Miloslav Beran

Molecular characteristics of chronic myelogenous leukemia in blast crisis

We have studied the expression of c-abl and c-myc in leukemic cells of patients in all clinical phases of chronic myelogenous leukemia. We demonstrate that an aberrant 8-Kb c-abl related transcript is present in the RNA of the leukemic cell from all patients with Ph+ CML and that the loss of both normal chromosome #9 is associated with the loss of the normal c-abl related transcripts. This represents direct evidence that the normal c-abl related transcripts derive from the normal c-abl gene locus on the normal chromosome #9, while the aberrant c-abl related transcript in Ph+ CML derives from the hybrid bcr-abl gene formed as a result of the t(9;22). We further demonstrate that trisomy 8 in some instances is associated with enhanced expression of the c-myc oncogene.

Combination of topotecan with cytarabine or etoposide in patients with refractory or relapsed acute myeloid leukemia: Results of a randomized phase I/II study

The topoisomerase I inhibitor topotecan has shown activity in acute myeloid leukemia (AML) and myelodysplastic syndromes. The present study was designed to assess whether topotecan with cytosine arabinoside (ara-C) or with etoposide (VP-16) should be studied in phase II trials in patients with refractory or relapsed AML.Patients with refractory or relapsed AML were assigned to one of 3 strata defined by expected CR rates of 7%, 20% and 40%, then randomly assigned to receive topotecan (d1-5) and ara-C (1 g/m2 over 2 hours; d1-5), topotecan (d1-5) followed by VP-16 (250 mg/m2 twice daily, d6-7), or VP-16 (250 mg/m2 twice daily d1-2) followed by topotecan (d3–d7). A dose-finding phase was conducted in the poorest stratum of each arm (topotecan starting dose: 1.0 mg/m2/day × 5). A Bayesian pre-phase II selection design was used to assess whether the CR rate with a given arm was sufficient to merit investigation in phase II.Thirty-seven patients, median age 58 years, were treated. Their median first CR duration was 28 weeks and 24% were primary refractory. Grade 3–4 mucositis occurred in the initial patients in the topotecan → VP-16, but not in the topotecan + ara-C or VP-16 → topotecan arms. Consequently, in subsequent patients, the topotecan dose was lower in the topotecan → VP-16 than in the other 2 arms (1.0 vs 1.25 mg/m2 daily × 5) and the VP-16 dose was lower in the topotecan → VP-16 arm (200 vs 250 mg/m2 twice daily × 2). One CR occurred (topotecan → VP-16 arm), and the treatment arms were terminated after 10, 15, and 12 patients were treated on the topotecan + ara-C, topotecan → VP-16, and VP-16 → topotecan arms, respectively. The principal cause of failure was insufficient anti-leukemia effect rather than death on study, and toxicity was minimal at the final doses used.We concluded that none of the combinations studied here warrants phase II evaluation in very poor prognosis AML salvage patients.

The addition of all-trans retinoic acid to chemotherapy may not improve the outcome of patient with NPM1 mutated acute myeloid leukemia

Background: Previous studies have suggested that NPM1 mutations may be a marker for response to all-trans retinoic acid (ATRA) given as an adjunct to intensive chemotherapy in older patients with acute myeloid leukemia (AML). Patients and Methods: We examined the impact of the addition of ATRA among patients with diploid cytogenetics treated on a randomized phase II study of fludarabineu2009+u2009cytarabineu2009+u2009idarubicineu2009±u2009G-CSFu2009±u2009ATRA with available data on their NPM1 mutation status. Between September 1995 and November 1997, 215 patients were enrolled in the study. Among them, 70 patients had diploid cytogenetic and are the subjects of this analysis. Results: The median age of the 70 patients was 66u2009years (range 23–87). Twenty (29%) of patients had NPM1 mutations. Among them 7 (35%) did and 13 (65%) did not receive ATRA in combination with chemotherapy. Complete remission (CR) was achieved in 71% of patients treated with ATRA as compared to 69% without ATRA (Pu2009=u20090.62). With median follow-up of 12.5u2009years, the overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were similar among patients who received ATRA compared to no ATRA regardless of NPM1 mutation status. Conclusion: The addition of ATRA to intensive chemotherapy did not affect the overall outcome of patients with AML regardless of NPM1 mutation status.

Critical issues in autologous bone marrow transplantation in adult acute leukemia

Background Adult acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignancies with great primary sensitivity to chemotherapeutic agents. In AML, a complete remission rate of 70% to 80% can be obtained (1-4); however, as with ALL, most patients usually die of recurrent disease within 1 yr of initial chemotherapy. The long-term survival of AML patients who achieve complete remission (CR) is about 20% at 5 yr (5-6). The addition of newer drugs such as 4’(9-acridinylaminomethanesulfon-manisidide) (mAMSA), etoposide (VP-16), and 5-azacytidine to the treatment regimens and the more effective use of older agents, e.g. high-dose cytosine arabinoside (Ara-C), have yielded only marginal improvements. Aggressive treatment approaches after induction therapy in the form of consolidation and early or late intensification of treatment have yet to demonstrate major survival improvements. Although recent studies suggest that early intensification of treatment in remission improves survival (7-9), late intensification has prolonged median remission duration but has not influenced the percentage of patients cured (1011). Because of logistics, late intensification selects patients who are in a continuous complete remission for a prolonged time period and therefore have a greater chance of being cured. Most studies evaluating the role of maintenance chemotherapy over an extended period of time indicate that, at best, it has a positive effect on remission duration but so far it shows no convincing effect on the survival and cure rate (12-18). The long-term outcome of adult ALL has also been dismal, though in most recent studies it appears somewhat better than that of AML. Treatment strategies employing intensive induction chemotherapy with cycling maintenance therapy and alternate regimens of aggressive chemotherapy given over prolonged periods of time have yielded long-term disease-free survival of close to 40% (19, 20). The emergence of therapy-resistant leukemic cell populations, which results in relapse and disease that does not respond to therapy, is most often blamed for therapy failure. Studies with hematological animal tumors treated with radiation and certain drugs have shown that a definite and often steep dose-response curve exists (21-23). Recognition of these phenomena led to the application of high-dose chemoand radiotherapy. Resistance can be overcome by escalation of the dose of chemotherapy alone (24) or in conjunction with supralethal total body irradia-

Analysis of nuclear m-AMSA content by DNA fluorochrome competition.

Spectrofluorometry and flow cytometry were used to measure cellular uptake of the anti-leukemic drug 4-(9-acridinylamino) methane sulfon-m-anisidide (m-AMSA). Because of its very low intrinsic fluorescence, we used m-AMSA to quench DNA-fluorescence induced by the vital DNA fluorochrome Hoechst 33342. Maximum fluorescence was obtained with cells incubated in the fluorochrome alone. Subsequent incubation of cells in increasing concentrations of m-AMSA resulted in a gradual decrease in fluorescence. Upon incubation in drug-free medium, the quenching phenomenon was reversible, consistent with rapid exit of m-AMSA from the cells. The novel competitive fluorescence assay for cellular uptake for m-AMSA showed a better correlation to nuclear accumulation of the drug, than to its overall cellular accumulation, which may be important in assessment of cellular resistance to m-AMSA, with possible low nuclear accumulation of the drug. When this competitive fluorescence technique for measurement of cellular m-AMSA concentration was applied in flow-cytometric setting, subpopulations of normal human white blood cells were detected with distinctly different fluorescence patterns, indicating differences in cellular m-AMSA uptake. The potential use of this technique is to detect differences between cell subpopulations with different drug uptake abilities.