Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3fl mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3fl mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3fl deletion does not cooperate with oncogenic KrasG12D expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

Ex Vivo Simulation of the Action of Antileukemia Drugs by Measuring Apoptosis-Related mRNA in Blood

Background: In conventional bioassays, isolated cells are suspended in culture media, incubated in vitro for several days, and then characterized with respect to any cellular changes. In developing new molecular tests under physiological ex vivo conditions, we quantified the production of mRNAs for p21 and PUMA (p53 up-regulated modulator of apoptosis), which are involved in cell cycle arrest and apoptosis, respectively. Methods: We stimulated human whole blood with a chemotherapeutic drug (cytarabine, daunorubicin, mitoxantrone, aclarubicin, etoposide, or idarubicin) for 4 h and then quantified mRNA by assessing mRNA recovery and cDNA-synthesis efficiency in each sample. We also used immunoassay and flow cytometry to investigate nucleosome and annexin V, respectively, as apoptosis markers. Results: Ex vivo mRNA analysis yielded more positive results than nucleosome and annexin V analyses. The concentrations of cytarabine- and daunorubicin-induced p21 and PUMA mRNAs were significantly lower in acute myelogenous leukemia (AML) patients than in healthy controls (P <0.0001), whereas idarubicin induced significantly greater responses in AML patients than in controls (P = 0.01). The patients had different mRNA-response patterns, which were largely classifiable into 4 groups. Prednisone enhanced cytarabine or mitoxantrone induction of p21 and PUMA mRNAs in 3 (2.6%) of 114 reactions. All 15 patients who achieved complete remission had received at least one drug that produced positive mRNA responses, whereas we observed a lack of mRNA response to the clinically used drugs in all 3 cases in which the therapy failed to induce any hematologic improvement. Conclusion: This study introduced ex vivo mRNA analysis as a candidate platform for drug-sensitivity tests in leukemia.

Prevention and treatment of meningeal leukemia in children

The prevention of meningeal leukemia has long been a keystone in its cure. The need was recognized when it became apparent in the 1950s and 1960s that meningeal relapse heralded hematologic relapse and a fatal course and that its incidence increased as systemic chemotherapy became more effective in controlling hematologic and visceral leukemia. Evasion of a biologic safety net, the blood-CSF barrier, is required to prevent meningeal leukemia. Three methods are used: meningeal radiotherapy, intrathecal administration of antileukemia drugs, and high-dosage intravenous antileukemia drugs. Recent and current clinical studies reflect a continuing dialogue about which methods are preferable and under what circumstances. For prevention of meningeal leukemia, extended intrathecal therapy and intensive systemic chemotherapy appear to be as effective as radiotherapy for most patients. For treatment of overt meningeal leukemia, meningeal radiotherapy may be necessary. However, its administration compromises subsequent systemic chemotherapy so that delay may be advisable to allow intensive systemic chemotherapy for control of concurrent hematologic and visceral leukemia, whether clinically evident or not. For patients with meningeal leukemia at diagnosis, cranial irradiation may be delayed or possibly omitted if evidence of disease is minimal and intrathecal and systemic chemotherapy are intensive. For those who develop meningeal leukemia while on therapy or after its completion, cranial or craniospinal irradiation is probably required as well as intensive intrathecal and systemic chemotherapy. Hopefully, current and future studies will dispel the uncertainties and better quantitate risks and benefits of alternative methods. Whatever method is used, careful attention to technical details is required to assure optimal efficacy at the least possible expense in immediate toxicity and late sequelae.

Prevention and treatment of meningeal leukemia in children

The prevention of meningeal leukemia has long been a keystone in its cure. The need was recognized when it became apparent in the 1950s and 1960s that meningeal relapse heralded hematologic relapse and a fatal course and that its incidence increased as systemic chemotherapy became more effective in controlling hematologic and visceral leukemia. Evasion of a biologic safety net, the blood-CSF barrier, is required to prevent meningeal leukemia. Three methods are used: meningeal radiotherapy, intrathecal administration of antileukemia drugs, and high-dosage intravenous antileukemia drugs. Recent and current clinical studies reflect a continuing dialogue about which methods are preferable and under what circumstances. For prevention of meningeal leukemia, extended intrathecal therapy and intensive systemic chemotherapy appear to be as effective as radiotherapy for most patients. For treatment of overt meningeal leukemia, meningeal radiotherapy may be necessary. However, its administration compromises subsequent systemic chemotherapy so that delay may be advisable to allow intensive systemic chemotherapy for control of concurrent hematologic and visceral leukemia, whether clinically evident or not. For patients with meningeal leukemia at diagnosis, cranial irradiation may be delayed or possibly omitted if evidence of disease is minimal and intrathecal and systemic chemotherapy are intensive. For those who develop meningeal leukemia while on therapy or after its completion, cranial or craniospinal irradiation is probably required as well as intensive intrathecal and systemic chemotherapy. Hopefully, current and future studies will dispel the uncertainties and better quantitate risks and benefits of alternative methods. Whatever method is used, careful attention to technical details is required to assure optimal efficacy at the least possible expense in immediate toxicity and late sequelae.

Intravenous mercaptopurine: life begins at 40.

PURPOSE This review is intended to provide a concise portrayal of the background, rationale, and current use of intravenous infusions of mercaptopurine (6MP) in patients with acute leukemia. DESIGN After a brief description of the mode of action of 6MP and the formulation, pharmacokinetics, and history of its intravenous administration, the rationale for current use of intravenous 6MP infusion is explained. Subsequently, the review summarizes and discusses clinical experience with intravenous 6MP alone and in combination with intravenous methotrexate (Mtx) and cytarabine (Ara-C). RESULTS Although still an investigative drug, intravenous 6MP has been used for 40 years and currently is being administered extensively to children with previously untreated acute lymphoid leukemia (ALL) in frontline protocol studies. The reasons are the better and more consistent bioavailability of intravenous versus oral MP, higher blood and CSF levels, compliance, and preliminary evidence suggesting superior remission experience for intravenous Mtx and 6MP than for Mtx alone. The apparent lack of late adverse sequelae with 6MP as compared with other antileukemia drugs adds to this interest. CONCLUSION The new life of intravenous 6MP at age 40 years illustrates the need for continued investigation of significant anticancer drugs as insights and technology progress.

Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia during therapy.

PURPOSE A protocol was designed to test the hypothesis that fludarabine infusion before arabinosylcytosine (cytarabine [ara-C]) would increase the accumulation of the active metabolite ara-C triphosphate (ara-CTP) in acute myelogenous leukemia (AML) blasts during therapy. PATIENTS AND METHODS Patients (n = 5) received 1 g/m2 of ara-C infused intravenously (IV) for 2 hours, followed at 20 hours by 30 mg/m2 of fludarabine for 30 minutes. At 24 hours, another identical dose of ara-C was infused. To determine the optimal duration of ara-C infusion following fludarabine, five additional patients were treated on an amended protocol in which the ara-C infusion was extended to 3 g/m2 infused over 6 hours. RESULTS Comparison of ara-CTP pharmacokinetics in circulating AML cells demonstrated that the area under the curve (AUC) of ara-CTP increased significantly (median, 1.8-fold; range, 1.6 to 2.4; P = .004) after fludarabine infusion. Neither the median plasma ara-C concentrations, the levels of its deamination product arabinosyluracil, nor the rate of ara-CTP elimination from circulating blasts was affected by fludarabine infusion. However, the rate of ara-CTP accumulation by AML cells was increased by a median of 2.0-fold (range, 1.8 to 2.2; P = .001) after fludarabine; the peak occurred within 1 hour of the end of the infusion. In vitro incubation of these cells with arabinosyl-2-fluoroadenine (F-ara-A) before ara-C also produced a median 1.7-fold increase in the ara-CTP accumulation rate. Pharmacology studies in patients receiving 6-hour infusions of ara-C demonstrated that the rate of ara-CTP accumulation was potentiated beyond 2 hours, but not for 6 hours. CONCLUSION Infusion of fludarabine before ara-C augments the rate of ara-CTP synthesis in circulating AML blasts during therapy. Evaluation of 6-hour ara-C infusions demonstrated that potentiation of ara-CTP synthesis is maximal up to 4 hours in most patients; this pharmacologically optimized regimen should be considered for combination with other antileukemia drugs.