Hisayuki Yokoyama

Toxic effects of sorafenib when given early after allogeneic hematopoietic stem cell transplantation.

To the editor: We read with interest the article by Metzelder et al showing sorafenib had antileukemic activity and could be given safely to patients with FLT-3 mutated AML relapsing after allogeneic stem cell transplantation (ASCT).1 Because sorafenib delays progression of renal cell carcinoma, we administered this drug to patients who had progression of metastatic kidney cancer after an ASCT. Besides the classic sorafenib hand-foot syndrome, we also observed new-onset, biopsy-confirmed chronic graft-versus-host disease (cGVHD) of the skin (including one case of sclerodermoid cGVHD) and exacerbations of preexisting chronic skin GHVD in 4 of 7 patients treated with 400 mg of sorafenib given orally twice daily. Metzelder et al also noted cGVHD occurred in 2 of 4 patients receiving sorafenib after ASCT, although the temporal association of this drug with cGVHD is unclear from their study. Although the authors speculate sorafenib might be effective when given prophylactically after an ASCT to reduce FLT-3 mutated AML leukemia burden, it is important to note that the initiation of sorafenib in the 4 patients in their study was delayed months after the transplantation (87-322 days). In vitro and murine findings from our laboratory raise the concern that sorafenib may result in substantial toxicity and increase the risk of GVHD when this drug is administered early after a T cell–replete ASCT. Using a major histocompatibility complex (MHC)–matched murine model of ASCT, we explored whether sorafenib would slow tumor progression potentially facilitating GVT effects in mice with established RENCA tumors. Balb/C mice conditioned with 950cGy total body irradiation received either a T cell–depleted (bone marrow alone) or T cell–replete (bone marrow plus splenocytes) ASCT from MHC-matched, minor antigen–mismatched B10.d2 donors. Nontransplanted tumor-bearing Balb/C control mice that received sorafenib by oral gavage (60 mg/kg/day) had no evidence of drug toxicity and had slower tumor growth which improved survival compared with mice not receiving sorafenib (median survival 49 vs 34 days, respectively; P = .04). In recipients of a T cell–depleted ASCT, sorafenib by oral gavage was not associated with overt toxicities and also delayed tumor progression and improved survival compared with nonsorafenib controls (median survival 42 vs 31 days; P < .01). Remarkably, T cell–depleted transplant recipients that received sorafenib had no evidence of organ toxicity or GVHD at autopsy. In contrast, there was a surprising and significant increase in clinical GVHD (Figure 1) and histologically confirmed severe skin and liver GVHD (P = .0023) with a trend toward shortened survival when sorafenib was administered to recipients of a T cell–replete SCT (Figure 1). Blood samples showed a nonsignificant increase in the percentage of CD3+ T cells in mice that received a T cell–replete ASCT with sorafenib versus without sorafenib (62% ± 29% vs 28% ± 10%; P = .26). Figure 1 Sorafenib worsens GVHD and shortens survival when given after a T cell–replete allogeneic SCT in mice with RENCA tumors. GVHD score and survival in tumor bearing mice undergoing allogeneic SCT using BM + splenocytes with or with sorafenib given ... Although the exact mechanism through which this agent enhances GVHD in vivo remains under investigation, correlative in vitro studies using human peripheral blood mononuclear cells obtained from healthy volunteers revealed OKT3-induced T-cell proliferation increased significantly in the presence of sorafenib (median stimulation index 4.6; range, 1.8-10.8; P = .035). These preclinical murine studies and early observations in humans raise the concern that sorafenib may exacerbate GVHD, and imply the early or prophylactic use of this agent after a T-cell replete ASCT for FLT-3-ITD–positive AML should be pursued with caution and should be given only in the context of a clinical trial.

Allogeneic hematopoietic stem cell transplant following chemotherapy containing L-asparaginase as a promising treatment for patients with relapsed or refractory extranodal natural killer/T cell lymphoma, nasal type

The prognosis of advanced extranodal NK/T cell lymphoma (ENKTL) is poor. Allogeneic hematopoietic stem cell transplant (allo-HSCT) has been suggested to be a promising treatment for this disease, but its utility has yet to be established. Here we retrospectively analyzed five cases of ENKTL treated with allo-HSCT in our institute. After induction chemotherapy, disease status at allo-HSCT was second CR in three patients and refractory in two patients. All patients received a myeloablative conditioning regimen, and GVHD prophylaxis consisted of tacrolimus or cyclosporine with short-term methotrexate. Only one patient who received conventional induction chemotherapy developed severe complications, which needed long-term treatment, while others who received chemotherapy containing l-asparaginase did not have severe complications. There were no cases of treatment-related mortality, and all patients survived without disease for a median follow-up period of 1911 days. These results suggested that allo-HSCT following l-asparaginase-containing induction chemotherapy might improve the outcome of advanced ENKTL.

Safety and efficacy of rasburicase (SR29142) in a Japanese phase II study

The purpose of this study was to investigate the safety profile of SR29142 when administered as a single agent both prior to chemotherapy and during treatment, and to compare the efficacy of SR29142 administered at two dose levels in adult Japanese patients with leukemia or lymphoma. During this open‐label, multicenter, phase II study, patients received SR29142 for 5 days, administered at either 0.15 or 0.20 mg/kg per day. Chemotherapy was started 4–24 h after the first infusion of SR29142. The primary end‐point was overall response rate, defined as the normalization of plasma uric acid to 7.5 mg/dL or less, from 48 h after the first infusion to 24 h after the last infusion of SR29142. SR29142‐related adverse events including hypersensitivity (allergic) reactions were assessed. Overall, 50 patients received SR29142 at either 0.15 mg/kg per day (n = 25) or 0.20 mg/kg per day (n = 25) followed by chemotherapy. The overall response rate was 100.0% (95% confidence interval, 86.3–100.0%) with 0.15 mg/kg and 96.0% (95% confidence interval, 79.6–99.9%) with 0.20 mg/kg. Both dose levels of SR29142 were equally effective at reducing plasma uric acid levels. In six patients, seven drug‐related adverse events of grade 1/2 occurred before chemotherapy. SR29142‐related, hypersensitivity‐associated reactions occurred in three patients, and rash, anorexia, application site pain and pyrexia occurred in one patient each; only five patients (10%) showed anti‐SR29142 antibodies by day 29. In conclusion, SR29142 is effective at reducing plasma uric acid levels with a tolerable safety profile as a single agent both prior to chemotherapy and during treatment. (Trial register: ClinicalTrials.gov, NCT00631579.) (Cancer Sci 2009; 100: 357–362)

CD26, together with cell surface adenosine deaminase, is selectively expressed on ALK-positive, but not on ALK-negative, anaplastic large cell lymphoma and Hodgkin's lymphoma

CD26/dipeptidyl peptidase IV is a cell surface antigen with multiple biological functions. Although its involvement in tumor biology has been suggested, the significance of its expression in malignant lymphoma has not been clarified in detail. This study examined the expression of CD26 and cell surface adenosine deaminase (ADA) in 42 cases of Hodgkins lymphoma (HL) and T-cell lymphoma by immunohistochemistry on frozen sections. CD26 was expressed in three of 14 cases of HL, in four of eight cases of anaplastic large cell lymphoma (ALCL), in two of nine cases of peripheral T-cell lymphoma, in one of six cases of lymphoblastic lymphoma and in none of three cases of adult T-cell lymphoma/leukemia. Expression of cell surface ADA was fully correlated with the expression of CD26 and expression of CD26/ADA in ALCL and HL was also completely correlated with the expression of p80 and epithelial membrane antigen. Of 10 CD26-positive patients, seven had fever and elevated CRP at initial diagnosis and over a median follow-up of 61 months (range, 7 – 152 months) only three survived. This study suggested that CD26 is selectively expressed on ALK-positive, but not on ALK-negative, ALCL and HL. This is also the first report to demonstrate that ADA is coexpressed with CD26 on the cell surface of malignant neoplasms in vivo.

Autoimmune neutropenia in pregnant women causing neonatal neutropenia

Autoimmune neutropenia (AIN) can occur during pregnancy. However, neonatal neutropenia occurring in an infant born to a mother with AIN has only rarely been documented. Recently, we have experienced two cases of AIN during pregnancy, both of which caused severe yet transient neonatal neutropenia (< 0·3 × 109/l), probably as a result of transplacental maternal anti‐neutrophil autoantibodies. The anti‐neutrophil antibodies seemed to be against antigens other than NA1/NA2 because the autoantibodies did not bind to neutrophils of specific NA types selectively in the granulocyte indirect immunofluorescence test. Although AIN is a relatively uncommon disease, neonatal neutropenia caused by maternal AIN may not be quite as rare.

Synergistic Effect of Arsenic Trioxide and Flt3 Inhibition on Cells with Flt3 Internal Tandem Duplication

Flt3 internal tandem duplication (Flt3-ITD) is a prevalent mutation in acute myeloid leukemia (AML). Flt3-ITD constitutively activates various signaling pathways, including a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Arsenic trioxide (ATO) and MEK inhibition were recently reported to interact synergistically to induce apoptosis in AML cells. In this study, we aimed to clarify whether ATO and Flt3 inhibition would be a mor specific and efficient therapy for Flt3-ITD cells.We demonstrate that the combination of ATO and an Flt3 inhibitor,AG1296, profoundly inhibits the growth of Flt3-ITD cells and induces their apoptosis. We further revealed that this combined treatment potently inhibits the ERK activity that might be responsible for cell growth. Moreover, using the Chou-Talalay method, we observed a synergistic growth-inhibitory effect for ATO and AG1296 in Flt3-ITD cells (BaF3-Flt3-ITD, MV4-11, and PL-21 cells), but not in Flt3 wild-type cells (RS4-11 and NB4 cells), for almost all dose ranges tested. Our results provide an experimental basis for a specific and efficient therapy for Flt3-ITD cells that involves combined treatment with Flt3 inhibitors and ATO.

Clonal evolution from trisomy into tetrasomy of chromosome 8 associated with the development of acute myeloid leukemia from myelodysplastic syndrome

Tetrasomy 8, though rare, is usually associated with trisomy 8, a far more common chromosomal abnormality in acute myeloid leukemia (AML). Yet the clonal relationship between trisomy 8 and tetrasomy 8 in the cases with these chromosomal abnormalities has been unclear. Here, we report a case of a 17-year-old male, diagnosed as having a myelodysplastic syndrome (MDS). Chromosome analysis showed the presence of trisomy 8. Five years later, he developed overt AML exhibiting tetrasomy 8 only. After chemotherapy, the blast cells in the bone marrow decreased to 3.4%, and the karyotype showed trisomy 8 alone. Fluorescence in situ hybridization using a probe specific for chromosome 8 showed that the percentages of cells exhibiting 2/ 3 /4 signals were 7.8/89.2/2.0 at the MDS stage, 20.5/36.1/41.0 when overt AML developed and 24.0/72.1/2.4 after chemotherapy. These results suggested that tetrasomy 8 is derived from the AML clone, possibly evolved from the MDS clone with trisomy 8. To our knowledge, this is the first detailed case report of clonal evolution from trisomy 8 into tetrasomy 8 associated with the development of AML from MDS.

Induction of erythroid-specific genes by overexpression of GATA-2 in K562 cells

GATA transcription factors have been shown to play important roles in hematopoiesis. GATA-2 is expressed in stem and progenitor cells, and has been speculated to control the proliferation and maintain the immaturity of these cells. To examine whether the function of GATA-2 is changeable according to the differentiation stage, we established GATA-2 overexpress-ing subclones of K562, which is a leukemic cell line committed to the erythroid lineage. Via an increase in the GATA-2 expression level, the expression levels of erythroid-specific genes including a-, β-, and γ-globin were increased compared to control cells, while the expression level of GATA-1 was unchanged. Expression of the transferrin receptor was also increased in GATA-2 overexpressing K562 cells when examined by flow cytometry. In addition, the heme content of GATA-2 over-expressing K562 cells was more than 2 times higher than control cells. Chromatin immunoprecipitation analysis showed that GATA-2 protein binding to the GATA element in α-globin LCR was increased in GATA-2 overexpressing K562 cells. These findings suggest that GATA-2 could induce erythroid-specific genes without competition with GATA-1 when expressed in erythroid-committed cells, and thus further suggest that temporal and spatial regulation may be important for displaying specific functions of GATA-2.

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

High Expression of YB-1 Gene in Erythroid Cells in Patients with Refractory Anemia

It has been shown that aberrant expression of a transcription factor, GATA-1, leads to maturation arrest and transformation of erythroid cells. We previously reported that a multifunctional protein, YB-1, was expressed strongly in the spleen of a GATA-1 mutant mouse, which was filled with transformed erythroblasts. This finding suggested that YB-1 has roles in ery-thropoiesis. In this study, we examined in vivo expression of YB-1 messenger RNA (mRNA) in bone marrow erythroid cells and erythroid leukemic cell lines. During erythroid differentiation of erythroid leukemic cell lines, the expression level of YB-1 mRNA was highest at the early phase of differentiation and then decreased. In human bone marrow cells, the in vivo expression level of YB-1 mRNA was higher in glycophorin A-positive cells than in glycophorin A-negative cells. An interesting finding was that expression of YB-1 was higher in erythroblasts in myelodysplastic syndrome-refractory anemia (MDS-RA) than in normal cells. The findings suggested that YB-1 functions in the early stage of erythropoiesis and that aberrant expression of this protein may induce hematological diseases such as MDS.