The value of interphase fluorescence in situ hybridization for the detection of translocation t(12;21) in childhood acute lymphoblastic leukemia.

Translocation t(12;21)(p13;q22) is the most frequent cytogenetic abnormality in childhood acute lymphoblastic leukemia (ALL) and is generally associated with favorable prognosis. In this report, we assessed the value of dual-color interphase fluorescence in situ hybridization (FISH) for the detection of t(12;21). Fifty-three patients were screened for ETV6/CBFA2 fusion by means of FISH, using two cosmid probes mapped on ETV6 and on CBFA2, respectively. The cut-off value (mean + three standard deviations) for positivity established on control patients was 9.3%. A comparison between FISH and molecular methods [reverse-transcriptase polymerase chain reaction/Southern blot (RT-PCR/SB)] was possible in 52 patients: 34 of 52 (65.4%) showed negative results with both approaches, and 13 of 52 (25%) were positive; 5 of 52 (9.6%) showed discrepancies: four patients who were positive using RT-PCR/SB were negative using FISH. Conversely, one patient negative when using RT-PCR/SB was positive with FISH. Further investigations on this patients, cytogenetically characterized by add(12p), showed an atypical breakpoint on ETV6, located 5' to the common breakpoint. Compared with RT-PCR and SB, dual-color interphase FISH with the cosmid probe set proved to be highly specific but showed limited sensitivity.

Haematopoietic stem cell transplantation for sickle cell anaemia: the first 50 patients transplanted in Belgium.

Fifty patients affected by sickle cell anaemia underwent transplantation of HLA-identical haematopoietic stem cells (bone marrow, 48; cord blood, 2). Two groups of patients were considered for transplantation. Group 1 included 36 permanent residents of a European country who, retrospectively, met the inclusion criteria accepted at a consensus conference held in Seattle in 1990, wherein children were selected because they already had evidence of a morbid course. Group 2 included 14 patients who were transplanted earlier, had not received more than three blood transfusions and were transplanted because they had decided to return to their country of origin. Kaplan-Meier estimates of overall survival, event-free survival and disease-free survival at 11 years of the whole grafted population are 93, 82 and 85%, respectively. In group 1, overall survival, EFS and DFS were 88, 76 and 80% and in group 2, 100, 93 and 93%, respectively. Clinical manifestations of the disease, as well as disease associated haemolytic anaemia, disappeared in all successfully treated patients. Recovery of spleen function was present in seven out of 10 evaluated patients. Adverse events (death, absence of engraftment, mixed chimerism and relapse) occurred more frequently in group 1 than in group 2 (25% vs 7%, P< 0.001). Acute graft-versus-host disease (GVHD) was present in 20 patients (grade I or II, 19; grade III, 1), chronic GVHD in 10 (limited, 7; extensive, 3). One patient developed an acute myeloid leukaemia. Gonadal dysfunction was present in all patients (six boys and eight girls) transplanted close to or after puberty, although transient in one adolescent girl.

2-Chlorodeoxyadenosine with or without daunorubicin in relapsed or refractory acute myeloid leukemia.

2-Chlorodeoxyadenosine (2-CdA) is a purine analogue which has proved to be active in acute myeloid leukemia (AML), especially in children. In adults, results yielded by 2-CdA alone or with ara-C were less encouraging. Here we report on the efficacy of 2-CdA with or without daunorubicin (DNR) in 19 relapsing or refractory adult AML patients, with a median age of 57 years. 2-CdA was administered as a continuous infusion to all patients at a dose of 0.1 mg/kg per day for 7 days. For 14 patients, DNR was added at a dose of 50 mg/m2 per day on days 5, 6, and 7. Antileukemic activity was observed in all the patients, but no single complete remission was achieved. One patient had a long-lasting partial response (response rate=5%). The remaining patients died of progressive AML (n=7), uncontrollable infection with persistent disease (n=10), and cerebral hemorrhage (n=1). Median survival from start of 2-CdA therapy was 56 days. Long-lasting neutropenia and transfusion-dependent thrombopenia were encountered in all 16 evaluable patients. Grade 4 hepatic toxicity occurred in one patient. Other side effects included nausea in six, mucositis in three, and mental disturbances in three patients. Compared with 2-CdA alone, the addition of DNR to 2-CdA changed neither the response rate nor the toxicities. In conclusion, our data do not support the use of 2-CdA +/- DNR for relapsing or refractory adult AML patients, at least as used in the present regimen.

A prospective study of minimal residual disease in childhood B-lineage acute lymphoblastic leukaemia: MRD level at the end of induction is a strong predictive factor of relapse.

We prospectively investigated minimal residual disease (MRD) in 51 children with B-lineage acute lymphoblastic leukaemia (ALL) treated according to the Fralle 93 protocol. PCR follow-up was performed in children in morphological and cytogenetic complete remission, provided an immunoglobulin (IgH) gene rearrangement could be detected using FR 3/J(H) amplimers. MRD was studied according to our previously described methodology, with a few modifications including the use of a consensus J(H) probe to control for PCR efficiency variations. Out of the initial 51 patients, 34 were assessable for MRD at the end of induction at the time of analysis. MRD levels were as follows: > 1/10(3) in 26%, 1/10(3) to 1/10(4) in 50% and < 1/10(4) or not detectable in 24%. With a median follow-up of 20 months there were five relapses, all of which occurred in the group of patients with MRD > 1/10(3). To date, none of the patients with MRD < or = 1/10(3) (good molecular responder) has relapsed. Classification according to molecular response at the end of induction did not correlate with the conventional risks groups: there were no statistically significant differences between good and bad molecular responders. Of particular interest is the absence of correlation between WBC at diagnosis and MRD level at the end of induction. We conclude that classification of patients into good and bad molecular responders using PCR seems to be a better prognostic indicator than conventional risk factors in childhood B-lineage ALL. Patients with MRD level > 1/10(3) have a particularly poor outcome and should always be considered for alternative therapeutic strategies in the future, whereas in good molecular responders belonging to poor or intermediate risk categories, treatment de-escalation might be contemplated.

Long-term culture of autologous transplanted bone marrow for acute myeloid leukaemia: evidence for an in vitro haemopoietic defect and lack of correlation with the speed of engraftment.

Haematological recovery after autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is often delayed and available laboratory assays cannot accurately predict speed of engraftment. By using the long-term bone marrow culture (LTBMC) method, we attempted to define the haemopoietic defect underlying this slow engraftment, and assessed the usefulness of LTBMC in predicting engraftment. Cryopreserved bone marrow (BM) harvested from three different groups (AML autografts, n = 18; autografts for non-leukaemic diseases, n = 23; normal donors. n = 10) were cultured and their growth was compared and correlated with the speed of engraftment. In the AML autografts, non-adherent and adherent progenitor production was significantly reduced compared with normal BM during the whole culture period (P < 0.01). None of the LTBMC parameters was found to correlate with engraftment after autologous BMT. The non-leukaemic autografts showed progenitor production intermediate between normal and AML autografts. Their progenitor content at the end of the culture period (reflecting the stem cell pool) was not statistically different from normal BM. In this group, most of the progenitor cell contents, during LTBMC correlated with neutrophil (rs = -0.618 to -0.879, P < 0.01) and platelet (rs = -0.479 to -0.707, P < 0.02) recovery. The conclusion drawn from these results is that AML autografts are defective in their ability to sustain in vitro haemopoiesis, but this in vivo defect does not correlate with the slow haemopoietic recovery after autologous BMT. In contrast, LTBMC of autografts for non-leukaemic diseases, whose defect affects the stem cell pool to a lesser extent than BM in AML correlates with the speed of engraftment.

2-Chlorodeoxyadenosine therapy in Waldenstrom's macroglobulinaemia.

Despite some encouraging first results, experience with 2-chlorodeoxyadenosine (CdA) in the treatment of Waldenström's macroglobulinaemia (WM) has not as yet been very extensive. The present paper reports a clinical trial of the use of CdA in 18 patients having previously treated (n = 13) or untreated (n = 5) WM. CdA was administered by continuous intravenous infusion at a dose of 4 mg/m2/day for 7 days (5 patients) or as 2-h intravenous infusions at a dose of 5.6 mg/m2/day for 5 days (13 patients). Partial response was obtained in 7 cases. In this small series, no correlation could be found between response to CdA and patient characteristics at inclusion. During the first course of therapy, grade 4 neutropenia (< 0.5 x 10(9)/L) and thrombocytopenia (< 25 x 10(9)/L) developed in respectively 4 and 6 cases. In comparison with earlier reports haematological toxicity was more severe and the overall response rate lower in the present series of patients.

A single course of 2-chloro-deoxyadenosine does not eradicate leukemic cells in hairy cell leukemia patients in complete remission.

The nucleoside analog 2-chlorodeoxyadenosine (2-CdA) has recently emerged as a most promising treatment for hair-cell leukemia (HCL). The response rates are high regardless of prior therapy, and the duration of complete responses (CR) after a single course of treatment is longer than with any other therapeutic agent. We investigated the presence of minimal residual disease (MRD) in ten HCL patients treated in our institution with 2-CdA. The presence of residual leukemic cells was investigated in patients in CR following one course of treatment, using the polymerase chain reaction (PCR) and heavy-chain immunoglobulin genes (IgH), or TCR delta derived clonospecific probes. Eight patients achieved a complete remission after a single course of treatment, as evaluated at 6 months. Among these patients, seven are still in CR with a median follow-up of 12 months (range, 6-20 months) and one has relapsed after 15 months. Using PCR, all the evaluable patients remaining in CR showed persistent evidence of detectable MRD with no sign of decrease over the observation period. From this small series, we conclude that a single course of 2-CdA does not eradicate HCL and that persistence of residual leukemic cells appears to be common in patients in complete morphologic remission. Whether persistence of MRD will have an impact on long-term outcome, or whether HCL patients in morphologic CR with persistent MRD will remain so, is a matter of longer follow-up.

Cellular uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages.

Pefloxacin, like other fluoroquinolones, accumulates in macrophages and several other types of nucleated cells (but not in erythrocytes). Upon fractionation of macrophage homogenates by isopycnic centrifugation in sucrose gradients, fluoroquinolones are not found associated with any specific cellular structure. We have compared the activities of pefloxacin and roxithromycin against intracellular Staphylococcus aureus in mouse J774 macrophages. Pefloxacin was significantly more active for equivalent intracellular drug concentrations (i.e. expressed by reference to the respective MICs of the drugs as determined in broth), suggesting differences in intracellular availability and/or capacity of the drugs to express their activity in the intracellular environment. The difference was enhanced by incubating the cells in acidic medium. We have also examined the cellular pharmacokinetics and intracellular distribution of pefloxacin in uninfected and Legionella pneumophila infected guinea pig macrophages. In contrast to uninfected cells from which pefloxacin was quickly released, macrophages infected with legionella retained approximately 20-30% of the accumulated pefloxacin after a 60-min wash-out. Cell fractionation studies indicated that the drug remaining in cells was associated with components of high buoyant density. These fractions also contained [3H] if cells had been incubated with [3H] labelled legionella (by in-vitro exposure to [3H]-thymidine, before phagocytosis). These results suggest that part of the intracellular pefloxacin becomes associated with legionella, or with legionella-containing cytoplasmic structures.

Cellular uptake and subcellular distribution of roxithromycin and erythromycin in phagocytic cells.

The intracellular accumulation and subcellular distribution of 14C-labelled roxithromycin and erythromycin has been studied in macrophages and polymorphonuclear neutrophils of both human and animal origin. Roxithromycin was consistently and significantly more accumulated than erythromycin, reaching intracellular/extracellular concentration ratios between 14 (in polymorphonuclear neutrophils) and 190 (in alveolar macrophages from smokers). Uptake was reversible, insensitive to anaerobiosis and to the presence of an aminoglycoside, but inhibited by acid pH. Upon subcellular fractionation by isopycnic centrifugation in sucrose gradients., half the roxithromycin or erythromycin recovered in cell homogenates was found associated with the lysosomes in macrophages, and about one third with azurophil granules in polymorphonuclear leucocytes. Inasmuch as cellular uptake is a necessary, albeit not sufficient, condition for antimicrobials to kill or inhibit the growth of intracellular bacteria the properties of roxithromycin may give it a distinct advantage over other antimicrobial agents.

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al. (C. de Duve, T. de Barsy, B. Poole, A. Trovet, P. Tulkens, and A. Van Hoof, Biochem. Pharmacol. 23:2495-2531, 1974), in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

Cellular pharmacology of detorubicin and doxorubicin in L1210 cells.

Detorubicin (DET), a semi-synthetic analog of daunorubicin, releases at neutral pH doxorubicin (DOX) upon hydrolysis. DET enters faster than DOX into the cultured L1210 cells and reaches higher intracellular levels. When the cells are incubated for 120 min at pH 6.5, in spite of its rapid hydrolysis, one third of the intracellular fluorescence was due to undegraded DET. DET, like the other anthracyclines studied, is found associated intracellularly only to the lysosomes and to the nuclei. Unchanged DET is found mainly inside the lysosomes where the drug is stabilized, while DOX is found essentially associated to the nuclear DNA of L1210 cells. DET can therefore be viewed as an hydrophobic prodrug of DOX characterized, however, by a distinct subcellular localization in L1210 cells.

Cellular pharmacokinetics of spiramycin in cultured macrophages.

To gain a better understanding of the antibacterial and antiprotozoal activity of spiramycin as well as the characteristic conditions of cellular defence, we studied its accumulation and intracellular localization in cultured macrophages. Within two hours spiramycin in its active form is accumulated intracellularly by macrophages to a concentration 10 to 20 times that found in the extracellular medium; it is released slowly by the cells when they are incubated in antibiotic-free medium. After differential or isopycnic centrifugation, a bimodal localization of spiramycin was found; one part could be associated with the soluble cytosolic fraction and another with organelles sedimenting at a density of 1.17 g/ml, which represent part of the lysosomal population and perhaps the phagosomes.

Plasma levels of doxorubicin after IV bolus injection and infusion of the doxorubicin-DNA complex in rabbits and man. Comparison with free doxorubicin.

When injected rapidly IV into rabbits, the plasma levels of free DOX decreased biphasically and the drug was distributed in a volume greater than the body volume. When given as a DNA complex, the area under the concentration versus time curve was increased 10-fold and the distribution volume reduced more than 100-fold. The DOX-DNA complex infused both in rabbits and in human patients reached steady state-concentrations 10 and 20 times higher, respectively, than free DOX infusion, and the distribution volumes were reduced accordingly. These results confirm that the observed lower cardiotoxicity of the DOX-DNA complex arises despite higher plasma concentrations of the drug.

Cellular pharmacokinetics of aclacinomycin A in cultured L1210 cells. Comparison with daunorubicin and doxorubicin.

Aclacinomycin, which is more lipophilic than daunorubicin and doxorubicin, is taken up and released more rapidly and extensively by L1210 cells. After 5 h of incubation 91% of aclacinomycin is found in the nuclei of L1210 cells and the drug present in the post-nuclear fraction is distributed between the lysosomes and the cytosol. After an incubation of 5 h aclacinomycin decreases the density of the lysosomes. This effect is not observed either with doxorubicin or daunorubicin or when the cells are incubated with aclacinomycin for only 30 min.

Enzymatic characterization and analytical fractionation of L1210 cells.

The enzymatic characterization and analytical fractionation of L1210 cells have been performed in view of studying the cellular pharmacology of antitumoral drugs. Several enzymatic activities were detected and their assay conditions optimized. After a gentle homogenization to preserve as much as possible the integrity of the nucleus and cytoplasmic organelles, homogenates were fractionated by differential and isopycnic centrifugation. On the basis of pH dependency, effect of detergents and distributions after cell fractionation, enzymatic activities and biochemical constituents can be classified in several groups and by analogy to other organs or cultured cells, attributed to distinct cellular components. N-Acetyl-beta-glucosaminidase, alpha-L-fucosidase, alpha-D-mannosidase detected at acid pH and cathepsin D are therefore proposed as markers of lysosomes; inosine diphosphatase and uridine monophosphatase as markers of the plasma membrane, while phosphoglucomutase and neutral pyrophosphatase on one hand and galactosyl transferase and alpha-D-mannosidase at pH 6.0 on the other hand are attributed respectively to the cytosol and the Golgi apparatus.

Uptake of the daunorubicin-DNA complex in cultured fibroblasts.

The uptake and fate of the daunorubicin-DNA complex have been studied in cultured rat embryo fibroblasts. 125I-DNA was digested by the cells and appeared as low molecular weight fragments in the incubation medium. Subcellular fractionation of fibroblasts, previously incubated with the complex, showed that daunorubicin was localized to nuclei and lysosomes. At a high incubation concentration (17.5 microM), the accumulation of the free drug exceeded that of the complex. However, at a lower concentration (1.75 microM), the accumulation of the complex was as high as that of the free drug. The results are consistent with an uptake of the complex into cultured rat embryo fibroblasts by endocytosis. However, it cannot be excluded that the complex partly dissociates in the incubation medium, and that daunorubicin and DNA thereafter enter the cells separately.

DNA-binding parameters of daunorubicin and doxorubicin in the conditions used for studying the interaction of anthracycline-DNA complexes with cells in vitro.

Affinity constants of daunorubicin and doxorubicin for DNA at 37 degrees C and in presence of 10% serum were determined by an optical method and calculated from Scatchard plots. Values from 0.10 to 0.12 and from 0.13 to 0.16 X 10(6) M-1 were obtained for daunorubicin and doxorubicin, respectively. According to these affinity constants, the amounts of free drugs were calculated for various concentrations of daunorubicin-DNA or doxorubicin-DNA and for various molar ratios. In a large range of concentrations there is rather stable concentration of both free drugs, and these concentrations are inversely proportional to the nucleotides/drug ratio. The amount of free drug present in the medium is low as long as the concentration of daunorubicin-DNA or doxorubicin-DNA is higher than 1 microgram/ml (expressed as drug concentration). At lower concentrations, however, the percentage of free drug increases very sharply.