A prospective study of cyclosporine A treatment of patients with low-risk myelodysplastic syndrome: presence of CD55(-)CD59(-) blood cells predicts platelet response.

Although immunosuppressive therapy using antithymocyte globulin or cyclosporine A (CSA) is effective in selected patients with low-risk myelodysplastic syndrome, the response rates reported so far are inconsistent, and the indication of immunosuppressive therapy for myelodysplastic syndrome has not been clearly defined. We treated 20 myelodysplastic syndrome patients (17 refractory anemia cases [RA], 2 RA with excess blasts, and one RA with ringed sideroblasts) with 4 mg/kg per day of CSA for 24 weeks. Among the 19 patients evaluated, 10 showed hematologic improvement; 8 patients showed an erythroid response, 6 showed a platelet response, and one showed a neutrophil response. Most patients with hematologic improvement continued CSA thereafter, and the progressive response was observed until the latest follow-up (median, 30 months). Most toxicities associated with CSA usage were manageable, and no patient had developed acute leukemia up to this point. Short duration of illness, refractory anemia with minimal dysplasia determined by bone marrow morphology, and the presence of paroxysmal nocturnal hemoglobinuria-type cells were significantly associated with the platelet response. A minority of RA patients who did not possess such predictive variables achieved an isolated erythroid response. In conclusion, CSA may be a therapeutic option for patients with RA who do not have adverse prognostic factors.

[Benefits of mycophenolate mofetil for refractory graft-versus-host disease].

We retrospectively evaluated the efficacy of mycophenolate mofetil (MMF) in the treatment of steroid-resistant acute and chronic graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. Thirteen patients, ten men and three women, consisted of 5 cases of acute myelogenous leukemia, 2 of acute lymphoblastic leukemia, 2 of chronic myelogenous leukemia, 2 of lymphoblastic lymphoma, and 1 case each of adult T-cell leukemia and peripheral T-cell lymphoma. The transfusions consisted of 5 peripheral blood, 7 bone marrow and 1 cord blood from 3 mothers, 4 siblings and 6 unrelated donors with conditioning treatments, including 8 total-body irradiation-based regimens, and 2 busulfan plus cyclophosphamide and 2 reduced-intensity regimens. GVHD prophylaxis included FK506 plus methotrexate (MTX) and/or antithymocyte globulin for 9 patients, and cyclosporine and MTX for 4 patients. All patients were treated with second-line MMF for steroid-refractory acute and/or chronic GVHD, and 11 patients improved. The adverse events were tolerable except for one patient in whom grade 3 neutropenia forced discontinuation of treatment. No case of non-relapse mortality occurred. We consider that MMF is beneficial and well tolerated for treatment of steroid-refractory GVHD.

[Molecular targeting therapy for multiple myeloma].

Molecular target therapy is the most progressive and promising anticancer therapy in last decade. Multiple myeloma is also one of the major therapeutic targets for using molecular based technology. The recent availability of clinical data regarding thalidomide-, lenalidomide-, and bortezomib-based regimens has provided new, effective treatment options for patients with both newly diagnosed and relapsed/refractory multiple myeloma. We are expecting that future clinical trials can be designed to achieve a high likelihood of success based on molecular studies, cell-signaling, and correlative science studies. Studies with these agents also provide new insight into the cancer biology underlying multiple myeloma in humans.

Pleocytosis after hemopoietic stem cell transplantation.

Frequency and clinical significance of cerebrospinal fluid (CSF) pleocytosis in hemopoietic stem cell (HSC) transplantation were surveyed. Cyclosporine (CSA)- or tacrolimus (FK506)-based regimens were used as graft-vs-host disease (GVHD) prophylaxis in allogeneic HSC transplantation. CSF pleocytosis with or without neurologic symptoms was detected in 12 of 25 patients receiving allogeneic HSC transplants but in none of 11 patients receiving autologous HSC transplants. Of the 12 patients with CSF pleocytosis, only one patient developed leukoencephalopathy later. There was a correlation between CSF cell numbers and trough levels of CSA but not with those of FK506. In patients receiving allogeneic HSC transplants, CSF pleocytosis may be relatively common and may reflect neurologic damage associated with calcineurin inhibitors.

Hematopoietic microchimerism in sheep after in utero transplantation of cultured cynomolgus embryonic stem cells.

BACKGROUND: Although directed differentiation of human embryonic stem (ES) cells would enable a ready supply of cells and tissues required for transplantation therapy, the methodology is limited. We have developed a novel method for hematopoietic development from primate ES cells. We first cultured cynomolgus monkey ES cells in vitro and transplanted the cells in vivo into fetal sheep liver, generating sheep with cynomolgus hematopoiesis. METHODS: Cynomolgus ES cells were induced to mesodermal cells on murine stromal OP9 cells with multiple cytokines for 6 days. The cells (average 4.8 x 10 cells) were transplanted into fetal sheep in the liver (n=4) after the first trimester (day 55-73, full term 147 days). The animals were delivered at full term, and two of them were intraperitoneally administered with human stem-cell factor (SCF). RESULTS: Cynomolgus hematopoietic progenitor cells were detected in bone marrow at a level of 1% to 2% in all four sheep up to 17 months posttransplant. No teratoma was found in the lambs. After SCF administration, the fractions of cynomolgus hematopoiesis increased by several-fold (up to 13%). Cynomolgus cells were also detected in the circulation, albeit at low levels (<0.1%). CONCLUSIONS: Long-term hematopoietic microchimerism from primate ES cells was observed after in vitro differentiation to mesodermal cells, followed by in vivo introduction into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation.

Severe hepatitis and complete molecular response caused by imatinib mesylate: possible association of its serum concentration with clinical outcomes.

A 40-year-old female with chronic myelogeneous leukemia (CML) in the chronic phase was treated with imatinib mesylate (STI571) because of interferon resistance. She achieved complete cytogenetic response but not complete molecular response 3 months after STI571 administration. Six months later, she developed severe liver damage without evidence of actively infectious hepatitis A, B, C, G, E, TT virus, Epstein-Barr virus or cytomegalovirus. A significant serum level of STI571 (107 ng/ml) was detected, although she had not taken the drug for 6 days. Liver biopsy demonstrated massive hepatic necrosis, consistent with drug-induced hepatitis. She achieved complete molecular response, although she did not take STI571 for 47 days after the development of hepatitis. These results suggest that both hepatitis and molecular response were associated with the serum STI571 concentration.

Involvement of the esophagus and stomach as a first manifestation of varicella zoster virus infection after allogeneic bone marrow transplantation.

A 46-year-old man with myeloproliferative disorder received a stem cell transplant from an HLA-identical unrelated donor. Eight months status post transplantation, during the course of tacrolimus therapy, the patient developed severe epigastric pain and fever. FGS findings showed eruptions with blisters in the esophagus and ulcers in the stomach. Biopsy specimens revealed acidophilic inclusion bodies in the nuclei. Varicella zoster virus (VZV) DNA copies were detected in the serum. No skin lesions were observed prior to hospital admission. The diagnosis of visceral VZV infection was made and the gastric and esophageal lesions were successfully healed with acyclovir (ACV). Severe abdominal pain is one of the most important signs of VZV infection for recipients of stem cell transplantation.

[Successful treatment of hairy cell leukemia prolymphocytic variant with 2'-deoxycoformycin].

The hairy cell leukemia prolymphocytic variant, a subtype of hairy cell leukemia, is an extremely rare disease, especially in Japan. We report a case in which treatment with 2'-deoxycoformycin (DCF) improved the clinical features of the disease. The patient, a 70-year-old female, was first treated with 2-chlorodeoxyadenosine, but showed only transient improvement in the hematological findings. DCF was then administered every week. Following the start of this treatment, the leukemia cell count rapidly decreased and the platelet count simultaneously increased. This effect of DCF has so far been long term. More clinical studies are needed to confirm the therapeutic value of DCF.

[Treatment with a proteasome inhibitor, bortezomib, for thalidomide-resistant multiple myeloma].

A proteasome inhibitor with a new molecular target (PS-341: bortezomib) was recently developed, and its efficacy in the treatment of refractory multiple myeloma has been reported in the United States. Here, we present a 54-year-old Japanese male patient with refractory multiple myeloma resistant to thalidomide. In 1998, the patient was diagnosed as having multiple myeloma (IgG-kappa) and underwent chemotherapy, autologous hematopoietic cell transplantation and interferon therapy, but the disease recurred. In December 2002, thalidomide and high-dose dexamethasone therapy was initiated, and while this combination therapy was effective at first, the multiple myeloma became unresponsive. On 23 June 2003, bortezomib therapy with the following regime was therefore started: 2.2 mg (1.3 mg/m2) of bortezomib was injected intravenously on days 1, 4, 8 and 11, and after a one-week break, another cycle was performed. Starting on day 8 of the administration, the serum total protein, IgG, serum calcium and LDH levels decreased rapidly, and after day 45 of the administration, blood transfusion was no longer needed. Since this is the first report of the use of bortezomib in the treatment of refractory multiple myeloma in Japan, further monitoring of this patient will provide extremely valuable information for developing a therapy against this disease.

Morphologic analysis in myelodysplastic syndromes with del(5q) treated with lenalidomide. A Japanese multiinstitutional study.

Lenalidomide is known to be effective in myelodysplastic syndromes (MDS) with del(5q) in improving anemia and suppressing del(5q) cells. MDS with del(5q) shows increase of nonlobulated megakaryocytes. However, histopathology of MDS with del(5q) treated with lenalidomide has not been fully studied. We investigated the morphologic changes in lenalidomide treated low- or intermediate-1-risk MDS with del(5q). All of evaluable patients showed high proportion of nonlobulated megakaryocytes. The nonlobulated megakaryocytes were markedly decreased in 6 patients during therapy in parallel with suppression of del(5q) cells. Our analysis suggests that single allele deletion of common deleted region inhibits nuclear lobulation of megakaryocytes.

Lenalidomide plus dexamethasone treatment in Japanese patients with relapsed/refractory multiple myeloma.

We conducted a multicenter, open-label study to investigate the safety, efficacy, and pharmacokinetics of lenalidomide in Japanese patients with relapsed or refractory multiple myeloma The study was composed of the "monotherapy phase", a dose-escalation phase, to determine the tolerability to single agent lenalidomide and the "combination phase" to determine the safety and obtain preliminary data on the efficacy of lenalidomide plus dexamethasone. The primary end points were the tolerability to 25 mg lenalidomide and safety. Nine and six patients were enrolled in the monotherapy phase and the combination phase, respectively. Since 25 mg of monotherapy treatment did not satisfy the DLT criteria, this dose was employed in the combination phase. The major adverse event was myelosuppression. At the planned interim analysis (median study duration, 26.3 weeks), grade 3 or grade 4 neutropenia was observed with high frequency (66.7%). However, all adverse events observed were clinically manageable. In the combination cohort, the overall response rate (> or =PR) was 100%. The pharmacokinetics of lenalidomide showed rapid absorption and elimination after both single and multiple doses. In conclusion, 25 mg of lenalidomide was given safely as a single agent or in combination with dexamethasone in Japanese patients. The good efficacy of the combination therapy was also demonstrated in this study.

Lenalidomide is active in Japanese patients with symptomatic anemia in low- or intermediate-1 risk myelodysplastic syndromes with a deletion 5q abnormality.

Lenalidomide is an immunomodulatory agent recently reported to be effective in the treatment of transfusion-dependent anemia due to low- or intermediate-1 risk myelodysplastic syndromes (MDS) associated with a deletion 5q (del 5q) cytogenetic abnormality. We conducted a multicenter, single-arm clinical trial to evaluate the safety and efficacy of lenalidomide in Japanese patients with anemia in low- or intermediate-1 risk MDS associated with the del 5q cytogenetic abnormality. Eleven patients (5 with transfusion-dependent anemia; 6 with transfusion-independent symptomatic anemia) received once daily oral administrations of 10 mg of lenalidomide for 21 consecutive days in a 28-day treatment cycle. The efficacy was assessed by the IWG criteria. At an interim analysis after > or =24 weeks of therapy, hemoglobin increase was noted in all 11 patients, with a median increase of 6.0 g/dL (range, 0.9-10.9) from the baseline. All transfusion-dependent patients achieved transfusion independence. Histopathologic and cytogenetic improvement was also noted. Neutropenia and thrombocytopenia were the most common adverse events related to lenalidomide. The adverse events were manageable, and no patients experienced serious adverse events or adverse events requiring treatment discontinuation. The results indicate that lenalidomide can be a useful agent for treating Japanese patients with anemia associated with low- or intermediate-1 risk MDS with the del 5q cytogenetic abnormality.

Granulocyte-colony stimulating factor for the treatment of ritodrine-induced neutropenia.

We report on three pregnant women with ritodrine-induced neutropenia who were successfully treated with granulocyte-colony stimulating factor (G-CSF). The neutropenia occurred after continuous intravenous infusion of ritodrine for preterm labor. Ritodrine was discontinued and G-CSF was administered. Neutrophil counts returned to normal an average of 4.3 days after the administration. No infectious morbidity or adverse side-effects occurred in the mothers or infants. G-CSF is one possible treatment in women with ritodrine-induced neutropenia.

Retrospective nationwide survey of Japanese patients with transfusion-dependent MDS and aplastic anemia highlights the negative impact of iron overload on morbidity/mortality.

OBJECTIVE: Myelodysplastic syndromes (MDS) and aplastic anemia (AA) are the most common anemias that require transfusion therapy in Japan. This retrospective survey investigated relationships between iron overload, chelation practices, and morbidity/mortality in patients with these diseases. METHOD: Medical histories of transfusion-dependent patients were assessed at transfusion onset, chelation onset, and study end. RESULTS: Data were collected from 292 patients with MDS, AA, pure red cell aplasia, myelofibrosis, and other conditions. Patients received a mean of 61.5 red blood cell units during the previous year. Fewer than half (43%) of patients had previously received deferoxamine (DFO) therapy. Only 8.6% received daily/continuous DFO. In all, 75 deaths were reported, with cardiac and liver failure noted in 24.0 and 6.7% of cases. Of these, 97% had ferritin levels >1000 ng/mL. Abnormal cardiac and liver function was observed in 21.9% (14/64) and 84.6% (11/13) of all patients assessed. Effective chelation with DFO resulted in improved serum ferritin, liver enzymes, and fasting blood sugar. CONCLUSIONS: Mortality is higher in heavily iron-overloaded patients, with liver and cardiac dysfunction being the primary cause. Daily/continuous chelation therapy was effective at reducing iron burden and improving organ function. Chelation therapy should be initiated once serum ferritin levels exceed 1000 ng/mL.

Histone deacetylase inhibitor depsipeptide (FK228) induces apoptosis in leukemic cells by facilitating mitochondrial translocation of Bax, which is enhanced by the proteasome inhibitor bortezomib.

Histone deacetylase (HDAC) inhibitors are promising candidates for molecular-targeted therapy for leukemia. In this study, we investigated the mechanisms of cytotoxic effects of depsipeptide (FK228), one of the most effective HDAC inhibitors against leukemia, using human myeloid leukemic cell lines HL-60 and K562. We found that FK228 activated caspase-9 and a subsequent caspase cascade by perturbing the mitochondrial membrane to release cytochrome c, which was almost completely blocked by overexpression of Bcl-2. The mitochondrial damage was caused by the translocation of Bax but not other pro-apoptotic Bcl-2 family proteins to the mitochondria. FK228 did not affect the interaction between Bax and Bax adaptor proteins such as 14-3-3theta and Ku70. FK228-induced apoptosis and mitochondrial translocation of Bax were markedly enhanced by the proteasome inhibitor bortezomib. The synergistic action of FK228 and bortezomib was at least partly mediated through conformational changes in Bax, which facilitate its translocation to the mitochondria. These results suggest that the combination of HDAC inhibitors and proteasome inhibitors is useful in the treatment of leukemia especially in the context of molecular-targeted therapy. The status of Bcl-2 and Bax may influence the sensitivity of tumors to this combination and thus can be a target of further therapeutic intervention.

The usefulness of magnetic resonance imaging (MRI) for disseminated trichosporosis of the gastrocnemius muscles.

A 30-year-old man with acute myeloid leukemia who was pancytopenic after undergoing intensive chemotherapy developed pyrexia and severe pain of both lower legs. We immediately started empiric therapy with cefepime, vancomycin, and fluconazole for febrile neutropenia. However, symptoms progressed. After 4 days, Trichosporon was isolated from venous blood cultures. MRI showed hyperintense lesions within both gastrocnemius muscles and demonstrated reactive vasodilatation and interstitial tissue edema, thought to be induced by hyperpermeability of vessel membranes due to the local fungal infection. Amphotericin B was very effective against this organism. Trichosporosis is a rare infectious disease generally occurring in immunocompromized hosts. To the best of our knowledge, this is first reported case of bilateral Trichosporon infection of lower leg muscles. Severe leg pain was one of the most important signs of fungal infection in this patient with hematologic malignancy.

Severe pulmonary complications in Japanese patients after bortezomib treatment for refractory multiple myeloma.

Bortezomib is a novel proteasome inhibitor with significant antimyeloma activity. Its frequent adverse effects are manageable, including gastrointestinal symptoms, peripheral neuropathy, and thrombocytopenia. Severe lung toxicity has not previously been reported. Between June 2004 and September 2005, 13 Japanese patients with multiple myeloma were treated with bortezomib in Toranomon Hospital, Juntendo University School of Medicine, and Jichi Medical School. Four of them developed severe pulmonary complications, and 2 died of respiratory failure without progression of underlying disease. To our knowledge, this is the first report on life-threatening pulmonary adverse effects after bortezomib therapy. Previous clinical studies on bortezomib, mostly in the United States and Europe, have shown low incidences of pulmonary adverse effects. Our study suggests that bortezomib can cause serious lung injury, and that its incidence might vary among different ethnicities. Clinicians need to be alert to the possibility.

Low-dose total body irradiation causes clonal fluctuation of primate hematopoietic stem and progenitor cells.

Due to high frequency of side effects caused by high-dose total body irradiation (TBI) the nonmyeloablative regimen together with cytotoxic agents is currently used especially for elderly patients. However, immediate and long-term effects of low-dose irradiation used in allogeneic transplantation on stem cells is less well known. We have studied the effect of low-dose 3 Gy TBI on the number of hematopoietic stem cell (HSC) clones contributing simultaneously to granulocyte production in rhesus macaque. The number of clones after 3 Gy TBI decreased markedly by 2 to 3 weeks after 3 Gy TBI, followed by a period of clonal instability, and recovery to almost pre-3 Gy TBI clonal diversity. The clones accounting for this recovery contributed before 3 Gy TBI, suggesting the profound initial impact of TBI was on a pool of progenitor cells, whereas most of the more primitive HSCs remained unaffected and were able to again contribute to hematopoiesis after recovery. Clonal fluctuation may indirectly suggest the presence of short-term/long-term HSC populations in rhesus macaque bone marrow as reported in a mouse model. The results indicate that even low-dose irradiation affects hematopoietic clonal dynamics and have implications for design of conditioning regimens for transplantation purposes.

Autologous gamete cryopreservation before hemopoietic stem cell transplantation.

BACKGROUND: Infertility after hemopoietic stem cell transplantation (HST) is a serious problem for young patients. Autologous gamete collection before HST may be a promising strategy to overcome infertility. MATERIAL/METHODS: From October 1988 to December 2003, six male and nine female patients with hematological malignancies had autologous gametes collected before HST. The data on autologous gamete collection were analyzed. RESULTS: Sperm could be collected from three patients. However, in two of the three, the numbers and motility of the sperm were severely depleted because they received chemotherapy for one and 11 cycles, respectively. Normal sperm was only collected from one patient with myelodysplastic syndrome who had no history of receiving chemotherapy. One or more oocytes could be collected in five of nine female patients, although the five received multiple cycles of chemotherapy. The successful oocyte collection was associated with an ovulation stimulant. CONCLUSIONS: Autologous oocye collection before HST may be possible, even if patients receive multiple cycles of chemotherapy. In contrast, autologous sperm collection before HST may be difficult after patients receive chemotherapy. Successful pregnancy using autologous gametes after HST remains extremely difficult, especially in female patients; however, it is important to give information on infertility and autologous gamete collection to patients scheduled for HST.

A soluble CAR-SCF fusion protein improves adenoviral vector-mediated gene transfer to c-Kit-positive hematopoietic cells.

BACKGROUND: Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). METHODS: We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF. RESULTS: Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following Ad5-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells. CONCLUSIONS: The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies.