Nobuyuki Takayama

Imatinib mesylate has limited activity against the central nervous system involvement of Philadelphia chromosome‐positive acute lymphoblastic leukaemia due to poor penetration into cerebrospinal fluid

Summary. A 32‐year‐old woman with relapsed Philadelphia chromosome‐positive acute lymphoblastic leukaemia was treated with imatinib mesylate (formerly STI571), a selective inhibitor of BCR/ABL tyrosine kinase. Although the initial marrow response was good and stably maintained, she subsequently relapsed with extensive infiltration of leukaemic cells into the central nervous system (CNS). After controlling her CNS disease with additional intrathecal chemotherapy, we measured the concentration of imatinib in cerebrospinal fluid (CSF) and blood simultaneously. The concentration of imatinib in CSF was about 92‐fold lower than that in blood. These results suggest that imatinib poorly penetrates the blood–brain barrier and has limited activity against CNS leukaemia.

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation

We have prospectively evaluated the efficacy of real-time PCR-guided preemptive therapy for CMV diseases in allogeneic hematopoietic stem cell transplant recipients with grades II–IV acute GVHD. The dose of ganciclovir was adjusted according to the viral load determined by real-time polymerase chain reaction (PCR). On detecting CMV reactivation in the plasma, ganciclovir was initiated at a dose of 5 mg/kg body weight once daily, and the dose was increased to twice daily if viral load continued to increase after initiating ganciclovir. In 39 evaluable patients, CMV reactivation assessed by real-time PCR became positive in 30 (77%). One developed CMV gastroenteritis before PCR became positive. Thus the remaining 29 patients were treated preemptively with ganciclovir. The dose of ganciclovir was increased in 12 patients (41%) of preemptively treated patients for increasing viral load. CMV diseases were diagnosed in two patients (one gastroenteritis and one retinitis), and late CMV disease was diagnosed in one patient (gastritis). The treatment was generally well-tolerated, but three patients (10%) developed neutropenia (neutrophil count less than 1.0 × 109/l). In conclusion, real-time PCR-guided preemptive therapy with decreased dose of ganciclovir is feasible and does not increase the frequency of CMV diseases if the dose is adjusted according to the viral load.

All-trans and 9-cis retinoic acid enhance 1,25-dihydroxyvitamin D3-induced monocytic differentiation of U937 cells

Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (D3) are well known for inducing differentiation in many leukemic cell lines. The nuclear signalling pathways of RA and D3 are mediated through their cognate receptors, the retinoic acid receptor (RAR) and vitamin D3 receptor (VDR), respectively. Retinoid X receptor (RXR) is an auxiliary factor that forms a heterodimer with RAR and VDR, enabling their efficient transcriptional activation. 9-cis RA, a high-affinity ligand for RXR, greatly enhanced D3-induced CD14 expression in U937 cells, while RA alone did not induce CD14 expression. 9-cis RA also resulted in morphological changes of U937 cells to macrophage-like cells when combined with D3, while RA alone resulted in granulocyte-like cells. RA and D3 together enhanced c-fms expression, phagocytic activity, and acted synergistically to promote nitroblue tetrazolium reduction activity and inhibit proliferation. Northern analysis showed that U937 cells constitutively expressed RAR-alpha, VDR and RXR-alpha mRNAs. RA or D3 alone or in combination did not affect RAR-alpha and VDR expression, while 9-cis RA and 9-cis RA plus all-trans RA significantly reduced RXR-alpha expression. Interestingly, D3 could restore the down-regulation of RXR-alpha mRNA by 9-cis RA. These findings suggest that there is crossover of the nuclear signalling pathways of RA and D3. This may have clinical implications in that RA and D3 may be used in combination for differentiation-inducing therapy in acute myelogenous leukemia and myelodysplastic syndrome.

Serial quantification of minimal residual disease of t (8 ; 21) acute myelogenous leukaemia with RT-competitive PCR assay

The chromosomal translocation (8;21)(q22;q22) in the AML M2 subtype according to the FAB classification, results in the production of a novel fusion gene AML1/ETO. The chimaeric AML1/ETO transcript is useful for the detection of minimal residual disease (MRD). Recently, several studies on the detection of AML1/ETO transcripts in t(8;21) AML have been reported. However, the clinical significance of a small number of AML1/ETO transcripts by a reverse transcription–polymerase chain reaction (RT‐PCR) remains to be elucidated. We have developed a novel quantitative RT‐competitive PCR assay and evaluated the clinical usefulness of this method by the monitoring of MRD in eight patients with t(8;21) AML. In four patients in first continuous complete remission (CR) the value of MRD was always <0.1 fg of the competitor dose throughout their courses, whereas in four relapsed patients there was an increase in the value of MRD to <0.1 fg of the competitor dose before cytogenetic relapse. We conclude that the detection of the presence of cells with AML1/ETO fusion transcripts by our RT‐competitive PCR assay may be useful to monitor disease progression and to predict subsequent relapse.

Novel mutation in the PML/RARα chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia

OBJECTIVE All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.

Thalidomide for the treatment of refractory multiple myeloma: association of plasma concentrations of thalidomide and angiogenic growth factors with clinical outcome.

Recent reports showed that thalidomide has anti‐angiogenic activity and is effective for the treatment of refractory multiple myeloma (MM). We examined the relationship between the clinical efficacy and adverse effects of thalidomide and the plasma concentrations of this drug as well as angiogenic growth factors in refractory MM. Ten out of twenty‐four evaluable patients (42%) showed more than 25% reduction of M‐protein, and eight (33%) achieved more than 50% reduction. These changes were associated with restoration of anemia and recovery of normal immunoglobulin level. Somnolence and headache, constipation, peripheral neuropathy and skin rash were frequently observed, but were well tolerated. However, grade 2–4 severe neutropenia was also observed in nine cases. These adverse effects other than neutropenia occurred more frequently in the patients with higher plasma concentrations of thalidomide (≥2.0 μg/ml at 12 h after the last administration) and were readily alleviated by dose reduction. In contrast, neutropenia developed regardless of the plasma concentration. Plasma concentrations of angiogenic growth factors were frequently elevated before treatment. After thalidomide treatment, these growth factor levels tend to decrease to near‐normal ranges in responders but were still high in most non‐responders. After thalidomide treatment, plasma vascular endothelial growth factor (VEGF) level was significantly reduced in responders (P=0.025), but not in non‐responders (P=0.37). Reduction of plasma VEGF level might be an important indicator for anti‐myeloma effect of thalidomide.

Retinoid Resistance in Leukemic Cells

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients who received continuous treatment with all-trans RA relapsed and develop RA-resistant disease. The detailed mechanisms for this development of RA resistance by APL cells are still unclear. Several possible mechanisms have been considered to explain in vitro resistance to RA. One obvious explanation is the generation of new mutations in the retinoid receptors. However, UF-1 cells (the first permanent APL cell line with RA-resistant features) had no point mutations in the ligand-binding domain of the RAR-alpha gene. Another potential mechanism for clinical RA resistance is the pharmacologic alteration in the metabolism of all-trans RA. Continuous treatment with all-trans RA in APL is associated with a progressive reduction of the plasma concentrations of RA. Induction of cytochrome P-450, cellular RA-binding protein (CRABP) and P-glycoprotein resulted in lower plasma and cellular levels of active retinoids. Thus, acquired resistance to RA may be explained at least in part by drug metabolism in leukemic cells.

Granisetron plus dexamethasone versus granisetron alone in the prevention of vomiting induced by conditioning for stem cell transplantation: A prospective randomized study

This prospective randomized study compared the efficacy and toxicity of granisetron and dexamethasone to those of granisetron alone for antiemetic control in patients receiving high-dose chemotherapy with or without total body irradiation (TBI) for stem cell transplantation. Patients were divided randomly into 2 groups. Groups received granisetron twice daily at a dose of 40 µg/kg with or without 4 mg dexamethasone (GS group and G group, respectively), starting 30 minutes before each dose of chemotherapeutic agent or TBI, or 12 hours after the first dose if TBI or a drug was given once a day. Fifty patients were evaluated for the analysis. During the first 24 hours of conditioning, 23 of 25 patients (92.0%) in the GS group achieved complete control of emesis (no emetic episodes over the course of a day), compared with 72.0% in the G group (P = .06). For patients receiving TBI on the first day of conditioning, complete emetic control was achieved in all patients (100.0%) in the GS group, compared with 63.2% in the G group (P = .02). The same degree of emetic control was maintained throughout the conditioning period in 38.8% of the GS group and 29.9% of the G group (P = .10). Adverse reactions were observed more frequently in the GS group (68.0% versus 5.0% in the G group). These reactions included insomnia, headache, flushing, and hyperglycemia. None of the events were serious. We conclude that granisetron with dexamethasone seems superior to granisetron alone for the prevention of emesis resulting from the conditioning regimen; however, the more frequent side effects may limit the wide use of this combination.

Long-Term Follow-up of Allogeneic Bone Marrow Transplantation after Reduced-Intensity Conditioning in Patients with Chronic Myelogenous Leukemia in the Chronic Phase

Although allogeneic transplantation is a curative therapy for chronic myelogenous leukemia (CML), treatment-related mortality is still a major cause of death after transplantation, especially in older patients. We investigated the safety and efficacy of reduced-intensity conditioning consisting of low-dose (600 cGy) total body irradiation and cytosine arabinoside (1 g/m2) together with a continuous infusion of granulocyte colony-stimulating factor and cyclophosphamide (120 mg/kg) in patients with CML in the chronic phase. Fractionated splenic irradiation (5 Gy) was also administered as part of the conditioning treatment. Eight patients older than 40 years underwent allogeneic bone marrow transplantation from an HLA-matched sibling following this conditioning. Regimen-related toxicities (equal to or greater than grade III) were not observed. Rapid restoration of 100% donor chimerism was confirmed by fluorescence in situ hybridization methods in 5 sex-mismatched transplant recipients. One patient died from severe acute graft-versus-host disease and another fromPneumocystis carinii pneumonia early in the course of transplantation. A sustained engraftment was achieved in 5 long-term survivors; in 1 case, the graft was rejected but the Philadelphia chromosome and BCR/ABL-negative autologous hemopoiesis were restored. After a minimum follow-up period of 60 months, 6 patients, including the patient with restored autologous hemopoiesis, were still alive and in remission with 100% donor chimerism. Six years after the transplantation, 1 patient experienced a cytogenetic relapse, which was successfully treated with donor lymphocyte infusions. In summary, this reduced-intensity conditioning resulted in a cure with markedly reduced regimen-related toxicities in this relatively older cohort of patients with CML.

Allele-specific activation of the c-myc gene in an atypical Burkitt's lymphoma carrying the t(2;8) chromosomal translocation 250 kb downstream from c-myc

The genetic structure and regulation of the c-myc gene was comprehensively studied for the first time in Burkitts lymphoma with t(2;8) translocation. In a Burkitts lymphoma cell line, KOBK101, the immunoglobulin kappa-encoding gene on chromosome 2, accompanied by its enhancer, was translocated to the pvt-1 locus located about 250 kb downstream from c-myc on chromosome 8. Only the c-myc allele on the translocated chromosome carried aberrant SalI and KpnI sites in the first intron, so the two c-myc alleles and their transcripts were analyzed separately. The c-myc allele on the untranslocated chromosome conserved the normal c-myc sequence and was transcriptionally silent. In contrast, the c-myc allele on the translocated chromosome was actively transcribed at three- to fivefold higher levels, as compared with non-malignant B-cell lines. Additionally, it carried predominant multiple mutations consisting of 64 nucleotide substitutions, three short deletions, and a one-base insertion, most of which clustered in the first exon and intron. The 24-base deletion in the first intron completely overlapped the binding site of a putative negative transcriptional factor of the 138-kDa phosphoprotein, MIF. Thus, the multiple mutations and the deregulated, allele-specific expression of c-myc were associated with the chromosomal translocation in cis. Together activation by the long-distance immunoglobulin kappa enhancer, and the alleviation of negative regulation by the mutations, seemed to cause the allele-specific activation of c-myc.