High-Dose Methotrexate and Continuous Infusion Ara-C in Children's Non-Hodgkin's Lymphoma: Phase II Studies and Their Use in Further Protocols

Twenty-three children with refractory or relapsed non-Hodgkin's lymphoma (NHL) received high-dose methotrexate (HD-MTX), and 9 received Ara-C by continuous intravenous infusion, as phase II studies. They all had previously received a protocol including vincristine, adriamycin, cyclophosphamide, IV push Ara-C, asparaginase, intrathecal MTX, and cranial irradiation, and had failed to respond or had relapsed. HD-MTX was given at the dose of 6 g/m² or more with leucovorin rescue, Ara-C at the dose of 100 mg/m² /day by continuous infusion over 10 days. Among the 22 evaluable patients receiving HD-MTX, 10 responses (7 CR; 3 PR) were observed. Among the 9 patients receiving Ara-C, 4 responded (1 CR; 3 PR). Toxicity in those previously heavily treated patients was acceptable. These two drugs are now successfully included in childhood NHL treatment protocols.     

1-β-d-arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation

We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1--d-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induced apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced intergenomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in <1.0 g of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 M Ara-C for 4 h, which increased with 10 and 50 M Ara-C. Incubation with 100 M Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 M MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 M MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 M MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 M for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 M) of TXL. Although continuous exposure to 1.0 M TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which non of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.     

Clearance of microsphere-entrapped 5-fluorouracil and cytosine arabinoside from the vitreous of primates

Experiments were conducted with biodegradable microspheres containing antimetabolites to assess the release of the drugs from the microspheres into the vitreous cavity of primates. Microspheres containing a mixture of radiolabeled and cold cytosine arabinoside (Ara-C) or 5-fluorouracil (5-FU) were prepared using a solvent evaporation process. The copolymers of poly (lactic) and poly (glycolic) acid (8515) and drug was dissolved in a mixture of chloroform and acetone. The solutions were then emulsified in an aqueous solution of polyvinyl alcohol and stirred for 24 hours to evaporate the organic solvent. A 0.1 mL aliquot of a suspension of the microspheres was then injected into one eye of eight African Green monkeys. Half received 250±10g of Ara-C and the others 375±15g of 5-FU. The concentration in the vitreous was then measured by removing a 0.1 mL sample of vitreous at 1, 2, 4 and 11 days after injection. Both drugs released from microspheres were still detectable in the eye 11 days after injection and the clearance kinetics were similar for both drugs. The results indicate that the microspheres appear promising as a slow drugdelivery system for future investigations in conjunction with these and other antimetabolites suitable for the treatment of PVR.Presented at ARVO meeting, Sarasota, FL Spring 1990, USA     

Cell kinetic studies after high dose Ara-C and adriamycin treatment in a slowly growing rat leukemia model (BNML) for human acute myelocytic leukemia

The effect of high-dose cytosine arabinoside (Ara-C; 1-β-d-arabinofuranosyl cytosine) injections (200 mg/kg i.v.) on cell cycle perturbation was investigated in a slowly growing rat leukemia (BNML) which is a realistic model for human acute myelocytic leukemia. Flow cytometric analysis showed an initial decrease of cells in S phase from 26 to 13% and a subsequent accumulation of up to 50% at 10–14 h after injection. The low number of S phase cells during the first 8 h might be due to a combination of cell kill in S phase and a block at the $\text{G}{}_1\text{S}$ boundary. The results make it very likely that the origin of the accumulated S phase cells is the resting compartment and that these recruited cells enter the proliferation phase as a synchronized cell population. By repeating the Ara-C injection at the time of accumulation of cells in S phase, a similar synchronized wave of recruited cells to that after the first Ara-C injection was observed.Flow cytometric analysis after Adriamycin (7.7 mg/kg i.v.) treatment, which has been shown to be cytotoxic for BNML cells, showed no changes in cell cycle distribution. It was concluded that Adriamycin might have the same toxicity for cells in all of the different cell cycle phases. The application of these data with respect to effective tumor load reduction is discussed in a second report [10].     

Diagnosis and individualized therapy of neoplastic meningitis

Neoplastic meningitis is a diffuse dissemination of tumor cells into the cerebrospinal fluid (CSF) and/or leptomeninges. It occurs in approximately 5–10% of malignant diseases, most often in breast cancer, lung cancer, melanoma or B-cell lymphoma. Symptoms of neoplastic meningitis are head or back pain, cranial nerve palsies, diffuse radicular symptoms or psychiatric disturbances. MRI shows nodular contrast enhancement lining CSF spaces. Positive CSF cytology requires optimal sampling and processing. Treatment must be individually shaped: the CSF dissemination may be treated with intrathecal chemotherapy with methotrexate or cytarabinoside (Ara-C). Liposomal Ara-C is distributed over the entire CSF space even after lumbar application and maintains cytotoxic levels for at least 2 weeks. Radiotherapy should be applied only to symptomatic solid spinal manifestations or fast progressing cranial nerve palsies. Systemic chemotherapy is needed to control solid manifestations or, in the case of substances entering the CSF, to support intrathecal chemotherapy.     

16例高白细胞APL诱导缓解治疗临床分析

目的评价在诱导缓解治疗阶段应用柔红霉素、阿糖胞苷(DA方案)和全反式维甲酸(ATRA)双诱导治疗初诊高白细胞急性早幼粒细胞白血病(APL)的临床疗效及不良反应。方法回顾性分析2002～2011年深圳市人民医院收治的16例在诱导缓解治疗阶段均接受柔红霉素、阿糖胞苷和全反式维甲酸方案双诱导治疗的高白细胞急性早幼粒细胞白血病患者资料,对患者的临床缓解情况及治疗方案的不良反应进行分析。结果柔红霉素、阿糖胞苷联合全反式维甲酸方案双诱导治疗高白细胞急性早幼粒细胞白血病的临床完全缓解率达87.5%,1例死于肺部严重感染,1例死于颅内出血,3例患者出现维甲酸综合征且经治疗后好转。结论在诱导缓解阶段应用柔红霉素、阿糖胞苷联合全反式维甲酸方案双诱导治疗初诊高白细胞急性早幼粒细胞白血病完全缓解率高,不良反应少,是一种很好的治疗选择。     

急性单核细胞白血病细胞系THP-1侧群细胞的分选、鉴定与富集

目的 探讨人急性单核细胞白血病细胞系THP-1中是否存在侧群(SP)细胞,鉴定其是否具有白血病干细胞(LSCs)的生物学特性,并研究从THP-1细胞系中富集SP细胞的方法.方法 THP-1细胞用Hoechst33342荧光染料染色后,分别应用荧光显微镜和流式细胞术(FCM)检测是否存在SP细胞及SP细胞的形态和比例.分选SP和非侧群(NSP)细胞,FCM检测其表面抗原表达及细胞周期;实时荧光定量PCR检测耐药基因ABCG2、ABCB1和凋亡基因Bcl-2、Bax在两亚群细胞中的表达差异.将THP-1细胞用不同水平阿糖胞苷(Ara-C)处理24h,染色后检测SP细胞比例的变化.结果 1.荧光显微镜观察到在THP-1细胞中存在拒染的SP细胞,形态与NSP细胞无明显区别.FCM检测结果显示,试验管SP细胞比例为(1.81±0.99)％,对照管可被维拉帕米完全拮抗.2.FCM分选SP细胞的纯度达到(96.71±0.90)％;SP细胞大部分处于静止期,Go/G1期细胞比例达(84.04 ±4.98)％,而NSP细胞则处于增殖期,S/G2/M期细胞比例为(56.38±1.48)％;CD34+、CD34+ CD38-细胞在SP亚群中的比例要高于NSP亚群(P ＜0.05);SP细胞ABCG2及ABCB1表达水平显著高于NSP细胞(P均＜0.05).SP细胞Bcl-2表达水平(0.99±0.08)显著高于NSP细胞(0.29±0.13)(P＜0.05),而Bax表达值(0.68±0.05)与NSP细胞(0.85±0.09)表达水平差异无统计学意义(P＞0.05),但Bcl-2/Bax比值显著高于NSP细胞亚群(P＜0.05).3.经与Ara-C共培养24h后,SP细胞的比例显著提高,且随着Ara-C水平增大,SP细胞比例也越高.结论 人急性单核细胞白血病细胞系THP-1中存在少量SP细胞,较之NSP细胞更具有干细胞的生物学活性.与一定水平Ara-C共培养可有效富集THP-1细胞系中的SP细胞,可作为LSCs研究的切入点,对进一步研究白血病有重要意义.     

大剂量阿糖胞苷强化治疗急性髓性白血病的疗效观察

目的探讨大剂量阿糖胞苷强化治疗急性髓性白血病的临床疗效。方法将我院2002年6月至2005年6月收治的40例急性髓性白血病患者随机分为强化治疗组和对照组,两组均以DA、HA或DAE方案诱导至完全缓解,巩固后强化治疗组采用大剂量阿糖胞苷静脉滴注治疗3个疗程,对照组不采用阿糖胞苷治疗,比较两组患者强化治疗后1年、3年、5年的无病生存情况,不良反应发生率和总治疗有效率。结果强化治疗组在1年、3年、5年内的无病生存率分别为85．0％（17／20）、60．0％（12／20）、30．0％（6／20）;对照组在1年、3年、5年内的无病生存率分别为20．0％（4／20）、50％（10／20）、0;强化治疗组1年、3年、5年内的无病生存率均显著高于对照组（P〈0．05）。强化治疗组治疗总有效率为85％,显著高于对照组（P〈0．05）。两组之间不良反应的发生率无显著统计学差异（P〉0．05）。结论采用大剂量阿糖胞苷强化治疗能显著提高急性髓性白血病患者化疗后1年、3年、5年内的无病生存率,远期疗效好,可作为急性髓性白血病缓解后的强化治疗药物。     

Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures

Tyrosine hydroxylase-positive nerve fiber formation occurs in two diverse morphological patterns in rat fetal ventral mesencephalic slice cultures; one is non-glial-associated and the other is glial-associated. The aim of this study was to characterize the non-glial-associated nerve fibers and its relation to migration of astrocytes. Organotypic slice cultures were prepared from embryonic days 12, 14, and 18 rat fetuses and maintained for 5, 7 or 14 days in vitro. Inhibition of cell proliferation using cytosine β-d-arabinofuranoside was conducted in embryonic day 14 ventral mesencephalic cultures. The treatment impaired astrocytic migration at 7 and 14 days in vitro. The reduced migration of astrocytes exerted a negative effect on the glial-associated tyrosine hydroxylase-positive nerve fibers, reducing the outgrowth from the tissue slice. The non-glial-associated outgrowth was, however, positively affected by reduced astrocytic migration, reaching distances around 3 mm in 2 weeks, and remained for longer time in culture. Co-cultures of fetal ventral mesencephalon and frontal cortex revealed the cortex as a target for the non-glial-associated tyrosine hydroxylase-positive outgrowth. The age of the fetal tissue at plating affected the astrocytes such that older tissue increased the length of astrocytc migration. Younger tissue at plating promoted the presence of non-glial-asscociated outgrowth and long radial-glia-like processes, while older tissue promoted migration of neurons instead of formation of nerve fiber network. In conclusion, inhibition of astrocytic proliferation promotes the persistence of long-distance growing tyrosine hydroxylase-positive nerve fibers in ventral mesencephalic slices cultures. Furthermore, the long-distance growing nerve fibers target the frontal cortex and are absent in cultures derived from older tissue.     

Toxicity of cytostatic drugs to normal bone marrow cells in vitro

In this study we compared how different concentrations and periods of incubation of anthracyclines, amsacrine, and cytosine arabinoside would affect normal hematopoietic bone marrow cells in terms of interindividual differences in toxicity, the age of the donor, and the proliferative capacity of the bone marrow. Bone marrow was obtained from 36 donors in connection with bone marrow transplantation. After separation the mononuclear cell fraction was incubated with doxorubicin, 4-epidoxorubicin, daunorubicin, idarubicin, aclarubicin, mitroxantrone, amsacrine, and cytosine arabinoside for 1 h, for 3 h, or continuously. The cells were thereafter cultured in soft agar and CFU-GM were counted after 10–12 days. The results showed a large interindividual variation in toxicity for all drugs tested. Daunorubicin, idarubicin, aclarubicin, and mitoxantrone had a pronounced cytotoxic effect after 1 h of incubation. Doxorubicin and 4-epi-doxorubicin showed the greatest cytotoxic effect after 3 h and were also more toxic to normal bone marrow cells from donors over 40 years of age. Ara-C had a low cytotoxic effect after 1 and 3 h of incubation, even at high concentrations, but exerted a pronounced degree of toxicity during continuous incubation. Daunorubicin, idarubicin, and ara-C also showed increased toxicity to cell samples with a low proliferating capacity in the control. The conclusions drawn from these results are that interindividual variation, proliferation capacity, incubation conditions, and the age of the donors are factors of importance in the toxicity of drugs to normal hematopoietic bone marrow cells. Received: 12 January 1997 / Accepted: 24 November 1997