Abrupt cessation of immunosuppression in a patient with persistent acute lymphoblastic leukaemia following allogeneic transplantation

A 20-year-old man with acute lymphoblastic leukaemia (ALL) in second complete remission underwent sibling donor transplant. Post-transplant there was haematological and immunophenotypic evidence of persisting disease. Cyclosporine was abruptly discontinued and following this the leukaemic clone disappeared. Discontinuation of immunosuppression may be of benefit in leukaemia which persists following transplant.     

Predictive value of MTT assay as an in vitro chemosensitivity testing for gastric cancer： One institution＇s experience

AIM：To investigate the predictive clinical value of in vitro 3-（4,5-dimethylthiazolyl-2）-2, 5-diphenyltetrazolium bromide （MTT） assay for directing chemosensitivity in patients with gastric cancer.
METHODS：Results of a total of 353 consecutive patients with gastric cancer treated with MTT-directed chemotherapy or physician’s empirical chemotherapy from July 1997 to April 2003 were reviewed and analyzed retrospectively.
RESULTS：The overall 5-year survival rate of MTT- sensitive group （MSG） and control group （CG） was 47.5% and 45.1%, respectively. The results of subgroup analysis with Cox proportional-hazards model were favorable for the MSG-sensitive group. However, no statistically significant difference in survival rate was observed between the two groups.
CONCLUSION：Individualized chemotherapy based on in vitro MTT assay is beneficial, but needs to be confirmed by further randomized controlled trials.     

BALB／C小鼠脾淋巴细胞活性Alamarblue测定法

目的在比较Alamarblue法与MTT法的基础上探讨Alamarblue法用于测定小鼠淋巴细胞细胞体外活性的可行性及实验条件。方法Alamarblue法、MTT法分别测定不同浓度的BALB／C小鼠脾淋巴细胞在不同的作用时间下的还原率及OD值。结果使用Alamarblue法,作用时间为4、6、20h,细胞浓度和还原率之间呈正性线性相关;作用时间为0、2h,细胞浓度和还原率之间线性关系不明显。最大细胞接种浓度（2×10^5个／ml）,作用时间为20h,还原率为78．3％,仍未达到100％。Alamarblue法与MTT法显著正相关（r=0．998,P〈0．05）。高低两种细胞浓度时,Alamarblue法组内差异分别为0．0152、0．024。结论小鼠脾淋巴细胞增殖或细胞毒试验中,Alamarblue法可以间接反映淋巴细胞的量。Alamarblue法与MTT法有良好的相关性。在细胞接种浓度较低时,Alamarblue法更稳定。     

Clinical evaluation of chemosensitivity testing for patients with colorectal cancer using MTT assay

PURPOSE: Colorectal cancer is one of the tumors most refractory to treatment by chemotherapy. The chemosensitivity test should be performed to individualize the chemotherapy for patients with colorectal cancer, which is less sensitive for anticancer drugs. The present study was designed to determine the chemosensitivity in fresh human colorectal cancer, using highly purified tumor cells, and the correlation of this sensitivity with clinical response. METHODS: We determined the chemosensitivity for cisplatin, mitomycin C, adriamycin, and 5-fluorouracil in vitro in 93 fresh human colorectal cancers using the MTT assay and performed chemotherapy according to results of the MTT assay. RESULTS: Inhibition rate of tumor cells for cisplatin was higher than those for other drugs. Fifteen patients who have evaluable lesions received chemotherapy according to results of the MTT assay. Clinical responses were obtained in 5 of 15 patients, and the inhibition rate for cisplatin was higher in responders than in nonresponders. CONCLUSIONS: It is suggested that the chemotherapy according to results of the MTT assay is effective in patients with colorectal cancer.     

In vitro cytotoxicity of nelarabine, clofarabine and flavopiridol in paediatric acute lymphoblastic leukaemia

The in vitro efficacies of three new drugs--clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) - were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188-fold lower than that of NEL. B-lineage, but not T-lineage lines, were >7-fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25-fold and 113-fold higher than ARAC in T- and B-lineage, respectively. T-ALL cells were eightfold more sensitive to NEL than B-lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross-resistance of CLOF, NEL and FP was observed with many front-line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T-ALL, a subset of patients with B-lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B-lineage than T-ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.     

Comparative analysis of methotrexate polyglutamates in lymphoblast preparations from bone marrow and blood, and the contribution of residual red blood cells

Purpose: Blasts isolated from bone marrow aspirates or blood samples of patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were compared for uptake of methotrexate (MTX) and formation of MTX polyglutamates (MTX-Glu _n ). Red blood cells (RBC) from the same patient samples were also analyzed. Methods: Blasts were isolated by standard density centrifugation. RBC were prepared from the pellet of the same centrifugation. MTX-Glu _n were analyzed by means of HPLC and radiochemical quantification. Results: In lymphoblasts isolated from blood, the distribution patterns of MTX-Glu _n were the same as in bone marrow lymphoblasts, but the total amount of MTX-Glu _n accumulated in blood lymphoblasts was reduced by 41%–51% when compared to the same number of bone marrow lymphoblasts of the same patient. RBC accumulated MTX but no formation of MTX-Glu _n occurred. Conclusions: The determination of MTX and MTX-Glu _n in lymphoblasts isolated from blood samples of patients with common ALL provides qualitative information on the capacity of the blasts to form MTX-Glu _n since distribution patterns of MTX and MTX-Glu _n parallel that of bone marrow lymphoblasts. The amounts of MTX-Glu _n accumulated, however, were much lower in blood lymphoblasts. Blood lymphoblasts are therefore not useful for a quantitative analysis of MTX-Glu _n . The contribution of RBC to MTX and MTX-Glu _n in vitro is only marginal and residual RBC in lymphoblast preparations from bone marrow can therefore be ignored. Received: 19 November 1999 / Accepted: 19 January 2000     

Early UK experience in the use of clofarabine in the treatment of relapsed and refractory paediatric acute lymphoblastic leukaemia

Clofarabine is a second-generation purine nucleoside analogue, which has shown promising activity in relapsed and refractory paediatric acute lymphoblastic leukaemia (ALL). This report summarizes the early United Kingdom experience of clofarabine for the treatment of paediatric ALL in 23 patients, outside of the context of a clinical trial. Our results demonstrated that clofarabine-based chemotherapy regimes were effective and well-tolerated in this heavily pre-treated group, with an overall response rate of 67% when used in combination regimes. Responses were seen in both B and T cell disease and in patients with adverse cytogenetics.     

细胞衰老影响MTT法药效评价及其改进方法

目的MTT法是评价细胞增殖的常规方法,但有时有一定的假阳性率。本文从细胞衰老角度进行研究,探讨假阳性率产生的原因和改进方法。方法使用能引起细胞衰老的抗肿瘤药物博来霉素和博宁霉素,处理人子宫颈癌HeLa细胞和人乳腺癌MCF-7细胞,采用MTT、SRB、台盼蓝染色计数法和克隆形成率等方法检测细胞增殖的抑制作用;衰老相关的β-半乳糖苷酶染色确定衰老细胞。结果低浓度的博宁霉素处理MCF-7细胞,明显引起细胞衰老。MTT法测得的IC50值最大,SRB和台盼蓝染色计数法测得的IC50值次之,克隆形成率法最为敏感,测得的IC50值最小,达到了nM级。结论细胞衰老明显影响一些抗肿瘤药物的MTT评价结果,使用克隆形成率法是最为可靠的克服细胞衰老影响的方法。     

The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) occurs at all ages but is the most common cancer of childhood. The current treatment of paediatric ALL is highly successful with up to 90% children being cured. In contrast, survival rates for adult ALL are significantly lower at around 40%. The discovery and characterisation of genetic abnormalities have increased our understanding of the biology of the disease and provided important prognostic and predictive markers which have improved patient outcome. Not only is the spectrum of these aberrations vast but, due to advances in technology, continually expanding. A wide range of chromosomal and genomic abnormalities have been reported as being associated with patient outcome but only a subset are currently used to risk stratify patients. This review highlights the main genetic abnormalities which are used to manage patients with B-cell precursor ALL and discusses the evidence which has been accumulated on several newly described genomic abnormalities.     

Treatment of young patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia using increased dose of imatinib and deintensified chemotherapy before allogeneic stem cell transplantation

The main outcomes of the Programa Español para Tratamiento de Hemopatías (PETHEMA)‐acute lymphoblastic leukaemia (ALL)‐Ph‐08 trial were described and compared with those of the historical PETHEMA‐CSTIBES02 trial. The trials differed in imatinib dose (600 vs. 400 mg/d) and amount of chemotherapy (one vs. two consolidation cycles) before stem cell transplantation (SCT). All patients (n = 29) enrolled in the ALL‐Ph‐08 trial achieved complete remission (CR) (vs. 90% in CSTIBES02), and SCT was performed in CR in 90% (vs. 78%). The reduction in early death, relapse before SCT and transplant‐related mortality observed in the ALL‐Ph‐08 trial resulted in an improved 2‐year event‐free survival (63% vs. 37%, P = 0·009).     

Acute respiratory distress syndrome following intrathecal methotrexate administration: a case report and review of literature

 Acute Respiratory distress syndrome (ARDS) is a rare complication following intrathecal (IT) injection of methotrexate (MTX) in adult acute lymphoblastic leukemia (ALL) patients. A 19-year-old man with ALL developed strikingly acute respiratory failure during central nervous system (CNS) prophylaxis with IT MTX administration and cranial irradiation. Histopathologic study of the lungs revealed a pattern of diffuse alveolar damage with interstitial cellular infiltration. His symptoms were relieved soon following treatment with corticosteroids and the pulmonary infiltrates resolved gradually. Pulmonary symptoms did not recur as he was continuously treated with oral corticosteroids. Received: 31 January 2000 / Revised: 16 March 2000 / Accepted: 8 May 2000     

Two groups of Philadelphia chromosome-positive childhood acute lymphoblastic leukemia classified by pretreatment multidrug sensitivity or resistance in in vitro testing

The development of effective chemotherapy is imperative for children with Philadelphia chromosome-positive (Ph) acute lymphoblastic leukemia (ALL) because of the poor prognosis of this condition. Initial cellular drug resistance is thought to be an important cause of induction failure and early relapse. We carried out in vitro tests using a methyl-thiazol-tetrazolium assay on bone marrow samples from 274 children with newly diagnosed ALL. Sixteen children (5.8%) had Ph-positive results of cytogenetic analysis. We examined in vitro drug resistance to 14 agents and found that leukemic cells in Ph ALL were significantly more resistant than were cells in non-Ph ALL to melphalan, bleomycin, etoposide, mitoxantrone, L-asparaginase, and vinblastine. With the prednisolone, L-asparaginase, and vincristine (PAV) combination of drugs, 10 of the 16 Ph patients with ALL (62.5%) showed relative resistance (RR) (sensitivity to only 1 or to none of the 3 drugs) at initiation of treatment. These 10 patients experienced significantly poorer event-free survival (EFS) than did the 6 patients with supersensitivity (SS) (defined as sensitivity to all 3 or to 2 of the 3 drugs, P = .019). Leukemic cells from RR patients were found to be multiresistant to 12 drugs with 2.0- to 58.4-fold RR compared with cells from SS patients. This PAV sensitivity delineates initially sensitive and resistant groups. Of these, the SS subgroup of Ph ALL patients may be curable with chemotherapy and stem cell transplantation. For EFS improvement in the RR group, it may be necessary to use a new chemotherapy approach from initiation.     

Congenital leukaemia: the Dutch experience and review of the literature

We reviewed Dutch patients and those described in the literature with congenital leukaemia in the past 25 years, with the intention to obtain an overview of the characteristics of this rare disease. Among the 117 patients reviewed, acute myeloid leukaemia (AML) was more frequent (64%) than acute lymphoblastic leukaemia (ALL, 21%). Most patients had a high leukaemic cell load with hepatosplenomegaly, leukaemia cutis and hyperleucocytosis. Cytogenetic abnormalities were found in the majority of the patients tested (72%); 11q23 abnormalities were found in less than half of them (42%). The probability of overall survival at 24 months was only 23%. When congenital AML and ALL were compared, clinical characteristics and overall survival were not significantly different. However, in patients at risk, the probability of event-free survival (EFS) and disease-free survival (DFS) were significantly higher in AML than in ALL, 43% versus 13% and 68% versus 0% respectively. Among the congenital AML cases, six spontaneous remissions have been described. In conclusion, the clinical characteristics of congenital leukaemia differ from those of leukaemia in older children and prognosis is generally poor. Once complete remission is achieved, patients with AML fare better than those with ALL. Chemotherapy for congenital leukaemia needs improvement to increase the sustained remission rate.     

Cytotoxicity of adriamycin,idarubicin, and vincristine in acute myeloid leukemia: Chemosensitization by verapamil in relation to P-glycoprotein expression

Summary A 4-day colorimetric tetrazolium dye (MTT) assay was used to assess the cytotoxicity of adriamycin (ADM), vincristine (VCR), and idarubicin (IDA) in blasts isolated from 37 patients with newly diagnosed and pretreated acute myeloid leukemia (AML). The effect of verapamil (VRP) as a chemosensitizer was studied in relation to the expression of the membrane efflux pump P-glycoprotein (PGP) as determined by a semiquantitative flow-cytometric procedure. A slight positive correlation was found between the fraction of cells expressing PGP and the ID₅₀ values for ADM and VCR, but not between cellular PGP content and sensitivity to IDA. The overall data showed no significant sensitization effect of VRP. However, in specimens with more than 10% cells expressing PGP, 2M VRP sensitized cells to ADM and VCR significantly. The median of sensitization ratios (SRs), i.e., the ratios of cytotoxic drug ID₅₀ in the absence/presence of VRP, were 1.89 and 2.0, respectively. No sensitizing effect of VRP on the cytotoxicity of IDA was observed. Related to the clinical status, the median fraction of PGP-positive blasts was elevated fourfold in pretreated patients (n=16) in comparison to patients with de novo AML (n=19). No differences in ID₅₀ values were observed between newly diagnosed and pretreated patients. However, SRs for ADM and VCR were higher in samples of pretreated patients compared with de novo AML. PGP-mediated cellular drug resistance may thus be circumvented in leukemic blasts by application of chemosensitizers or, potentially, alternative anthracyclines.     

Advances in understanding the pathogenesis of CNS acute lymphoblastic leukaemia and potential for therapy

Central nervous system acute lymphoblastic leukaemia (CNS ‐ALL ) is a major clinical problem. CNS ‐directed ‘prophylactic’ chemo‐ or radio – therapy is associated with significant early and long‐term toxicity. Moreover, greater than a third of the relapses occur in the CNS . To design specific, more effective and less toxic therapy and for personalized precise adjustment of prophylactic therapy there is a need for better understanding of the biology of this disease. Specifically, the precise neurotropic mechanisms of ALL are currently unclear, as is the pathogenesis of CNS relapse. Here we review and contrast the recent findings with earlier studies of pathogenesis of CNS leukaemia. We also describe the challenges in research of this devastating complication of ALL .     

In vitro improvement of chlorambucil-induced cytotoxicity by deflazacort and 6-methylprednisolone in B-cell chronic lymphocytic leukaemia

Abstract: We examined whether CLL cell chemosensitivity to in vitro exposure to chlorambucil (CLB) might be improved by the presence of deflazacort (DFZ) in comparison to 6-methylprednisolone (PDN). The PDN lethal dose (LD)₅₀ values were low in 5 samples, intermediate in 4 and high in 7. Low, intermediate and high DFZ-LD₅₀ values were detected in 3, 2 and 11 samples, respectively. The CBL-LD₅₀ mean values were significantly reduced at all PDN and at the 4 highest DFZ concentrations. However, a dose-response effect was seen only in the DFZ group. Both CLB-DFZ and CLB-PDN interactions were analysed in 16 samples at 25 different dose-combinations, resulting in 400 comparisons between expected and observed leukaemic cell survival (LCS) values for each group. In particular, 45.75% and 40% dose combinations were synergistic in CLB-DFZ and CLB-PDN groups, respectively. A relatively higher number of antagonistic interactions were observed among CLB-PDN dose combinations, while analogous number of additive interactions were detected. At concentrations of CLB × 1 μg/ml the phenomenon of synergism, regardless of the steroid concentration, did occur more frequently. On the other hand, a more elevated number of antagonistic interactions were counted at CLB 100 μg/ml. In conclusion, both DFZ and PDN synergize in vitro with CLB, especially at low concentrations of the alkylating agent.     

Personal practice: how we manage the risk of bleeding and thrombosis in children and young adults with acute lymphoblastic leukaemia

Bleeding and thrombosis are not infrequent problems in children receiving treatment for acute lymphoblastic leukaemia (ALL). The exact frequency varies with age, co-morbidity and treatment schedule, but the risk is highest in the first few weeks of treatment when disease and treatment-related haemostatic abnormalities prevail. Recommendations for prevention and management are lacking due to a weak evidence base, resulting in considerable variation in practice. This article describes our personal practice in this area with reference to the available literature on the subject.     

Clearance of marrow infiltration after 1 week of therapy for childhood lymphoblastic leukaemia: clinical importance and the effect of daunorubicin

At the commencement of UKALL XI, a national MRC trial for childhood lymphoblastic leukaemia (ALL), the therapy included a bolus of daunorubicin (DR) on the first 2 d of the protocol. This component of treatment was subsequently withdrawn because of concern about long-term cardiotoxicity. All children both before and after this change of policy had their marrow status at the end of the first week assessed by central review as part of the trial to examine the clinical importance of the rate of disease clearance. This also afforded an opportunity to observe the effect of DR on gross residual disease at an early stage of therapy.
1419 children were studied: 342 received DR ('recipients'), 1077 did not. 44% of the recipients completely cleared their marrow of blast cells after 8 d compared with 13% of the non-recipients (χ²=158.2, P <0.0001). The difference in the proportion with massive residual disease (>80% blasts) was less impressive but there was still a difference in favour of DR recipients (DR 9%, no DR 15%; χ²=7.7, P =0.006). The rate of disease clearance correlated with disease-free survival for both recipients and non-recipients, but there was no significant difference in outcome when comparing the two groups with each other, either in terms of disease-free or relapse-free survival.
DR accelerated the rate of blast cell disappearance from the marrow but the difference this made to disease free survival is small or non-existent. It appears to be the relative speed of response to a given therapeutic regimen that is prognostically important rather than the absolute rate of response when comparing one treatment with another.