Transplantation of marrow cells from children with standard risk-acute lymphoblastic leukemia at the end of therapy into NOD/SCID mice for detecting residual leukemic cells with in vivo growth potential

In the present work, we developed a strategy for detecting residual leukemia in the marrow of children with standard risk-acute lymphoblastic leukemia (sr-ALL) at the end of therapy, based on the capacity of human leukemic cells for growing in the NOD/SCID mice marrow microenvironment. Mononuclear (MN) marrow cells from 62 patients were injected into sublethally irradiated NOD/SCID mice and the engraftment kinetics and composition of the human grafts were determined periodically. The presence of human leukemic cells with immunophenotypes and clonal DNA markers similar to those of the original leukemic clone was studied.     

非小细胞肺癌病人源性NOD/SCID小鼠和BALB/c裸鼠移植瘤模型的建立及比较

目的：构建人非小细胞肺癌的NOD/SCID小鼠和BALB/c裸鼠移植瘤模型,并探讨移植瘤成瘤性与小鼠品系间的关系,为后续建立优良的动物模型奠定基础。方法：取手术切除获得的23例非小细胞肺癌组织,1 h内移植于NOD/SCID小鼠或BALB/c裸鼠皮下,观察移植瘤成瘤情况,测量移植瘤体积,绘制生长曲线图,计算成瘤率、成瘤潜伏时间和成瘤时间,分析并比较移植成瘤组和未成瘤组所对应患者的相关临床病理指标,并取移植成瘤组和所对应患者的组织标本进行病理学分析。结果：NOD/SCID小鼠皮下移植瘤模型成瘤率（55.6%）,BALB/c裸鼠皮下移植瘤模型（20%）,两者间差异无统计学意义（P > 0.05）,但前者的成瘤潜伏时间和成瘤时间均短于后者（P < 0.05）。患者相关临床病理指标中鳞癌、TNM分期和淋巴结转移情况与移植瘤成瘤建模无明显相关（P=0.109、0.153、0.077）,NOD/SCID小鼠和BALB/c裸鼠皮下移植瘤模型均能保持病人肺癌肿瘤组织的形态学特征。结论：成功构建了NOD/SCID小鼠和BALB/c裸鼠皮下移植瘤模型,NOD/SCID小鼠更适宜用于肺癌病人源性皮下移植瘤模型的建立,非小细胞肺癌移植瘤模型的建立为进行体外抗肿瘤药物的筛选提供了良好的研究工具。     

Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/Scid mice

We describe a new human tumor xenotransplant animal model that is highly efficient for engraftment, does not need host conditioning and is suitable for in vivo studies of human tumors. Pieces of 61 freshly operated primary breast tumors were implanted into 172 irradiated and 228 nonconditioned NOD/Scid mice. A high mortality was observed in irradiated but not in nonconditioned recipients. More than 90% of analyzed implanted breast cancer specimens engrafted in the NOD/Scid mice irrespective of pretreatment. The tumors were vascularized within 3 days of implantation and maintained original histomorphology as well as expression patterns of tumor markers (cytokeratin and MUC1) and cytokines (tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4) and IL-10) released by adjacent stromal cells. A majority of tumors grew slowly, locally infiltrating host tissue, whereas some grew aggressively, developing large, fatal tumor masses and metastases within regional lymph nodes. Tumor progression in mice correlated with stage, grade, proliferation index and hormone receptor status of primary tumors. The reproducible growth behavior and preservation of characteristic features suggest that this new xenotransplant model is relevant and can be recommended for testing new anticancer therapies.     

MicroSPECT/CT imaging of primary human AML engrafted into the bone marrow and spleen of NOD/SCID mice using In-DTPA-NLS-CSL360 radioimmunoconjugates recognizing the CD123/CD131 epitope expressed by leukemia stem cells

Engraftment of primary human acute myeloid leukemia (AML) specimens into the bone marrow (BM) of NOD/SCID mice has been used to study leukemia biology and new treatments for the disease. CSL360 is a chimeric IgG₁ monoclonal antibody that recognizes CD123 (IL-3 receptor α-subchain) expressed in the absence of CD131 (β-subchain), an epitope that is displayed by leukemia stem cells (LSCs). We are studying CSL360 modified with diethylenetriaminepentaacetic acid (DTPA) for complexing ¹¹¹In and 13-mer nuclear translocation sequence (NLS) peptides to enable nuclear importation in LSCs for Auger electron radioimmunotherapy (RIT) of AML. We demonstrate that microSPECT/CT imaging using ¹¹¹In-DTPA-NLS-CSL360 revealed engraftment of primary human AML specimens into the BM and spleen of NOD/SCID mice. Our results suggest that microSPECT/CT imaging is a powerful tool which enables non-invasive assessment of the engraftment of AML into NOD/SCID mice and in the current study specifically probes an epitope displayed by the LSC subpopulation. The targeting of ¹¹¹In-DTPA-NLS-CSL360 to sites of AML engraftment in the NOD/SCID mouse model is encouraging for future RIT studies. Ultimately, SPECT imaging could be applied in AML patients to assess the delivery of ¹¹¹In-DTPA-NLS-CSL360 to sites of leukemia and be combined with Auger electron RIT using the same agent targeting the LSC population as a “theranostic” pair.     

Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.

Primitive malignant progenitors defined as nonobese diabetic/severe combined immunodeficient (NOD/SCID) leukemia-initiating cells or NOD/SL-IC from patients with acute myeloid leukemia (AML) can be detected and quantitated in sublethally irradiated NOD/SCID mice. However, there is variability in the levels of bone marrow (BM) engraftment obtained after intravenous injection of cells from different AML samples. In the current study, AML cell engraftment in standard NOD/SCID mice was compared to that obtained with NOD/SCID mice transgenic for the human growth factor genes Steel factor (SF), interleukin-3 (IL-3) and granulocyte macrophage-colony-stimulating factor (GM-CSF) (N/S-S/GM/3) as well as beta 2 microglobulin-null NOD/SCID (N/S-beta 2m(-/-)) mice. Three of the eight AML samples that failed to engraft in standard NOD/SCID animals showed easily detectable and up to 70-fold increased in the number of leukemic cells in BM 8-12 weeks post-transplantation in each of the N/S-beta 2m(-/-) and N/S-S/GM/3 mouse strains. In two of the four AML samples studied at limiting dilution, the frequency of NOD/SL-IC detected was increased six- and seven-fold. Thus, in these novel mouse strains a broader spectrum of AML patient samples can be evaluated for their progenitor content and potentially studied for their response to innovative therapeutics in vivo.     

急性淋巴细胞白血病伴CD13表达免疫表型分析

目的：探讨急性淋巴细胞白血病（acutelymphocytic leukemia,ALL）伴CD13表达的免疫表型特点,对包括CD33在内的其他抗原进行相关性分析.方法：对85例初诊ALL患者进行免疫学分型,以是否表达My把B-ALL和T-ALL分为My＋ALL和My-ALL,进行组间分析.结果：全部85例B-ALL和T-ALL患者均高表达B系和T系相关抗原（100%,100%）.所有的B-ALL均不表达T-ALL相关抗原,所有T-ALL均不表达B-ALL相关抗原.CD13的表达率为31%（B-ALL31.4%,T-ALL 28.6%）.My＋B-ALL和My＋T-ALL患者的CD13阳性细胞的中位数高于My-B-ALL和My-T-ALL患者,P值分别为0.013和0.04.My＋B-ALL患者的CD15阳性细胞的中位数高于My-B-ALL患者,P=0.000 1.结论：白血病免疫分型对于白血病的诊断、治疗和预后判断均有很大帮助,其在临床诊断中的推广将有助于患者的诊断和治疗.CD13在My＋ALL中表达较高（31%）,白血病细胞在恶性演变过程中不同时期表现出特征反映,其临床意义有待于重新评价.     

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.     

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.     

Diphtheria toxin/human B-Cell activating factor fusion protein kills human acute lymphoblastic leukemia BALL-1 cells: An experimental study

Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.     

大剂量甲氨蝶呤治疗成人急性淋巴细胞白血病的临床观察

探讨使用大剂量甲氨蝶呤(HD -MTX)在成人急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)维持强化治疗过程中的疗效和毒副反应,采用甲氨蝶呤(MTX)1~3 g/m2,24 h持续静脉滴入,用来成人ALL的强化治疗,并用四氢叶酸钙(CF)解救。结果呈缓解状态86 .0%(49/57),骨髓复发7 0%(4/57),中枢神经系统白血病(CNS L)1 .8%(1/57),死亡5. 2%(3/57)。初步研究结果提示,HD- MTX治疗成人ALL疗效肯定,相对骨髓抑制较轻,黏膜皮肤损害较突出,毒副反应可以耐受。     

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.     

成人急性淋巴细胞白血病免疫表型特点分析

目的研究成人急性淋巴细胞白血病(ALL)患者中不同亚型的各种白血病细胞免疫表型分布特点。方法采用当前国际通用的四色流式细胞术图像分析系统检测并综合分析76例ALL患者的免疫表型及其发生规律与特点。结果①76例ALL免疫表型系列来源可分为三种不同亚型,其中T-ALL亚型5例(占6.57%)、B-ALL亚型68例(89.48%)、T和B细胞混合型(T/B-ALL)亚型3例(3.95%)②ALL早期抗原表达特点:在B-ALL亚型中CD38、CD34、HLA-DR呈高表达;在T-ALL亚型中,CD38和CD34呈高表达,但HLA-DR不表达;在T/B-ALL亚型中,HLA-DR表达,CD34和CD38不表达。③按免疫分型相关抗原的敏感性和特异性分析:在B-ALL中,特异性抗原cCD79a占91.18%,敏感性抗原CD19表达占97.06%;在T-ALL中,特异性抗原cCD3和敏感性抗原CD7均表达为100%;在T/B混合型ALL中,cCD3、cCD79a、CD19均表达,CD7表达2例;④伴髓系抗原交叉表达分析:在B-ALL中,伴髓系抗原表达占21例(30.88%);在T-ALL中,伴髓系抗原表达1例(20%);T/...     

A new set of monoclonal antibodies against acute lymphoblastic leukemia

Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.     

Treatment of acute lymphoblastic leukemia in adults

Thirty-seven adult patients with acute lymphoblastic leukemia were treated with a protocol called HEAV'D including Adriamycin, Vincristine, L-Asparaginase, Cyclophosphamide and Dexamethasone. The complete remission rate was 70 per cent. The median duration of remission was more than 30 months. Patients with initial leukocyte count below 20 000/mmc experienced a longer remission (median not reached) than patients with a leukocyte count above 20 000/mmc (median 10 months).     

青少年急性淋巴细胞白血病治疗后多发性骨坏死(附1例)

目的:报告1例急性淋巴细胞白血病(ALL)青少年治疗后并发多发性骨坏死及其治疗,并复习相关文献。方法:1例15岁的青少年ALL接受了BFM方案化疗,皮质激素(泼尼松和地塞米松,并将地塞米松换算为泼尼松的等效剂量)的累积剂量为7876mg/m2。结果:治疗结束后6个患者出现肩关节和髋关节骨坏死。ALL诊断和骨坏死诊断的相隔时间为41个月。病人有长期疼痛和活动受限。已建议行手术治疗。结论:接受强烈ALL化疗的青少年是发生ON的高危人群,未来的研究应在保证疾病控制的前提下,尽量减少治疗相关ON的发生。     

PD-1 在急性T淋巴细胞白血病细胞中的表达及其临床意义

[摘要] 目的：探讨PD-1 分子在急性T淋巴细胞白血病（T-ALL）患者来源的肿瘤细胞（T-ALL细胞）中的表达及其临床意义。方法：选用2015 年12 月江苏省中医院血液科提供的1 例T-ALL细胞、4 例健康志愿者提供的PBMC和博生吉医药科技（苏州）有限公司提供的人293T/PD-1 细胞,将T-ALL细胞经尾静脉注射到B-NDG小鼠构建T-ALL细胞异种移植瘤模型,用流式细胞术检测移植瘤小鼠脾中获得的细胞是否主要是由T-ALL细胞组成。用流式细胞术检测T-ALL细胞中PD-1 蛋白的表达,用RT-PCR进一步验证T-ALL细胞中PD-1 mRNA表达水平。对T-ALL细胞中PD-1 基因进行SNP测序,以检测PD-1 基因DNA序列是否发生改变。在体外使用PD-1 抑制剂研究其对T-ALL细胞增殖、凋亡以及相关因子mRNA表达水平的影响。结果：成功构建小鼠TALL细胞异种移植瘤模型,用流式细胞术确认了该例疾病是T-ALL。T-ALL 细胞中PD-1 mRNA和蛋白均高表达（均P<0.01）。PD-1 基因的第5 个外显子的一个碱基由胞嘧啶突变成胸腺嘧啶。在体外PD-1 抑制剂对T-ALL细胞的增殖和凋亡均无明显影响;PD-1 抑制剂上调抑癌蛋白IGFBP3 mRNA表达,降低促癌蛋白SULT1A3 mRNA表达（均P<0.01）。结论：PD-1 在T-ALL细胞中高表达,PD-1可作为临床上T-ALL诊断及治疗的靶点。     

Characterization of thymocytes expressing the common acute lymphoblastic leukemia antigen

We studied the relationship between CALLA + thymocytes and two known markers of T-lymphocyte differentiation, Tdt and the sheep erythrocyte receptor. Thymocytes were studied using double fluorochrome analysis (with monoclonal anti-CALLA antibody and anti-Tdt) before and after E rosette separation. We found that approx. 4% of unseparated thymocytes were CALLA + and that most CALLA + cells were also Tdt+. After E rosettes separation CALLA + Tdt + cells were found mostly in the ER- fraction (20% of ER- cells) while only 1.0% of ER + cells were CALLA +. The expression of CALLA on ER? Tdt + thymocytes suggests that CALLA may defined cells early in T-cell differentiation.     

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。     

紫草素调节有氧糖酵解代谢诱导急性淋巴细胞白血病细胞凋亡

目的:探讨紫草素通过调节白血病细胞能量代谢影响细胞增殖与凋亡的具体机制。方法:采用MTS法检测细胞增殖,流式细胞术检测细胞凋亡;采用Western blotting 法检测细胞凋亡相关蛋白的表达,包括cleaved-caspase 8、cleaved-PARP、cleaved-caspase 3。利用丙酮酸激酶（PK）活性检测试剂盒评估紫草素对PK酶活性的影响;利用海马能量代谢检测仪检测Reh细胞经紫草素处理后其有氧糖酵解代谢水平的变化。结果:紫草素以时间剂量依赖性的方式抑制白血病细胞增殖,并诱导白血病细胞凋亡,凋亡率与药物剂量呈正相关关系（P＜0.01）。紫草素激活caspase级联反应,凋亡相关蛋白cleaved-caspase 8、cleaved-PARP及cleaved-caspase 3的表达随紫草素的剂量增加而增加。紫草素抑制白血病细胞PK酶的活性,并抑制有氧糖酵解代谢的代偿能力（P＜0.001）。结论:紫草素抑制急性淋巴细胞白血病Reh细胞有氧糖酵解代谢的代偿能力,并诱导Reh细胞以caspase依赖性的方式凋亡。