Sadao Tokimasa

Polycomb Group Gene rae28 Is Required for Sustaining Activity of Hematopoietic Stem Cells

The rae28 gene (rae28), also designated as mph1, is a mammalian ortholog of the Drosophila polyhomeotic gene, a member of Polycomb group genes (PcG). rae28 constitutes PcG complex 1 for maintaining transcriptional states which have been once initiated, presumably through modulation of the chromatin structure. Hematopoietic activity was impaired in the fetal liver of rae28-deficient animals (rae28 −/−), as demonstrated by progressive reduction of hematopoietic progenitors of multilineages and poor expansion of colony forming units in spleen (CFU-S12) during embryonic development. An in vitro long-term culture-initiating cell assay suggested a reduction in hematopoietic stem cells (HSCs), which was confirmed in vivo by reconstitution experiments in lethally irradiated congenic recipient mice. The competitive repopulating units (CRUs) reflect HSCs supporting multilineage blood-cell production. CRUs were generated, whereas the number of CRUs was reduced by a factor of 20 in the rae28 − / − fetal liver. We also performed serial transplantation experiments to semiquantitatively measure self-renewal activity of CRUs in vivo. Self-renewal activity of CRUs was 15-fold decreased in rae28 − / −. Thus the compromised HSCs were presumed to reduce hematopoietic activity in the rae28 − / − fetal liver. This is the first report to suggest that rae28 has a crucial role in sustaining the activity of HSCs to maintain hematopoiesis.

A congenital mutation of the novel gene LRRC8 causes agammaglobulinemia in humans.

A girl with congenital agammaglobulinemia and minor facial anomalies lacked B cells in peripheral blood: karyotypic analysis of white blood cells showed balanced translocation, t(9;20)(q33.2;q12). In the current study, we isolated a novel gene, leucine-rich repeat-containing 8 (LRRC8), at the translocation site on chromosome 9. It has four transmembrane helices with one isolated and eight sequentially located leucine-rich repeats (LRRs) and constitutes a new protein family. It is expressed on T cells as well as on B-lineage cells. Translocation truncates the LRRC8 gene, resulting in deletion of the eighth, ninth, and half of the seventh LRR domains located close to the C-terminal. The truncated form of the LRRC8 gene is transcribed with sequences from the noncoding region adjacent to the truncated seventh LRR. Protein products derived from the truncated gene are coexpressed on white blood cells with the intact LRRC8 protein from the untranslocated allele. Transplantation experiments with murine bone marrow cells that were forced to express the truncated LRRC8 show that expression of the truncated protein inhibited B cell development. These results indicate that LRRC8 is responsible for the B cell deficiency in this patient and is required for B cell development.

Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allogeneic stem cell transplantation

Human herpesvirus 6 (HHV-6) infection and disease are serious complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Ganciclovir (GCV) is effective against HHV-6 in vitro but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. We retrospectively compared the HHV-6 reactivation rate in pediatric allo-SCT recipients with and without GCV prophylaxis. The HHV-6 reactivation rate at 3 weeks after allo-SCT in patients without prophylactic GCV administration was significantly higher than that in those receiving prophylactic GCV (11/28 vs 0/13, P < 0.01). Five of 36 patients without prophylactic GCV showed clinical manifestations including skin rash, interstitial pneumonitis, persistent thrombocytopenia, enterocolitis and thrombotic microangiopathy, respectively. HHV-6-associated symptoms were observed in one of the 13 patients receiving prophylactic GCV. This patient showed fever, diarrhea and graft rejection concomitantly with a sudden increase of HHV-6 DNA copy number. Patients who received GCV for treatment of HHV-6 infection showed an improvement in symptoms and/or decrease of HHV-6 copy number. Thus, GCV is effective for treating HHV-6 disease after allo-SCT in vivo.

Excellent outcome of allogeneic hematopoietic SCT with reduced-intensity conditioning for the treatment of chronic active EBV infection

Since we reported the first successful case of allogeneic hematopoietic SCT (allo-HSCT), we have performed allo-HSCT for 29 patients with chronic active EBV infection (CAEBV), using either myeloablative conditioning (MAC) allo-HSCT (MAST) or reduced-intensity conditioning (RIC) allo-HSCT (RIST). In this retrospective analysis we compared the outcomes after MAST and RIST to identify the optimal conditioning for patients with CAEBV. Of 29 patients, 11 underwent allo-HSCT with MAC, consisting of TBI (12 Gy), etoposide (900 mg/m2) and CY (120 mg/kg) or melphalan (210 mg/m2), and the remaining 18 patients received allo-HSCT after RIC, consisting of fludarabine (∼180 mg/m2) and melphalan (140 mg/m2) or CY (120 mg/kg), with/without antithymocyte globulin and low-dose irradiation. Donor sources were 8 related BM, 2 related peripheral blood, 5 CD34 selected cells from HLA-haploidentical donors, 8 unrelated BM and 8 unrelated cord blood. The 3-year-EFS rate was 54.5±15.0% for MAST group and 85.0±8.0% for RIST group, and the 3-year OS rate was 54.5±15.0% for MAST group and 95.0±4.9% for RIST group (P=0.016). Allo-HSCT after RIC seems to be a promising approach for the treatment of CAEBV.

Lack of the Polycomb-group gene rae28 causes maturation arrest at the early B-cell developmental stage

The rae28 gene (rae28) is a murine homologue of the Drosophila polyhomeotic gene, which is a member of the Polycomb-group genes. In this study, we examined the role of rae28 in lymphocyte development. Because homozygous rae28-deficient (rae28-/-) mice died in the perinatal period, we examined lymphocyte development by generating chimeric mice reconstituted with green fluorescence protein-labeled mutant fetal liver cells as well as in in vitro culture systems. We further examined RAE28 expression by reverse transcriptase polymerase chain reaction assay in human leukemic cells with B-lineage acute lymphoblastic leukemia (ALL). Severe B-cell maturation arrest was observed in rae28-/- between pro- and pre-B lymphocyte stages. B-cell development was also delayed in heterozygous neonates. Furthermore, interleukin-7-dependent colony-forming ability was impaired not only in homozygous lymphocytes but also in heterozygotes. Its human homologue, RAE28, is located on chromosome 12p13, which frequently is associated with chromosomal abnormalities and loss of heterozygosity in patients with hematologic malignancies. To determine whether a link exists between RAE28 and leukemia, we examined RAE28 expression in leukemic cells from pediatric patients with B-lineage ALL. RAE28 expression was not detected in four B-cell precursor ALL cases of a total of 43 examined, although RAE28 is normally expressed constitutively during the process of B-cell maturation as assessed in isolated cell populations. rae28 plays an important role in the early B-cell developmental stage in a gene dosage-dependent manner. Furthermore, the human RAE28 locus may provide a candidate gene causing the molecular pathogenesis of childhood B-cell precursor ALL.

Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors

Major dose-limiting factors of high-dose thiotepa (TEPA) and melphalan are life-threatening mucositis and neurotoxicity. To administer a maximum dose of these drugs safely and to obtain a maximum anti-cancer effect, a double-conditioning regimen with a single grafting, two cycles of administration of a combination of TEPA (300–600 mg/m2) plus melphalan (70–150 mg/m2) with a 1-week interval was attempted in 20 patients with pediatric advanced or chemotherapy-resistant solid tumors (seven rhabdomyosarcoma, four hepatoblastoma, three neuroblastoma and four other malignancy). Combinations of TEPA plus melphalan/busulfan (Bu) (8–10 mg/kg) and TEPA plus Bu were given to four and two patients with brain tumors, respectively. In an additional two patients, three cycles of drug administration were performed. According to the results of the dose-escalating study, the maximum tolerable doses of TEPA and melphalan for children aged 2 years old or older were 1000 mg/m2 and 280 mg/m2, respectively. Mucositis was dose-limiting. Renal toxicity was also dose-limiting in young children (<2 years old). there were two treatment-related deaths (7%) (fungal pneumonia and renal tubular acidosis). among 13 patients who received high-dose chemotherapy during cr, 10 are alive with no evidence of disease (15–59 months, median: 35 months) and in 13 evaluable patients without cr, six are alive without regrowth of the disease (14–59 months, median: 39 months). thus, these novel conditioning regimens allowed us to increase the dose intensity to nearly the maximum for each drug and seemed to reduce adverse effects compared to previously reported regimens with these drugs. with regard to the effect on outcome, the results of this study seem to be encouraging, but a further study on a larger number of patients is required.

Defective long-term repopulating ability in hematopoietic stem cells lacking the Polycomb-group gene rae28

Abstract:  The rae28 gene (rae28) is a member of a Polycomb‐group (PcG) complex 1, which is known to help maintain transcription states once these have been initiated, by generating heritable higher‐order chromatin structures. In this study, we examined the capacity of rae28‐deficient (rae28−/−) hematopoietic stem cells (HSCs) to generate long‐term marrow reconstitution. rae28−/− fetal liver cells containing 20 competitive repopulation units (CRUs) were able to support the survival of lethally irradiated congenic mice for as long as 6 months. The marrow reconstituted with the rae28−/− cells, however, could not increase HSCs efficiently. This was evidenced by its inability to reconstitute marrow in serial transplantation experiments, as well as by the reduction in HSC‐enriched Lin− c‐kit+ Sca‐1high+ subpopulation in the bone marrow cells. Moreover, the reconstituted marrow produced less than half of the peripheral blood cells in each of the lineages examined. We also monitored the mean stem cell activity (MAS). MAS of rae28−/− CRUs was progressively reduced after transplantation, and after 12 months it was reduced to one‐tenth of that of the wild‐type. These in vivo results clearly indicate that rae28 is indispensable for the long‐term repopulating ability of HSCs. We further referred to the plausible mechanisms underlying defective long‐term repopulating ability of rae28‐deficient HSCs and argued for its involvement in maintenance of cell proliferation capability as well as that in self‐renewal ability.

Serum levels of soluble adhesion molecules in stem cell transplantation-related complications.

To assess the involvement of vascular endothelial cell activation and damage in stem cell transplantation (SCT)-related complications, such as acute and chronic GVHD and thrombotic microangiopathy (TMA), we investigated the changes in serum levels of soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin (sE-selectin) in SCT. The soluble form of intercellular adhesion molecule-1 (sICAM-1) was also analyzed. In patients with acute GVHD (grades II–IV), serum levels of sE-selectin and sICAM-1 increased around onset of GVHD (day 30). While the increase of sE-selectin levels was transient, sICAM-1 levels remained high until day 60. In patients with extensive chronic GVHD, sVCAM-1 as well as sE-selectin levels significantly increased. The appearance of clinical symptoms was preceded by elevations of sVCAM-1 and sE-selectin levels on day 60, and sICAM-1 levels on days 30 and 60. For the analysis of TMA, to exclude the influence of GVHD, serum levels were measured in auto-SCT patients. Around the onset of TMA, sVCAM-1 and sE-selectin levels significantly increased in patients with TMA without an increase of sICAM-1 levels. These findings support the notion that activation and injury of endothelium are commonly involved in the pathogenesis of acute and chronic GVHD and TMA. Bone Marrow Transplantation (2001) 27, 977–982.

Antifungal prophylaxis with micafungin in patients treated for childhood cancer.

Invasive fungal infections (IFIs) remain a major cause of infectious morality in neutropenic patients receiving chemotherapy or hematopoietic stem cell transplantation (HSCT). Micafungin exhibits broad antifungal activity against both Aspergillus and Candida species. We performed a retrospective study to determine the efficacy and safety of prophylactic micafungin against IFI in pediatric neutropenic patients during chemotherapy or HSCT.

WT1 (Wilms tumor 1) peptide immunotherapy for childhood rhabdomyosarcoma: a case report.

Immunotherapy using a Wilms tumor (WT1) peptide has been undergoing clinical trials for adulthood leukemia and solid cancer with promising results. In this study, the authors used WT1 peptide vaccination to treat a 6-year-old girl with metastatic alveolar rhabdomyosarcoma. She received weekly intradermal injection with HLA-A*2404-restricted, 9-mer WT1 peptide against residual bone disease. After 3 months her bone disease disappeared, concurrent with an increase in the frequency of WT1-specific cytotoxic T lymphocytes (CTLs). A high proportion of WT1-specific CTLs with effector or effector memory phenotype were detected in peripheral blood of this patient. She is currently still on continued WT1 peptide immunotherapy in a disease-free condition for 22 months. WT1 peptide-based immunotherapy should be a promising option for high-risk rhabdomyosarcoma in childhood.