Takahiro Nagashima

Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan

Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation of cells to hypoxia, is frequently observed in numerous types of solid tumors. Dysregulation of HIF-1 induces tumor angiogenesis and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma. Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells. Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1 inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1 also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. [Mol Cancer Ther 2009;8(8):2329–38]

Paraproteinemia After Hematopoietic Stem Cell Transplantation

The frequency and clinical significance of paraproteinemia in patients receiving hematopoietic stem cell (HSC) transplants were assessed. Of 66 patients with hematologic malignancies, excluding multiple myeloma who received an allogeneic or autologous HSC transplant, paraproteins were detected in 12 patients using immunoelectrophoresis. None of the patients showed paraproteinemia before HSC transplantation. The class of paraproteins most commonly seen was IgG. In 9 of these 12 patients (75%), a paraprotein was detected continuously after HSC transplantation for an average duration of 464 days, while others demonstrated a transient appearance of the protein. Paraproteinemia after HSC transplantation was not related to the stem cell source, (allograft vs. autograft), age, gender, viral infection and graft-vs.-host disease (GVHD). None of the patients developed plasma cell dyscrasia after the appearance of the paraprotein, while 1 patient developed secondary acute lymphoblastic leukemia. These findings indicate that paraproteinemia after HSC transplantation may be caused by an aberrant immune reconstitution after both allogeneic and autologous HSC transplantation. A long-term follow-up of patients with paraproteinemia after HSC transplantation is needed to confirm this finding in a larger series of patients.

Evaluation of engraftment by ABO genotypic analysis of erythroid burst-forming units after bone marrow transplantation.

Six patients received an allogeneic bone marrow transplant from HLA-identical ABO-mismatched donors. ABO genotype of erythroid burst-forming units (BFU-E) from peripheral blood was analyzed using polymerase chain reaction with sequence specific primers (PCR-SSP). After bone marrow transplantation (BMT), engraftment of donor cells by ABO genotypic analysis of BFU-E was compared with ABO phenotypic analysis of red blood cells (RBCs). During the early stage after BMT, ABO genotype of BFU-E in the recipients converted to that of the donors. In contrast, mixed ABO phenotype of RBCs persisted for about 3 months. In one patient, autologous hemopoietic cell recovery was detected by the ABO genotypic analysis before clinical manifestation. ABO genotypic analysis of BFU-E is relevant for enagraftment after ABO-mismatched BMT.

Long-term results of dose-intensive chemotherapy with G-CSF support (TCC-NHL-91) for advanced intermediate-grade non-Hodgkin's lymphoma: a review of 59 consecutive cases treated at a single institute.

We evaluated the long-term outcome of very dose-intensive chemotherapy (TCC-NHL-91) for advanced intermediate-grade lymphoma, in which an eight-cycle regimen with 11 drugs was given with granulocyte colony-stimulating factor (G-CSF) support (total 18 weeks). Fifty-nine patients were treated during February 1, 1991 and March 31, 2001 (median age: 48 years). Forty-three patients (73%) were in a high-intermediate risk or high-risk group (HI/H) according to the age-adjusted International Prognostic Index (aa-IPI). Forty-six patients received 7 or 8 cycles of therapy. Ten of 15 patients over age 60 stopped before 7 cycles. Forty-three patients with an initial bulky mass or a residual mass received involved-field radiation. Overall, 56 patients (95%) achieved complete remission (CR). Grade 4 hematotoxicity was observed in all patients. With a median follow-up of 128 months, the 10-year overall survival (OS) and progression-free survival (PFS) rates were 76% and 61%, respectively. Neither aa-IPI risk factors nor the index itself was associated with response, OS, or PFS. One patient died of sepsis during the therapy and one died of secondary leukemia. This retrospective study suggests that the TCC-NHL-91 regimen achieves high CR, OS, and PFS in patients with advanced intermediate-grade lymphoma up to 60 years old and may be a valuable asset in the management of this disease. Further evaluation and prospective studies of the TCC-NHL-91 are warranted.

Rapid reduction in BCR-ABL1 transcript predicts deep molecular response in dasatinib-treated chronic-phase chronic myeloid leukaemia patients

We conducted a phase‐II study to evaluate the efficacy and safety of dasatinib in patients newly diagnosed with chronic‐phase chronic myeloid leukaemia (CML‐CP) in Japan (IMIDAS PART 2 study).

Wilms Tumor 1 Expression at Diagnosis Correlates With Genetic Abnormalities and Polymorphism But Is Not Independently Prognostic in Acute Myelogenous Leukemia: A Hokkaido Leukemia Net Study

&NA; The association between Wilms tumor 1 (WT1) expression, genetic abnormalities, and homozygous single nucleotide polymorphism (SNP) in the WT1 gene was evaluated in 252 patients with acute myelogenous leukemia. WT1 expression correlated with prognostic genetic abnormalities. Homozygous WT1 SNP rs16754 was associated with lower expression of WT1. However, WT1 expression had no prognostic effect in any cytogenetic group or SNP status. Background: The prognostic effect of Wilms tumor 1 (WT1) expression at the diagnosis of acute myelogenous leukemia (AML) has been controversial. The aim of the present study was to determine the correlations of WT1 expression at the diagnosis of AML with established prognostic alterations. Patients and Methods: We analyzed diagnostic bone marrow samples from 252 patients. WT1 expression, single nucleotide polymorphism (SNP) in the WT1 gene (rs16754), and Fms‐like tyrosine kinase receptor‐3 internal tandem duplication (FLT3‐ITD) mutation were analyzed for all patients. The nucleophosmin 1 (NPM1) mutation and CCAAT/enhancer‐binding protein‐&agr; (CEBPA) double mutation were analyzed for cytogenetically normal (CN)‐AML. The KIT mutation was analyzed for core‐binding factor AML. Results: Within the cytogenetically favorable prognosis group, WT1 expression in AML with inv(16) or t(15;17) was significantly greater than that in AML with t(8;21). In cases with CN‐AML, FLT3‐ITD and NPM1 mutations both correlated with greater expression of WT1, and the CEBPA double mutation was related to lower WT1 expression. The existence of both FLT3‐ITD and NPM1 mutations showed synergistically greater expression of WT1 in CN‐AML. SNP in the WT1 gene (rs16754) was significantly associated with lower expression of WT1. The WT1 levels were not prognostic factors in the total cohort or any cytogenetic group or stratified by SNP status. Conclusion: Because WT1 expression has correlated with known prognostic factors, the prognostic effect of WT1 levels could be misunderstood depending on the distribution of the collaborative mutations in each cohort. We have concluded that the prognostic significance of WT1 at the diagnosis of AML is weak compared with the other established prognostic factors.

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET‐based drug sensitivity test in which a CrkL‐derived fluorescent biosensor efficiently quantifies the kinase activity of BCR‐ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR‐ABL. Here, we validated the utility of the FRET‐based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty‐two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR‐ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET‐based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).

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.