Glucocorticoid sensitisation in Mixed Lineage Leukaemia-rearranged acute lymphoblastic leukaemia by the pan-BCL-2 family inhibitors gossypol and AT-101

Aim of the studyResistance to glucocorticoids (GCs) remains a major problem in the treatment of infants with acute lymphoblastic leukaemia (ALL) carrying Mixed Lineage Leukaemia (MLL) translocations. Despite intensive research, the mechanism(s) underlying GC resistance remain poorly understood. Recent studies suggested an important role for the pro-survival BCL-2 family member MCL1 in GC resistance in MLL-rearranged ALL.MethodsWe exposed GC-resistant MLL-rearranged SEMK2 cells to potent MCL1-inhibiting agents, including gossypol, AT-101, rapamycin, SU9516 and obatoclax (GX15-070) and determined GC sensitisation using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays. Using Western blotting we analysed the protein expression of most BCL-2 family members in MLL-rearranged SEMK2 cells after treatment with potent MCL-1 inhibiting agents.ResultsOnly gossypol and its synthetic analogue AT-101 induced GC sensitivity in MLL-rearranged ALL cells. Remarkably, the GC-sensitising effects of gossypol and AT-101 appeared not to be mediated by down-regulation MCL1 or other anti-apoptotic BCL-2 family members, but rather involved up-regulation of multiple pro-apoptotic BCL-2 family members, in particular that of BIM and BID.Concluding remarksIn conclusion, gossypol and AT-101 induce GC sensitivity in MLL-rearranged ALL cells, most likely mediated by the activation of BID and BIM without the necessity to down-regulate anti-apoptotic BCL-2 family members like MCL1. Hence, co-administration of either gossypol or AT-101 during GC treatment of GC-resistant MLL-rearranged ALL patients may overcome GC resistance and improve prognosis in this high-risk childhood leukaemia.     

The potential of clofarabine in MLL-rearranged infant acute lymphoblastic leukaemia

MLL-rearranged acute lymphoblastic leukaemia (ALL) in infants is the most difficult-to-treat type of childhood ALL, displaying a chemotherapy-resistant phenotype, and unique histone modifications, gene expression signatures and DNA methylation patterns. MLL-rearranged infant ALL responds remarkably well to nucleoside analogue drugs in vitro, such as cytarabine and cladribine, and to the demethylating agents decitabine and zebularine as measured by cytotoxicity assays. These observations led to the inclusion of cytarabine into the treatment regimens currently used for infants with ALL. However, survival chances for infants with MLL-rearranged ALL do still not exceed 30–40%.Here we explored the in vitro potential of the novel nucleoside analogue clofarabine for MLL-rearranged infant ALL. Therefore we used both cell line models as well as primary patient cells. Compared with other nucleoside analogues, clofarabine effectively targeted primary MLL-rearranged infant ALL cells at the lowest concentrations, with median LC₅₀ values of ∼25 nM. Interestingly, clofarabine displayed synergistic cytotoxic effects in combination with cytarabine. Furthermore, at concentrations of 5–10 nM clofarabine induced demethylation of the promoter region of the tumour suppressor gene FHIT (Fragile Histidine Triad), a gene typically hypermethylated in MLL-rearranged ALL. Demethylation of the FHIT promoter region was accompanied by subtle re-expression of this gene both at the mRNA and protein level. We conclude that clofarabine is an interesting candidate for further studies in MLL-rearranged ALL in infants.     

Glucocorticoid use in acute lymphoblastic leukaemia

Glucocorticoids (prednisone and dexamethasone) play an essential part in the treatment of acute lymphoblastic leukaemia (ALL), but their optimum doses and bioequivalence have not been established. Results of preclinical studies have shown that dexamethasone has a longer half-life and better CNS penetration than does prednisone. In prospective randomised trials, dexamethasone improved control of CNS leukaemia. At a prednisone-to-dexamethasone dose ratio of less than seven, dexamethasone (6-18 mg/m(2) per day) resulted in a better event-free survival than did prednisone (40-120 mg/m(2) per day), and high-dose dexamethasone (10-18 mg/m(2) per day) improved the outcome of T-cell ALL and high-risk ALL. However, dexamethasone caused more adverse effects, including infection, bone fracture, osteonecrosis, mood and behaviour problems, and myopathy. At a dose ratio greater than seven, the two drugs showed no difference in efficacy. Therefore, the efficacy of prednisone and dexamethasone is dose dependent and needs to be carefully assessed against the toxic effects. Moreover, although dexamethasone generally showed increased activity in ALL cells in vitro, the dose ratio of the two drugs that exerted equivalent cytotoxic effects differed substantially in samples from individuals. The selection of the type and dose of glucocorticoid should be based on the risk of relapse, treatment phase, and the chemotherapeutic drugs used concomitantly.     

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

Hypomethylation of the global genome, considered to be composed mainly of repetitive sequences, is consistently observed in cancers, and aberrant hypo- and hypermethylation of CpG islands (CGIs) in promoter regions are also observed. Since methylation alterations in unique promoter sequences and in other genomic regions have distinct consequences, we analyzed the relationship between the global hypomethylation and the hypomethylation of unique promoter CGIs using human gastric cancers. Seven of ten gastric cancer cell lines showed marked decreases in 5-methylcytosine content, which correlated with hypomethylation of the LINE1 repetitive sequence. Six of the seven cell lines showed hypomethylation in five or all of the six normally methylated CGIs in promoter regions of six genes, and this was associated with induction of aberrant expression. The remaining three cell lines without global hypomethylation showed promoter hypomethylation in one or none of the six CGIs. Frequent promoter hypomethylation, however, did not correlate with frequent promoter hypermethylation. In primary gastric cancers too, global hypomethylation was associated with hypomethylation of LINE1 repetitive sequence and promoter hypomethylation. Of 93 gastric cancers, 33 cancers with frequent promoter hypomethylation and 27 cancers with frequent promoter hypermethylation constituted different groups. These findings represent experimental evidence that frequent hypomethylation of normally methylated promoter CGIs is associated with global hypomethylation, and that these hypomethylations occur independently of frequent promoter CGI hypermethylation. (Cancer Sci 2004; 95: 58–64)     

Gene-specific promoter hypermethylation without global hypomethylation in follicular thyroid cancer

Genome-wide hypomethylation and hypermethylation at CpG promoters are common in cancer. To date, little is known about global methylation changes in follicular thyroid cancer (FTC). Two independent quantitative methods, bisulphite Pyrosequencing of Long Interspersed Nucleotide Elements-1 (LINE-1) and LUminometric Methylation Assay (LUMA) were used to quantify genome-wide methylation in 21 FTC and corresponding normal thyroid tissues. Unexpectedly global methylation was not found significantly altered in tumors compared to normal thyroid by either LINE-1 (p=0.57) or LUMA (p=0.42), whilst the promoter of a tumor suppressor that is often epigenetically dysregulated, RASSF1A was found to be significantly hypermethylated by Pyrosequencing (p=0.0001). Moreover, allelic imbalance at the RASSF1A locus was observed in 15/21 of the tumors. mRNA expression of RASSF1A was significantly lower in tumors compared to corresponding normal tissues (p=0.0002). In summary, the epigenetic inactivation of RASSF1A is a frequent event in FTC, but is not coupled to changes in global methylation.     

Spontaneous complete remission in acute lymphoblastic leukaemia

Case records of 78 patients of acute lymphoblastic leukemia have been reviewed. Complete remission occurred in seven cases following an episode of septicemia and supportive care.     

Bone-marrow relapse in paediatric acute lymphoblastic leukaemia

Marrow relapse is the major obstacle to cure for 10-15% of young patients with acute lymphoblastic leukaemia (ALL). Recent investigations into the biology of minimal residual disease indicate that many early relapses derive from residual cells present at first diagnosis, but some late relapses might represent new mutations in leukaemic cells not eliminated by conventional therapy. Treatment of marrow relapse involves higher doses and more intensive schedules of the drugs used for initial therapy with or without haemopoietic stem cell transplantation. In most reports, transplantation is better than continuation chemotherapy in early marrow relapse, but its role in later relapse is less clear. Current therapy cures 10% of patients with early marrow relapses and 50% of those with late relapses, but outcomes have changed little in the past two decades. Understanding the molecular biology of ALL underlies development of improved risk stratification and new therapies. Although better drugs are needed, introduction of new agents into clinical trials in paediatric disease has been difficult. Innovative trial designs and use of valid surrogate endpoints may expedite this process.     

Meningeosis leukaemica in adult acute lymphoblastic leukaemia

This review addresses diagnosis of CNS involvement, incidence and treatment of CNS disease at time of diagnosis, prophylaxis and treatment of CNS relapse and risk factors for meningeal recurrence in adult acute lymphoblastic leukaemia (ALL).At the time of diagnosis meningeosis leukaemica is present in about 6% (1–10%) of the adult ALL patients with a higher incidence in ALL subgroups T-ALL (8%) and B-ALL (13%). With the invention of early additional CNS directed therapy it no longer represents an unfavourable prognostic factor.In the absence of prophylaxis meningeal relapses occur in approximately one third of adults with ALL. A literature review including more than 4000 adult ALL patients showed for the different prophylactic treatment approaches the following CNS relapse rates: intrathecal therapy alone 13% (8–19%), intrathecal therapy and CNS irradiation 15% (6–22%), high dose chemotherapy 14% (10–16%), high dose chemotherapy and intrathecal therapy 8% (2–16%) and high dose chemotherapy, intrathecal therapy together with CNS irradiation 5% (1–12%). It became obvious that the early onset of intrathecal therapy and CNS irradiation and the continuation of intrathecal administrations throughout maintenance are essential. The most favourable results where achieved with high dose chemotherapy combined with intrathecal therapy and/or CNS irradiation. The majority of treatment regimens in adult ALL already include high dose chemotherapy in order to reduce the risk of bone marrow relapse.The outcome of patients with CNS relapse is still poor. Although a remission can be induced in the majority of patients (> 60%) it is usually followed by a bone marrow relapse and the survival is poor (< 5–10%). Bone marrow transplantation might be in adults at present the only curative approach.     

Effect of Aplidin in acute lymphoblastic leukaemia cells

The cytotoxic effect of Aplidin was investigated on fresh leukaemia cells derived from children with B-cell-precursor (BCP) acute lymphoblastic leukaemia (ALL) by using stromal-layer culture system and on four cell lines, ALL-PO, Reh, ALL/MIK and TOM-1, derived from patients with ALL with different molecular genetic abnormalities. In ALL cell lines Aplidin was cytotoxic at nanomolar concentrations. In the ALL cell lines the drug-induced cell death was clearly related to the induction of apoptosis and appeared to be p53-independent. Only in ALL-PO 20 nM Aplidin treatment caused a block of vascular endothelial growth factor (VEGF) secretion and downregulation of VEGF-mRNA, but Aplidin cytotoxicity does not seem to be related to VEGF inhibition since the sensitivity of ALL-PO cells to Aplidin is comparable to that observed for the other cells used. Aplidin induced a G(1) and a G(2) M block in ALL cell lines. In patient-derived leukaemia cells, Aplidin induced a strong cytotoxicity evidenced in a stroma-supported immunocytometric assay. Cells from children with genetic abnormalities such as t(9;22) and t(4;11) translocations, associated with an inferior treatment outcome, were sensitive to Aplidin to the same extent as that observed in other BCP-ALL cases. Aplidin exerted a strong cell killing effect (>88%) against primary culture cells from five relapsed ALL cases, at concentrations much lower than those reported to be achieved in plasma of patients receiving Aplidin at recommended doses. Taken together these data suggest that Aplidin could be a new anticancer drug to be investigated in ALL patients resistant to available therapy.     

Granular cell variant of acute lymphoblastic leukaemia

A rare morphological variant of acute lymphoblastic leukaemia in a ten years old child is described. Ninety five percent of the lymphoblasts had significant number of course granules in the cytoplasm. Diagnostic and prognostic significance of this variant is discussed.     

Immunological phenotypic pattern of acute lymphoblastic leukaemia in Egypt

We have performed immunophenotyping studies on 186 untreated cases of acute lymphoblastic leukemia (ALL) in an Egyptian population, using panels of monoclonal antibodies (mAb) and an avidin-biotin-immunoperoxidase detection system. Sixty-two of these cases were tested with a panel of mAb directed against the T-cell markers CD2, CD4, CD8, B-cell markers CD20, kappa and lambda, the common ALL antigen (common ALLa) and class II HLA antigens. The remaining 124 cases were also tested with additional markers of T- and B-cell precursors, namely CD7 and CD19. The common leukocyte antigen, T200, was used to exclude nonhemopoietic neoplasms. Cases that remained unclassifiable were further tested with a wider panel of T-cell markers, including CD1, CD2, CD3 and CD5. In some cases multiple mAb directed against the same antigens were used. The relative frequencies of common ALL and B ALL were calculated from the total number of cases and were found to be 39.2% and 3.2%, respectively. The proportions of T-cell and null leukemias were calculated from the better characterized subgroup of 124 cases, and were found to be 50% and 4.8%, respectively. In our series, the age distribution of common ALL revealed a peak at 2-5 yr, but this was partially obscured in the entire series by the high proportion of T-cell cases, which had an age peak between 4 and 12 yr of age. Our results demonstrate marked differences in the phenotypic pattern of ALL in Egypt compared to Western Countries, the predominant finding being a relative excess of T-cell ALL and a paucity of common ALL cases. At present it is not clear whether this results from an increased incidence of T-cell ALL or a decreased incidence of common ALL.