照组(0.90±0.13)比较差异无统计学意义,t=-2.442,P=0.071.不同浓度的(0.025、0.05、0.1、0.2和0.5 μmol/L)DS联合0.5 μmol/L Cu组处理nalm6细胞12 h后,线粒体膜电位水平明显降低,表现为JC-1多聚体/单体比值随浓度的增加而逐渐降低,分别为1.4±0.32、0.32±0.13、0.14±0.09、0.1±0.02和0.06±0.005,与对照组比较差异有统计学意义,P值均＜0.001.蛋白质印迹法显示,DS/Cu处理nalm6细胞12 h后能下调Bcl-2及Bcl-xl蛋白的表达.结论 DS/Cu对BALL具有增殖抑制及诱导其凋亡的作用,其作用机制可能是通过线粒体通路实现的.     

大剂量甲氨蝶呤治疗成人急性淋巴细胞白血病的药物代谢研究

目的：研究大剂量甲氨蝶呤（HD-MTX）治疗成人急性淋巴细胞白血病时的药物代谢作用及不同解救方案的价值。方法回顾性分析2003至2010年收治的124例成人患者（共190例次 HD-MTX 治疗）的临床和实验室资料。结果190例次患者的甲氨蝶呤血药浓度降至0.1μmol/L 以下的中位时间是72 h（48～342 h）,76例次（40%）发生延迟排泄;延迟排泄组患者的不良反应显著增高（P＜0.05）,其体质指数和甲氨蝶呤治疗第7日的血清肌酐水平均显著高于正常排泄组（分别为 P=0.046和 P＜0.001）。甲酰四氢叶酸钙联合左旋门冬酰胺酶解救不优于单用亚叶酸钙（P=0.849）。缩短甲氨蝶呤输注时间并提前亚叶酸钙的解救时机不能改善甲氨蝶呤的延迟排泄,但可减轻血液学不良反应。结论体质指数、血清肌酐水平影响甲氨蝶呤的代谢。适度缩短甲氨蝶呤输注时间和提前亚叶酸钙的解救时机或能改善甲氨蝶呤的排泄和不良反应。     

Cytogenetic studies on acute monocytic leukemia

Chromosome studies were performed on ten acute monocytic leukemias of which seven were poorly differentiated and three well differentiated. Chromosome abnormalities were found in 8 cases. Rearrangements of chromosome 11 were observed in 5/7 of the poorly differentiated cases. Although not specific, chromosome 11 rearrangements appear to be abnormally frequent in poorly differentiated acute monocytic leukemias.     

Genetic and epigenetic characterization of hypodiploid acute lymphoblastic leukemia

Purpose

To investigate the genetic and epigenetic landscape of hypodiploid (<45 chromosomes) acute lymphoblastic leukemia (ALL).

Methods

Single nucleotide polymorphism array, whole exome sequencing, RNA sequencing, and methylation array analyses were performed on eleven hypodiploid ALL cases.

Results

In line with previous studies, mutations in IKZF3 and FLT3 were detected in near-haploid (25–30 chromosomes) cases. Low hypodiploidy (31–39 chromosomes) was associated with somatic TP53 mutations. Notably, mutations of this gene were also found in 3/3 high hypodiploid (40–44 chromosomes) cases, suggesting that the mutational patterns are similar in low hypodiploid and high hypodiploid ALL. The high hypodiploid ALLs frequently displayed substantial cell-to-cell variability in chromosomal content, indicative of chromosomal instability; a rare phenomenon in ALL. Gene expression analysis showed that genes on heterodisomic chromosomes were more highly expressed in hypodiploid cases. Cases clustered according to hypodiploid subtype in the unsupervised methylation analyses, but there was no association between chromosomal copy number and methylation levels. A comparison between samples obtained at diagnosis and relapse showed that the relapse did not arise from the major diagnostic clone in 3/4 cases.

Conclusion

Taken together, our data support the conclusion that near-haploid and low hypodiploid ALL are different with regard to mutational profiles and also suggest that ALL cases with high hypodiploidy may harbor chromosomal instability.     

Detection of circulating ‘terminal transferase-positive’ cells does not predict relapse in acute lymphoblastic leukemia

Serial samples of peripheral blood were obtained from 35 children with ALL over a period of 18 months. The mononuclear cells were examined for TdT by indirect immunofluorescence using an unpurified anti-calf thymus TdT as the primary antibody. This analysis failed to distinguish those children who were destined to relapse (n = 9) from those who remained in continuous complete remission. Rather, the exhibition of fluorescence was linked to the co-existence of infection, with a negative predictive value of 0.91. Putative ‘TdT-positive’ cells were concentrated in the T-lymphocyte fraction and the very process of E-rosette formation seemed to contribute to this phenomenon. It appears as if the anti-TdT reagent recognizes not only TdT but also a variety of antigens which are expressed on or in immature and activated lymphocytes.     

TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT), underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest, but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs, but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement, while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally, adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL.