CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

PBSC collection from family donors in Japan: a prospective survey.

Severe adverse events (SAE) and late hematological malignancies have been reported after PBSC donation. No prospective data on incidence and risk factors have been available for family donors so far. The Japan Society for Hematopoietic Cell Transplantation (JSHCT) introduced therefore in 2000 a mandatory registration system. It defined standards for donor eligibility and asked harvest centers to report any SAE immediately. All donors were examined at day 30 and were to be contacted once each year for a period of 5 years. Acute SAEs within day 30 were reported from 47/3264 donations (1.44%) with 14 events considered as unexpected and severe (0.58%). No donor died within 30 days. Late SAEs were reported from 39/1708 donors (2.3%). The incidence of acute SAEs was significantly higher among donors not matching the JSHCT standards (P=0.0023). Late hematological malignancies in PBSC donors were not different compared with a retrospective cohort of BM donors (N:1/1708 vs N:2/5921; P=0.53). In conclusion, acute and late SAEs do occur in PBSC donors at relatively low frequency but risk factors can be defined.

Glyoxalase-I is a novel target against Bcr-Abl+ leukemic cells acquiring stem-like characteristics in a hypoxic environment.

Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl(+) leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl(+) stem cells is needed to cure leukemias caused by Bcr-Abl(+) cells. Human Bcr-Abl(+) cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl(+) sublines by selection in long-term hypoxic cultures (1.0% O(2)). Interestingly, HA-Bcr-Abl(+) cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher beta-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl(+) cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl(+) cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl(+) leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl(+) leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

NOD/Shi-scid IL2rgamma(null) (NOG) mice more appropriate for humanized mouse models.

"Humanized mice," in which various kinds of human cells and tissues can be engrafted and retain the same functions as in humans, are extremely useful because human diseases can be studied directly. Using the newly combined immunodeficient NOD-scid IL2rgamma(null) mice and Rag2(null) IL2rgamma(null) humanized mice, it has became possible to expand applications because various hematopoietic cells can be differentiated by human hematopoietic stem cell transplantation, and the human immune system can be reconstituted to some degree. This work has attracted attention worldwide, but the development and use of immunodeficient mice in Japan are not very well known or understood. This review describes the history and characteristics of the NOD/Shi-scid IL2rgamma(null) (NOG) and BALB/cA-Rag2(null) IL2rgamma(null) mice that were established in Japan, including our unpublished data from researchers who are currently using these mice. In addition, we also describe the potential development of new immunodeficient mice that can be used as humanized mice in the future.

Outcome of risk-based therapy for infant acute lymphoblastic leukemia with or without an MLL gene rearrangement, with emphasis on late effects: a final report of two consecutive studies, MLL96 and MLL98, of the Japan Infant Leukemia Study Group.

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0-60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

C1-C2 point monitoring of low-dose cyclosporin a given as a single daily dose in children with steroid-dependent relapsing nephrotic syndrome.

AIM: It has been reported that the pharmacokinetics of cyclosporin A (CsA) in children is different from that in adults. It appears that, in general, pediatric patients metabolize CsA more rapidly than adult patients, necessitating the use of higher dose of the drug in pediatric transplant recipients. In this context, we speculated that single-dose daily administration of low-dose CsA may be associated with a higher peak blood level without associated trough blood level elevation, and thereby yield better results and allow safer use of the drug than the conventional twice daily administration dosing used for the treatment of childhood idiopathic nephrotic syndrome (INS). METHODS: A total of 10 children with steroid-dependent relapsing INS (9 with biopsy-proven minimal-change disease) who showed steroid toxicity were enrolled in the study. The initial daily dose of CsA (Neoral) used was around 2.0 mg/kg, given as a single daily dose before breakfast. The dose was subsequently adjusted to achieve a C1-C2 point blood level between 600 - 800 ng/ml. The dose of the concomitantly administered prednisolone was tapered following the commencement of CsA. RESULTS: The mean daily CsA dosage, the mean C1-C2 point blood level and the mean trough blood level in the subjects were 2.2 +/- 0.8 mg/kg, 754.0 +/- 71.9 ng/ml and 42.7 +/- 29.2 ng/ml, respectively. At the latest observation, after a mean duration of 17 months (6 - 24 months) of CsA therapy, the minimum dose of prednisolone required for maintenance of clinical remission and the calculated relapse rate were significantly decreased as compared to the respective pretreatment values (0.52 +/- 0.46 mg/kg on alternate days, vs. 0.97 +/- 0.63 mg/kg on alternate days, and 0.28 +/- 0.32 times per six months, vs. 1.06 +/- 0.41 times per six months, respectively, p = 0.005). No significant change was observed in the mean estimated GFR value as compared to the pretreatment value (183.1 +/- 35.4 ml/min/1.73 m2vs. 185.4 +/- 39.3 ml/min/1.73 m2). No evidence of CsA nephrotoxicity was observed in a repeat renal biopsy performed around 12 months after the commencement of CsA therapy in two patients. CONCLUSIONS: Despite the limitations of the study, our results suggest that administration of low-dose CsA as a single daily dose with C1-C2 point blood level monitoring might be an equally effective and safe and, therefore, more cost-beneficial, protocol for the treatment of steroid-dependent cases of relapsing INS, as conventional twice-daily administration of CsA with trough blood level monitoring. Further studies to confirm the long-term efficacy and safety of this CsA treatment protocol in larger numbers of patients are, however, needed.

Therapy-related myelodysplastic syndrome in childhood: a retrospective study of 36 patients in Japan.

We report here a retrospective analysis of 36 children with therapy-related myelodysplastic syndrome (t-MDS) diagnosed between 1990 and 1999 in Japan. Their median age was 7.7 years and the median latency period for the development of t-MDS was 38.5 months. The primary tumors were hematologic in 15 of the cases and nonhematologic in 21. Chromosomal abnormalities were detected in 32/34(94%) patients: abnormalities of chromosomes 5and/or 7 in 41% and notably, 11q23 abnormalities in 31%. The prognosis of children with t-MDS was very poor as compared to children with primary MDS (5 year survival: 16% versus 54%, p<0.0001).

Mizoribine for the treatment of lupus nephritis in children and adolescents.

AIM: The optimal treatment for lupus nephritis in the pubertal age group is still unclear. We therefore retrospectively evaluated the efficacy and safety of mizoribine (MZR), a novel selective inhibitor of inosine monophosphatase dehydrogenase, developed in Japan for the treatment of lupus nephritis in pubertal patients, because MZR has been reported to have relatively less clinically toxicity than conventionally used drugs. METHODS: Over the past 12 years, we have treated 20 children with newly diagnosed, biopsy-proven lupus nephritis at our hospital. Of these, 10 patients who received a 2-year course of MZR in combination with prednisolone (PSL) as initial maintenance therapy and who could be observed for at least two years after the commencement of the MZR treatment, were enrolled in this study. MZR was given orally at the dose of 4 - 5 mg/kg per day in two divided doses. Changes in the clinical parameters, such as the urinary protein excretion, serum anti-dsDNA antibody titer, serum hemolytic complement activity (CH50) and serum creatinine, and in the frequency of disease flares defined as persistent worsening of those clinical parameters, were examined pre- and posttreatment for comparison, and the steroid-sparing effect of MZR was analyzed. RESULTS: As induction therapy, all the patients had received high-dose oral PSL or intravenous methylprednisolone pulse therapy. Five female patients who were diagnosed to have proliferative lupus nephritis (WHO class III or IV) were scheduled to receive an 8-week course of oral cyclophosphamide (CPA) combined with high-dose steroids prior to the commencement of MZR, but CPA needed to be discontinued in four of these patients because of clinical drug toxicity. The clinical parameters showed significant improvement after the 2-year treatment with MZR combined with PSL, as compared to the pretreatment values (mean urinary protein excretion: 1.6+/-1.9 g/day vs.0.1 +/-0.1 g/day, serum CH50 value: 12.6+/-5.4 U/ml vs. 34.5+/-7.7 U/ml, serum anti-dsDNA antibody titer: 141.8+/-128.2 IU/ml vs. 18.5+/-17.7 IU/ml, respectively (p <0.01)). The serum creatinine remained normal. Although the daily dose of the concomitantly administered PSL to maintain clinical remission could be decreased significantly in all the study participants (39.5+/-1.6 mg/day vs. 6.8+/-5.1 mg/day, p < 0.01), two patients developed flares while on treatment, which were successfully treated by transiently increasing the dose of PSL. The MZR therapy could be completed in all of the patients without significant clinical toxicity being reported. At their most recent follow-up (mean 4.3 years), none of the 10 patients had renal insufficiency, and complete remission without any need for further medication had been achieved in two patients. CONCLUSION: Although this case series is without controls, maintenance therapy with MZR administered in combination with PSL may be beneficial and clinically less toxic in at least a proportion of pubertal patients with lupus nephritis.

Mizoribine oral pulse therapy for patients with disease flare of lupus nephritis.

AIM: Mizoribine (MZR) is a newly developed immunosuppressive agent in Japan. To relieve disease flare of lupus nephritis, a prospective pilot study of oral MZR pulse therapy (a phase II trial) is conducted as an alternative therapy to dose up of corticosteroids. METHODS: Six Japanese patients with biopsy-proven lupus nephritis who experienced disease flare were prospectively evaluated. MZR at a dose of 5 - 10 mg/kg per day (up to 500 mg) in 1 or 2 divided daily doses was orally administered twice a week for 3 months. At the time of disease flare, 4 patients had refused to take dose up of corticosteroids, and the other 2 had complained of opportunistic infection. RESULTS: At presentation, urine protein excretion, serum hemolytic complement activity (CH50) and serum anti-dsDNA antibody were 1.9 +/- 0.6 g/day, 15.7 +/- 5.8 U/ml (normal 23 - 46 U/ml) and 164.8 +/- 184.0 IU/ml (normal < 12.0 IU/ml), respectively. Urine protein excretion and serum anti-dsDNA antibody decreased significantly following MZR oral pulse therapy (0.2 +/- 0.1 g/day and 29.7 +/- 23.4 IU/ml (p < 0.05), respectively), and serum CH50 recovered to normal (35.1 +/- 10.4 U/ml, p < 0.05). Moreover, a significant histologic improvement was observed in a patient who received repeat renal biopsies at pre- and post-treatment. Reported peak serum MZR levels enough to inhibit human mixed-lymphocyte reaction (3.0 - 6.0 microg/ml) were achieved in all patients. No serious adverse effects were observed. CONCLUSION: Although we had no control subjects in this series, MZR oral pulse therapy may be of benefit to a proportion of patients with disease flare of lupus nephritis as an alternative therapy to dose up of corticosteroids.

Successful T-cell-replete peripheral blood stem cell transplantation from HLA-haploidentical microchimeric mother to daughter with refractory acute lymphoblastic leukemia using reduced-intensity conditioning.

A 16-year-old girl with refractory acute lymphoblastic leukemia underwent reduced-intensity hematopoietic stem cell transplantation from her two-locus-mismatched haploidentical mother, who was microchimeric for the patient's hematopoietic cells. The conditioning regimen comprised melphalan, fludarabine, and low-dose total body irradiation. Non-T-cell-depleted peripheral blood stem cells were infused with graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus, prednisolone, and short-course methotrexate. Complete donor-type engraftment without evidence of residual leukemia was confirmed on day 22. Severe GVHD was not observed despite rapid cessation of immunosuppression. The patient remains well in continuous remission 15 months after transplant. This successful experience suggests that maternal hematopoietic stem cell transplants for children, in the presence of microchimerism, may be associated with hyporesponsiveness to the inherited paternal HLA antigens (IPA); preventing severe GVHD.

Successful hematopoietic stem cell transplantation for aplastic anemia following living-related liver transplantation.

A 1-year-old boy received a living-related liver transplantation (LRLT) from his HLA-haploidentical father to treat acute liver failure following non-A, non-B, non-C hepatitis. He subsequently developed pancytopenia and was diagnosed with aplastic anemia (AA). He was platelet transfusion dependent and developed two episodes of life-threatening intracranial hemorrhage despite immuno-suppressive therapy consisting of cyclosporin A, antithymocyte globulin, and anabolic steroids. He received combined hematopoietic stem cell transplantation (hSCT) with cord blood and bone marrow from an HLA-matched sibling. Conditioning consisted of cyclophosphamide (CY) 200 mg/kg and 7 Gy total lymphoid irradiation (TLI). Marrow engraftment was prompt and there was no significant graft-versus-host disease (GVHD).

Allogeneic hematopoietic stem cell transplantation for 27 children with juvenile myelomonocytic leukemia diagnosed based on the criteria of the International JMML Working Group.

Prognostic factors of juvenile myelomonocytic leukemia (JMML) have not been clarified because of its very low incidence and inaccuracy in the diagnosis. The purpose of this study was to evaluate children with JMML given an allogeneic hematopoietic stem cell transplantation (SCT) and the role of different variables potentially influencing outcome in a nationwide survey in Japan based on the newly proposed criteria by the International JMML Working Group. The study patients were 27 children who underwent SCT among 55 JMML patients retrospectively collected in the survey. The source of grafts was HLA-identical siblings in 12 cases, HLA-matched unrelated individuals in 10 and others in five. Total body irradiation was used in 18 cases. Event-free and overall survival (OS) at 4 years after SCT were 54.2 +/- 11.2% (s.e.) and 57.9 +/- 11.0% (s.e.), respectively. Six patients died of relapse and three of complications. Patients with abnormal karyotypes showed a significantly lower OS than those with normal karyotypes (P < 0.001). Patients below 1 year of age showed a significantly higher OS than those of 1 year of age or more (P = 0.02). Patients with grade 0-1 acute graft-versus-host disease (GVHD) or chronic GVHD had a more favorable OS than those without them, although they were not statistically significant (P > 0.05). Other variables studied were not associated with OS. A multivariate analysis of these factors yielded the abnormal karyotype as the only significant risk factor for lower OS (risk ratio: 11.0; 95% CI: 2.7-45.1).

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) stimulates primitive and definitive erythropoiesis in mouse embryos expressing hGM-CSF receptors but not erythropoietin receptors.

Although erythropoietin (EPO) and its receptor (EPOR) are crucial for the proliferation, survival, and terminal differentiation of erythroid progenitors, it remains to be elucidated whether EPOR-unique signaling is required for erythropoiesis. To address this issue, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR)-transgenic mice and heterozygous EPOR mutant mice were crossed by in vitro fertilization. In methylcellulose clonal culture of fetal liver (FL) cells of generated hGMR-expressing EPOR(-/-) embryos at embryonic day (E) 12.5 of gestation, hGM-CSF stimulated erythroid colony formation under serum-containing and serum-free conditions. Analysis of globin expression in individual erythrocyte-containing colonies formed from E12.5 FL cells showed that hGM-CSF supports primitive and definitive erythropoiesis even in EPOR(-/-) embryos. In comparison of activities between hGM-CSF and EPO in hGMR-expressing EPOR(+/+) embryos, the 2 substances supported the formation of similar numbers of erythroid colonies in clonal culture of E12.5 FL cells; enhanced adult, but not embryonic, globin synthesis; and induced increase of GATA-1 expression and decrease of erythroid Kruppel-like factor and cMyb expression in the FL cells. On the other hand, in E8.0 yolk sac erythropoiesis, both substances had a similar effect on erythroid colony formation, but hGM-CSF induced an increase of beta-major globin expression, while EPO did not. All together, the results of the present study demonstrated that hGM-CSF can stimulate the proliferation and differentiation of primitive and definitive erythroid cells independently of EPOR signal if they express hGMR, and the activity is comparable to that of EPO in definitive, but not primitive, erythropoiesis.

Intracranial aneurysms in Ehlers-Danlos syndrome type IV in early childhood.

Ehlers-Danlos syndrome type IV is of special interest to neurologists because of the risk of cerebrovascular complications. We describe a 5-year-old female with Ehlers-Danlos syndrome type IV, demonstrating multiple intracranial aneurysms and right middle cerebral artery stenosis. The diagnosis of Ehlers-Danlos syndrome type IV was confirmed by electron microscopic examination of a skin biopsy. To our knowledge, this is the youngest reported patient with intracranial aneurysms associated with the Ehlers-Danlos syndrome type IV. Ehlers-Danlos syndrome type IV should be considered in the differential diagnosis of cerebrovascular disorder and stroke in early childhood.