A. Ganser

Individual Patient Data–Based Meta-Analysis of Patients Aged 16 to 60 Years With Core Binding Factor Acute Myeloid Leukemia: A Survey of the German Acute Myeloid Leukemia Intergroup

PURPOSE To evaluate prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in adult patients with core binding factor (CBF) acute myeloid leukemias (AML). PATIENTS AND METHODS Individual patient data-based meta-analysis was performed on 392 adults (median age, 42 years; range, 16 to 60 years) with CBF AML (t(8;21), n = 191; inv(16), n = 201) treated between 1993 and 2002 in prospective German AML treatment trials. RESULTS RFS was 60% and 58% and OS was 65% and 74% in the t(8;21) and inv(16) groups after 3 years, respectively. For postremission therapy, intention-to-treat analysis revealed no difference between intensive chemotherapy and autologous transplantation in the t(8;21) group and between chemotherapy, autologous, and allogeneic transplantation in the inv(16) group. In the t(8;21) group, significant prognostic variables for longer RFS and OS were lower WBC and higher platelet counts; loss of the Y chromosome in male patients was prognostic for shorter OS. In the inv(16) group, trisomy 22 was a significant prognostic variable for longer RFS. For patients who experienced relapse, second complete remission rate was significantly lower in patients with t(8;21), resulting in a significantly inferior survival duration after relapse compared with patients with inv(16). CONCLUSION We provide novel prognostic factors for CBF AML and show that patients with t(8;21) who experience relapse have an inferior survival duration.

Treatment of Adult ALL According to Protocols of the German Multicenter Study Group for Adult ALL (GMALL)

The German Multicenter Study Group for Adult Acute Lymphoblastic leukemia (GMALL) has conducted 5 consecutive trials with more than 3000 patients since 1981. This article provides an overview on aims, treatment concepts, and results of these studies. It includes brief summaries on the development of prognostic models within the GMALL group and on approaches for prophylaxis of CNS relapse, and it summarizes specific treatment concepts for mature B-lineage acute lymphocytic leukemia.

Aggravation of vincristine-induced neurotoxicity by itraconazole in the treatment of adult ALL

In four of 14 patients with acute lymphoblastic leukemia (ALL) who received induction chemotherapy containing weekly injections of vincristine and simultaneous antifungal prophylaxis with itraconazole, unusually severe and early vincristine-induced neurotoxicity was observed. In these patients (three female, one male) paresthesia and muscle weakness of the upper/lower extremities and paralytic ileus occurred after the first or second vincristine injection. In one patient a laryngeal nerve paresis required mechanical ventilation. The neurotoxic complications were more serious than those seen in a previous series of 460 ALL patients under the identical cytostatic regimen but without itraconazole prophylaxis. The underlying mechanism is unclear. Interaction with the cytochrome P-450 system, reversal of multidrug resistance, and influence of estrogens are to be considered.

Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation

To analyze the value of real time RT-PCR for monitoring of bcr-abl expression in CML patients after allogeneic or autologous stem cell transplantation (SCT), we generated pairs of PCR-primers and TaqMan probes specific for either the b2a2- or the b3a2-variant of bcr-abl. Either variant could be detected specifically from cDNA from a single K562 (b3a2) and BV173 (b2a2) cell with the respective TaqMan probe. Bcr-abl expression was normalized by comparison with GAPDH expression, and samples were quantitated using standard cDNA dilutions from K562 or BV173 cells. In a retrospective analysis 13 patients with CML after allogeneic (n = 10) or autologous (n = 3) SCT including patients with relapsed or persistent CML were analyzed by both real-time and conventional nested RT-PCR. In addition chimerism was monitored by FISH analysis of sex chromosomes in three patients with relapsed disease. The bcr-abl/GAPDH ratio dropped at least 1000-fold in all seven patients evaluable prior to and after allogeneic SCT as estimated by real-time RT-PCR, and conventional RT-PCR became negative in 6/7 patients. In five patients with relapsed or persistent disease after allogeneic SCT the bcr-abl/GAPDH ratio eventually increased again, and real-time RT-PCR was as sensitive as conventional RT-PCR for detection of bcr-abl. Donor lymphocyte infusions (DLI) were given to all five patients, and the bcr-abl/GAPDH ratio dropped to undetectable levels in two patients both remaining in continuing molecular remission. In contrast, in three other patients the bcr-abl/GAPDH ratio decreased only or did not change significantly after DLI. In three patients undergoing autologous SCT the bcr-abl/GAPDH ratio dropped only 1.1 to 30-fold, and the patients were tested positive with real-time RT-PCR at all time points. These data demonstrate that real-time RT-PCR is valuable to quantitate bcr-abl expression in CML patients after transplantation.

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages

Safety and toxicity of amphotericin B in glucose 5% or intralipid 20% in neutropenic patients with pneumonia or fever of unknown origin: randomised study

Abstract Objective : To compare the feasibility of treatment, safety, and toxicity of intravenous amphotericin B deoxycholate prepared in either glucose or intralipid for empirical antimycotic treatment of neutropenic cancer patients. Design : Single centre stratified, randomised non-blinded phase II study. Setting : University hospital providing tertiary clinical care. Subjects : 51 neutropenic patients (leukaemia (35), lymphoma (11), solid tumours (5)) with refractory fever of unknown origin (24) or pneumonia (27). Interventions : Amphotericin B 0.75 mg/kg/day in 250 ml glucose 5% solution or mixed with 250 ml intralipid 20%, given on eight consecutive days then alternate days, as a 1-4 hour infusion. Main outcome measures : Feasibility of treatment, subjective tolerance (questionnaire), and objective toxicity (common toxicity criteria of the National Cancer Institute). Results : Study arms were balanced for age, sex, underlying malignancy, renal and liver function, and pre- and concomitant treatment with antibiotics and nephrotoxic agents. No statistically significant or clinically relevant differences were found between the treatment groups for: daily or cumulative dose and duration of treatment with amphotericin B; incidence and time of dose modifications or infusion duration changes related to toxicity; dose or duration of symptomatic support with opiates, antipyretics, or antihistamines; renal function; subjective tolerance; most common toxicity scores; course of infection; and incidence of treatment failures. Patients treated with amphotericin B in intralipid were given fewer diuretics (P<0.05) and therefore had more peripheral oedema (P<0.01) and needed less potassium supplementation (P<0.05) than patients given amphotericin in glucose. Acute respiratory events were more common in the intralipid arm (P<0.05). Conclusions : Amphotericin B 0.75 mg/kg/day in intralipid given on eight consecutive days then alternate days provides no benefit and is associated with potential pulmonary side effects possibly because of fat overload or an incompatibility of the two drugs.

Biologic effects of recombinant human interleukin-3 in vivo.

The biologic in vivo effects of recombinant human interleukin-3 (rhIL-3) were assessed in a phase I clinical study of 30 patients with advanced malignancy. On day 1 rhIL-3 was administered by a single intravenous (IV) bolus injection, followed by subcutaneous (SC) injections once daily from day 2 to 15; at least three patients were treated at each dose level (60, 125, 250, and 500 micrograms/m2). A transient decrease of eosinophil and monocyte counts was observed immediately after IV injection of rhIL-3, whereas the neutrophil count remained unaffected. Total WBC counts and neutrophil counts increased dose dependently up to threefold, whereas a 10-fold to 50-fold rise was observed in levels of circulating eosinophils and basophils. Platelet counts increased up to twofold. Patients developed moderate increases of serum levels of soluble interleukin-2 receptors, beta 2-microglobin, and immunoglobulin M (IgM), and of the acute phase reactants, C-reactive protein (CRP), fibrinogen, and haptoglobin. An increase in interleukin-6 (IL-6) serum levels was detected in patients treated by IV bolus rhIL-3. The serum half-life of IV injected rhIL-3 was 20 +/- 3 minutes; after SC administration, 210 +/- 15 minutes. Administration of rhIL-3 was generally well tolerated, with mild fever, headache, and local reactions at the injection site being the most frequent side effects. The primary course of the underlying malignant diseases was not significantly altered by administration of rhIL-3. The results indicate that rhIL-3 acts in vivo as a multilineage hematopoietic growth factor and a weak inflammatory mediator that may be used successfully to improve states of hematopoietic failure.

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo OLIGORIBONUCLEOTIDES AS THERAPEUTIC AGENTS

Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2′-modified to protect them against degradation by nucleases. 2′-Fluoro-2′-deoxyuridine/cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.

Improved multilineage response of hematopoiesis in patients with myelodysplastic syndromes to a combination therapy with all-trans-retinoic acid, granulocyte colony-stimulating factor, erythropoietin and α-tocopherol

Abstract Differentiation induction therapy is being tested in myelodysplastic syndromes to ameliorate maturation defects and to restore normal hematopoietic function. To this end, 17 patients (eight with refractory anemia, two with refractory anemia and ring sideroblasts, and seven with refractory anemia and excess of blast cells) were treated with a combination of all-trans-retinoic acid (ATRA), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and α-tocopherol for durations of 8–16 weeks. Absolute neutrophil counts increased in all patients; platelet counts increased in five patients with discontinuation of transfusion needs in two of four transfusion-dependent patients. Stimulation of erythropoiesis was seen in eight patients with an increase in hemoglobin concentration in three, a discontinuation of transfusion requirements in another three, and a significant increase in reticulocyte counts as the only parameter in two patients. Clinically important multilineage responses with increases of hemoglobin levels or discontinuation of transfusion needs were thus seen in six patients (35.3%) with three patients having a trilineage response. Serum erythropoietin concentrations did not differ significantly between responders and nonresponders, but the erythroid response was accompanied by a rise in the serum transferrin receptor levels. In the bone marrow, the myeloid-to-erythroid ratio and the maturation index of myeloid cells increased during therapy, while the percentage of blast cells did not change. Cytogenetic analysis demonstrated the persistence of the abnormal clones. Prior to therapy, nonresponders had a significantly higher serum TNF level than responders. Serum concentrations of TNF-α and soluble TNF-α receptor significantly increased during therapy, but mainly in the patients without an erythroid and platelet response. Soluble IL-2 receptor and soluble ICAM-1 concentrations both increased. This pilot study demonstrates that treatment with ATRA/G-CSF/EPO/tocopherol is well tolerated, leading to normalization of neutrophil counts in most, and to improvement of platelets and red blood cells in a significant subgroup of patients.

“Atypical” Leukemias: Preleukemia, Smoldering Leukemia and Hypoplastic Leukemia

The term “atypical leukemia” covers a wide range of heterogeneous disorders whose classification has hitherto been controversial. This controversy has arisen from the use of different criteria for the definition of the various entities. The application of morphological criteria has led to the separation of conditions without recognizable leukemic blasts, e.g., refractory anemia [11] and refractory anemia with ring sideroblasts [11], from subentities with a definite leukemic population, for which the terms refractory anemia with excess of blasts (RAEB) [38], oligoblastic leukemia [81], and hypoplastic acute leukemia [10, 78, 132] are used. Laying the emphasis on the disease stage at clinical presentation has allowed the differentiation of preleukemic states in which the diagnosis of leukemia has not yet been possible because of the lack of a definite leukemic cell population from overt acute leukemia [16,17, 62,114, 206]. Observation of the disease course has allowed separation of a further group of patients with a slowly progressing course, in whom the terms “subacute leukemia” [30] and “smoldering leukemia” [44, 74, 75, 83, 194] have been applied. The combination of morphological and anamnestic criteria, finally, has led to the definition of “subacute” and “chronic myelomonocytic leukemia” [11,210].