Novel therapies for children with acute myeloid leukaemia.

Significant improvements in survival for children with acute myeloid leukaemia (AML) have been made over the past three decades, with overall survival rates now approximately 60-70%. However, these gains can be largely attributed to more intensive use of conventional cytotoxics made possible by advances in supportive care, and although over 90% of children achieve remission with frontline therapy, approximately one third in current protocols relapse. Furthermore, late effects of therapy cause significant morbidity for many survivors. Novel therapies are therefore desperately needed. Early-phase paediatric trials of several new agents such as clofarabine, sorafenib and gemtuzumab ozogamicin have shown encouraging results in recent years. Due to the relatively low incidence of AML in childhood, the success of paediatric early-phase clinical trials is largely dependent upon collaborative clinical trial design by international cooperative study groups. Successfully incorporating novel therapies into frontline therapy remains a challenge, but the potential for significant improvement in the duration and quality of survival for children with AML is high.

Raised blast glutathione levels are associated with an increased risk of relapse in childhood acute lymphocytic leukemia.

A preliminary study has linked raised blast glutathione levels with chemoresistance in acute myeloid and lymphoblastic leukemia in adults and children. In this study, therefore, the relationship between leukemic blast glutathione levels and prognosis in childhood acute lymphoblastic leukemia (ALL) was investigated. A total of 77 childhood ALL samples were analyzed, 62 at initial presentation and 15 at relapse. A 20-fold interindividual variation in glutathione levels at presentation (median, 6.54 nmol/mg protein; range, 1.37 to 27.9) was demonstrated. The median level in T-lineage ALL was 2. 3-fold higher than in B-lineage ALL (Mann-Whitney test, P <.0001). There was a significant correlation between presenting white cell count (WBC) and glutathione level (Spearman rank correlation coefficient, rho = 0.45, P =.001). A high DNA index correlated with low glutathione levels (Mann-Whitney test, P =.013). There was no significant relationship between glutathione levels and in vitro drug sensitivity. Patients with glutathione levels above the median had a significantly greater risk of relapse (log-rank test statistic, 5.55; P =.018), and the overall survival rate was significantly reduced (log-rank test statistic, 4.38; P =.04). Multivariate analysis demonstrated that glutathione concentration was of independent prognostic value when assessed in conjunction with age, gender, WBC, and immunophenotype. The association of elevated blast glutathione levels with an increased risk of relapse suggests that glutathione-depleting agents may be of therapeutic value in patients who present with a high WBC.

Different expression of glutathione S-transferase alpha, mu and pi in childhood acute lymphoblastic and myeloid leukaemia.

Expression of three major classes of glutathione S-transferases (GSTs), i.e. alpha, mu and pi class, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry. In vitro cytotoxicity of 4-hydroxy-ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay. Expression of alpha, mu and pi class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTalpha P=026; GSTmu P=O009; GSTpi P=0.13). The expression of GSTalpha (1.4-fold, P=0.0004), GSTpi (13-fold, P = 0001) and to a lesser extent also GSTmu (1.1-fold, P=0.03) was higher in ALL compared with normal peripheral blood lymphocytes. Expression of GSTmu and GST7pi was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3-fold, P=0.0005 and 2-fold, P<0.0001). In contrast, GSTalpha was median 2-fold lower expressed in the AML samples (P< 0.0001). Expression levels of alpha, mu and pi class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL. One exception was a remarkably low expression of GSTalpha in IFOS-sensitive samples compared with a heterogenous expression in IFOS-resistant samples (P= 0.02). Expression of GSTpi, but not of GSTalpha or GSTmu, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P-gp. However, a high expression of both GSTpi and MRP was not associated with in vitro resistance to IFOS, DNR or PRED. The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL. Whether the high expression of GSTmu and GSTpi in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P < or = 0.0001) warrants further study.

Microtiter plate technique for the measurement of glutathione in fresh and cryopreserved lymphoblasts using the enzyme recycling method.

Glutathione is an intracellular, nonprotein thiol that appears to play an important role in protection of the cell against cytotoxic drugs (1) and has been implicated in the control of cell cycling (2,3) and apoptosis (4,5). It can exist in an oxidized (disulfide, [GSSG]) and a reduced (sulphydryl, [GSH]) form. In general, GSSG comprises less than 1% of the total intracellular glutathione. In circumstances of oxidative stress, GSH dimerizes to form glutathione disulfide (GSSG), making protons available to neutralize reactive oxygen species (Reaction 1). In vivo, reduction of GSSG is catalysed by glutathione reductase, efficiently regenerating high intracellular GSH levels (Reaction 2).