Shu Tamura

Expression and role of p27kip1 in neuronal differentiation of embryonal carcinoma cells

Abstract We examined the expression and the regulation of p21 waf1 and p27 kip1 cdk inhibitors in P19 mouse embryonal carcinoma (EC) cells following treatment with all- trans retinoic acid (ATRA) to induce neuronal differentiation. The levels of p27 mRNA and protein increased within 24 h of treatment with ATRA, reaching a plateau 4–5 days later prior to neurite formation. In contrast, levels of p21 expression remained low until after neurites were extensively formed. Induction of muscle differentiation from P19 cells by treatment with dimethyl sulfoxide caused only transient increases in p27 levels. In a mutant P19 cell line, RAC65, treatment with ATRA induced neither p27 accumulation nor neuronal differentiation, but p21 mRNA expression increased markedly. In contrast, treatment of RAC65 cells with 9- cis retinoic acid induced both p27 expression and neuronal differentiation. Correlation between p27 expression and neuronal differentiation was also observed in NT2/D1 human EC cells. Luciferase reporter assays showed that p27 promoter activity increased in ATRA-treated cells, consistent with the elevation of p27 mRNA levels. Arrest of neuronal differentiation of P19 cells by okadaic acid resulted in inhibition of p27 expression, whereas p21 mRNA expression was greatly enhanced. Conversely, inhibition of p27 expression by antisense p27 oligonucleotides resulted in blockade of neuronal differentiation. Taken together, these results strongly suggest that the expression of p27 is indispensable for neuronal differentiation of EC cells.

New Imaging Findings in a Patient with Central Nervous System Dysfunction after Bone Marrow Transplantation

Central nervous system disorders are an important complication of bone marrow transplantation (BMT). We have recently performed cerebral angiography to examine central nervous system dysfunction in a 22-year-old woman with acute lymphoblastic leukaemia who had undergone BMT. Angiography demonstrated multiple stenoses and occlusions in the peripheral branches of the anterior and middle cerebral arteries, a pattern similar to that seen in vasculitis. She was thought to most likely have cytomegalovirus (CMV) vasculitis, but other forms of vasculitis, such as angiitis-like-syndrome-associated graft-versus-host disease could not be excluded. This case suggests that CMV vasculitis may cause central nervous system dysfunction after BMT and that imaging studies may provide useful information about central nervous system disorders in these patients.

Clonal evolutions during long-term cultures of bone marrow from de novo acute myeloid leukaemia with trilineage myelodysplasia and with myelodysplastic remission marrow.

Summary. We previously established a long‐term bone marrow culture (LTBMC) system in which novel abnormal karyotypes could emerge in vitro prior to the appearance of the same karyotypes in vivo in patients with myelodysplastic syndrome (MDS). We extended our study to examine whether acute myeloid leukaemia (AML) transformed from MDS (MDS/AML) and de novo AML with trilineage myelodysplasia (AML/TMDS) show clonal evolution in LTBMC similar to that of typical AML or MDS. We also analysed the cytogenetic changes in cultures with bone marrows from AML with myelodysplastic remission marrow (AML/MRM) as well as chronic myeloid leukaemia (CML) to compare them with typical AML with respect to the liability of clonal evolution. Among the 34 AML cases, abnormal karyotypes were newly detected in four of seven MDS/AML, three of six AML/TMDS and three of three AML/MRM. Novel abnormal karyotypes were also observed in nine out of 13 CML cases after culture. In contrast, no other abnormal karyotypes were found after culture in 18 typical AML without myelodysplasia. These findings suggest that AML/TMDS and AML/MRM are different from typical AML and are similar to MDS/AML and CML in view of their potential for disease progression from latent multiple clones. Typical AML may develop from a single abnormal clone without any subclones.

Early detection of relapse and evaluation of treatment for mixed chimerism using fluorescence in situ hybridization following allogeneic hematopoietic cell transplant for hematological malignancies

In order to detect chimerism, fluorescence in situ hybridization (FISH) and cytogenetic analyses were performed on bone marrow cells from 47 patients with hematological malignancies following allogeneic hematopoietic cell transplant (HCT). The dual-color XY, major Bcr-Abl (M-Bcr-Abl), and specific alpha-satellite probes were used for sex-mismatched HCT, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) cases with karyotypic abnormalities before HCT, respectively. Donor cells were found using FISH analysis in all 32 cases examined within 2 months following HCT, confirming engraftment. In six cases, however, cytogenetic analysis failed to detect donor cells due to lack of metaphases. Relapse occurred in four of the six cases in which mixed chimerism was detected using FISH analysis after 6 months of HCT. In contrast, after 12 months of HCT, no relapse was found in 24 patients without host cells. For two patients with mixed chimerism, gradual reduction of immunosuppressants or donor lymphocyte infusion resulted in the disappearance of host cells as analyzed using FISH analysis. In three extramedullary relapse cases, however, cytogenetic relapse preceded morphological and FISH relapse. These findings suggest that FISH analysis is more useful for detecting residual host cells after HCT, and the combination of FISH and cytogenetic analyses provide a more detailed evaluation for HCT patients. The results also indicate that monitoring of mixed chimerism using FISH analysis after 6 months of HCT is important for allowing the early detection of hematological relapse.

Adult respiratory distress syndrome-like disorders after allogeneic bone marrow transplantation

BACKGROUND Adult respiratory distress syndrome-like respiratory disorders are a serious, but uncommon, complication of bone marrow transplantation. METHODS We measured various cytokines in 2 patients with respiratory disorders and 11 patients without respiratory problems after allogeneic bone marrow transplantation. RESULTS The patients with respiratory disorders had elevated levels of interferon-gamma and interleukin-2 in the aplastic phase, and elevation of tumor necrosis factor-alpha, intercellular adhesion molecule-1, and interleukin-8 at the time of leukocyte recovery. Both patients with respiratory disorders developed fever during the aplastic phase, whereas none of the patients without fever had respiratory disorders. Among patients who had fever during the aplastic phase but no respiratory disorders, there was no elevation of cytokines from the aplastic phase to the recovery phase. CONCLUSIONS Respiratory disorders may occur after bone marrow transplantation when an inflammatory response during the aplastic phase stimulates cytokines that cause vascular endothelial damage and increases the levels of chemokines and adhesive molecules along with elevation of the leukocyte count.

Detection of latent subclones with abnormal karyotypes by long-term bone marrow cultures in cases of myelodysplastic syndrome

Cytogenetic studies on non‐adherent cells from long‐term bone marrow cultures (LTBMC) were done for 23 patients with myelodysplastic syndrome (MDS) and compared with the karyotypes detected during their clinical courses. Of 14 cases with normal karyotype before culture, abnormal karyotypes were detected first in LTBMC from seven. Novel abnormal karyotypes were observed after LTBMC in two of four cases which had had both normal and abnormal karyotypes before culture. Abnormal karyotypes were also newly detected in four of five cases with only abnormal karyotypes before culture. Among 13 cases in which abnormal karyotypes were observed during the cultures, three patients showed the same karyotypes 1–11 months later in their clinical courses. These findings suggest that our LTBMC might be useful for evaluating the prognosis and choice of treatment for MDS patients.

Significance of trilineage myelodysplasia in de novo acute myeloid leukaemia during remission rather than at diagnosis

Patients with trilineage myelodysplasia (TMDS) in de novo acute myeloid leukaemia (AML) at diagnosis and remission were clinically evaluated between 1983 and 1996. AML with TMDS (AML/TMDS) was observed in 20 (12%) of 162 patients with de novo AML at diagnosis. Complete remission (CR) was achieved with combination chemotherapy in 12 (67%) of 18 AML/TMDS cases. This CR rate was relatively worse than the rate of 78% (106/136 cases) of AML without TMDS, but this difference was not significant. Disease‐free survival curves also showed no difference between AML/TMDS and AML without TMDS. During remission, eight (67%) of 12 AML/TMDS cases had myelodysplastic remission marrow (AML/MRM). AML/MRM was also seen in seven (7%) of 106 AML cases without TMDS. The actuarial disease‐free survival was significantly lower in AML/MRM than in AML without MRM (P = 0.0003). All of the AML/MRM cases exhibited early leukaemic relapse; median remission duration was only 9 months. Clonal changes occurred in two cases of AML/TMDS and five cases of AML/MRM at the time of relapse. These findings suggest that TMDS during remission predicts a poorer prognosis and early leukaemic relapse when compared with the absence of TMDS.

Application of long-term bone marrow cultures for studying the leukemic transformation of myelodysplastic syndromes.

Myelodysplastic syndrome (MDS) has been thought to be an identifiable early stage in multistep leukemogenesis. Considerable numbers of patients with MDS eventually develop acute myelogenous leukemia (AML), which is very much more difficult to manage than typical denovo AML. There are several differences in both the clinical and biological behavior of AML with and without prior MDS. We have established an unique long-term bone marrow culture (LTBMC) system which allows abnormal cells to grow in preference to normal cells, based on the method originally described by Dexter et al. Leukemic transformations occur more frequently in MDS patients with abnormal karyotypes, and particularly those with multiple abnormalities. New cytogenetic abnormalities were occasionally observed at the time of transition to AML. We have applied this LTBMC system for cytogenetic and molecular studies on the leukemic transformation from MDS. Among the 32 patients with MDS studied thus far, novel abnormal karyotypes were detected during the LTBMCs in 15 patients. Furthermore, 6 out of 23 novel karyotypes detected during the in-vitro cultures emerged in vivo, one to 11 months later in 4 patients. In addition, point mutation of the N-ras proto-oncogene was observed in 3 of 18 cases. The signal of the dot-blot hybridization was increased in one examined case after 2 weeks in culture. Thus, this LTBMC system may provide some promising information with respect to understanding the multistep process from the preleukemic stage to the development of overt leukemia as well as its prognostic relevance.

Comparison of the Survivals Between Bone Marrow Transplantation and Chemotherapy for Acute Leukemia in First Remission—A Japanese Single Institution Study

The outcome of sixty-four patients with acute leukemia in first remission who had been treated with either bone marrow transplantation (BMT) or conventional chemotherapy was retrospectively evaluated (a median follow-up of 37 months). Among them, 26 patients (age range; 14-42 years) received allogeneic BMT from HLA-identical siblings and 38 patients (age range; 13-43 years) who had no HLA-identical donors undertook the continued combination chemotherapy. Kaplan-Meier product-limit estimate of actuarial survival of acute myelogenous leukemia (AML) patients was 48.9% for the BMT group and 15.7% for the chemotherapy group (p = not significant, NS). For acute lymphoblastic leukemia (ALL) patients, the survival following BMT was 80.2% and was significantly higher than that of the chemotherapy group of 33.3% (p < 0.05). The disease-free survival of AML and ALL for the BMT group was 34.3% and 36.5%, respectively, which was higher than that of the chemotherapy group (16.7% and 23.4%, respectively (p = NS)). These findings in our Japanese single institution study suggested that BMT may be the treatment of choice for adult patients with acute leukemia in first remission if they had suitable donors and that more effective therapeutic regimens were necessary for patients without compatible donors in order to obtain the longer remission duration.