Hiroyuki Sugahara

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line

ABSTRACT Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ∼30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Constitutively activated Rho guanosine triphosphatases regulate the growth and morphology of hairy cell leukemia cells.

Hairy cell leukemia (HCL) is a rare type of chronic B-cell leukemia characterized by the hairy morphology of the leukemia cells. All of 5 HCL samples and an HCL-derived cell line, BNBH-I, showed serrated edges and hairlike projections in May-Grünwald Giemsa stain and protruding actin spikes and lamellipodia in phalloidin stain.These structures were hardly detected on B-cell chronic lymphocytic leukemia (B-CLL) and precursor B-cell acute lymphocytic leukemia (B-ALL) cells. Because Rho guanosine triphosphatases (GTPases) regulate the formation of these structures, we examined the expression levels and activation states of Rho GTPases in HCL cells. RhoA, Rac1, and Cdc42 were overexpressed and constitutively activated in HCL samples and BNBH-I cells but not in B-CLL or precursor B-ALL cells. Next we overexpressed dominant-negative (DN)- RhoA, DN-Rac1, and DN-Cdc42 in BNBH-I.As a result, each DN mutant repressed the growth of BNBH-I cells by more than 50% and inhibited actin spike formation, but only DN-Rac1 suppressed lamellipodia formation.We also found that enforced expression of constitutively active-RhoA, Rac, or Cdc42 in the proB-cell line Ba/F3 was sufficient to induce actin spike formation, whereas none of these molecules produced lamellipodia. These results indicated that constitutively activated Rho GTPases regulate the growth and unique morphology of HCL cells.

Constitutive activation of c-kit by the juxtamembrane but not the catalytic domain mutations is inhibited selectively by tyrosine kinase inhibitors STI571 and AG1296.

The c-kit receptor tyrosine kinase (KIT) is constitutively activated by 2 types of naturally occurring mutations, the Val559→Gly (G559) mutation in the juxtamembrane domain and the Asp814→Val (V814) mutation in the catalytic domain. We evaluated the effects of the tyrosine kinase inhibitors STI571 and AG1296 on BaF3 cells expressing wild-type KIT (KITWT) or activating mutants of KIT (KITG559 and KITV814) in the presence or absence of the KIT ligand, stem cell factor (SCF). Both STI571 and AG1296 inhibited SCF-dependent activation of KITWT and SCF-independent activation of KITG559 more efficiently, whereas SCF-independent activation of KITV814 was scarcely affected. Furthermore, both inhibitors inhibited SCF-dependent growth of BaF3-KITWT cells and, with higher potencies, SCF-independent growth of BaF3-KITG559 cells through the induction of apoptosis. In contrast, the inhibitors had little or no effect on SCF-independent growth of BaF3-KITV814 cells or on IL-3-dependent growth of BaF3-Mock cells. These results suggested that both inhibitors may be effective therapeutic agents for oncogenic KIT with the juxtamembrane domain mutation, but not with the catalytic domain mutation, and that the activation mechanism of the catalytic domain mutant KIT is complex and entirely different from that of the wild-type KIT or the juxtamembrane domain mutant KIT.Int J Hematol. 2002; 76: 427-435.

Improvement of recurrent urticaria in a patient with Schnitzler syndrome associated with B-cell lymphoma with combination rituximab and radiotherapy

Schnitzler syndrome is a rare condition defined by chronic urticaria, osteosclerotic bone lesions, and monoclonal IgM gammopathy. Schnitzler syndrome can precede the onset of a true lymphoproliferative disorder including Waldenström macroglobulinemia and rarely systemic marginal zone B-cell lymphoma. We describe a case of intractable chronic urticaria accompanied by a retroperitoneal neoplasm. IgM monoclonal gammopathy, lumber pain, intermittent fever, and elevation of C-reactive protein were the clues for the diagnosis of Schnitzler syndrome. An evaluation for malignancy using systemic computed tomography scan and fluorodeoxyglucose positron emission tomography revealed the retroperitoneal tumor, and a subsequent bone-marrow aspirate confirmed the diagnosis of B-cell lymphoma. Combined rituximab and radiotherapy ameliorated the skin symptoms. This case indicates that a detailed search for malignant neoplasms might be required for the long-term management of Schnitzler syndrome, and that B-cell lymphomas may contribute to the pathogenesis of this condition.

Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant

FLT3 tyrosine kinase domain (TKD) mutations are detected in ∼7% of acute myeloid leukemia patients, and suggested to correlate with poor prognosis and confer resistance to FLT3 inhibitors. To explore activation mechanism of FLT3 TKD mutation, we analysed critical tyrosine residues for the constitutive activation and downstream signaling of the mutant by generating a series of single Tyr → Phe substitution mutant of all 22 cytoplasmic tyrosine residues of murine FLT3 TKD-mutant (mFLT3Asp838Val). Tyr845Phe, Tyr892Phe and Tyr922Phe substitutions suppressed the phosphorylation of mFLT3Asp838Val itself, the activation of Erk1/2, STAT3 and STAT5, and the factor-independent cell proliferation and survival. In contrast, these three Tyr → Phe mutations partially suppressed but maintained the ligand-dependent activation and anti-apoptotic activity of wild-type FLT3, suggesting that these tyrosine residues were more critical for the constitutive activation and signaling of mFLT3Asp838Val. These three Tyr → Phe mutations also inhibited the constitutive activation of other FLT3 mutants bearing internal tandem duplication, Asp838Tyr or Ile839del. The suppression of mFLT3Asp838Val activation and signaling by these substitutions was partially recovered by shifting the culture temperature from 37 to 33°C, or by the introduction of Cdc37 and Hsp90. Taken together, Tyr845, Tyr892 and Tyr922 are the critical residues in mFLT3Asp838Val activation, possibly through stabilizing the active conformation of mFLT3Asp838Val.

Footwear exchange has no influence on the incidence of febrile neutropenia in patients undergoing chemotherapy for hematologic malignancies.

OBJECTIVE To determine whether footwear exchange affects the incidence of febrile neutropenia among patients undergoing chemotherapy for hematologic malignancies. DESIGN Open trial with historical comparison. SETTING The 12-bed high-efficiency particulate air-filtered hematology unit at Osaka University Hospital, Suita, Japan. PATIENTS Those with hematologic malignancies who underwent chemotherapy from January 1997 through January 2003. Footwear exchange was discontinued in January 2000. METHODS The surveillance system was based on the National Nosocomial Infections Surveillance System of the Centers for Disease Control and Prevention. Rates of febrile neutropenia were calculated for neutropenic patient-days (ie, days with neutropenia < 500/microL). RESULTS From January 1997 through December 1999 and from February 2000 through January 2003, 58 and 54 patients endured 237 and 184 neutropenic periods following chemotherapy, and their total neutropenic days were 3,123 and 2,503, respectively. They showed episodes of febrile neutropenia 89 and 68 times, respectively. Infection rates were 28.5 and 27.2 per 1,000 neutropenic patient-days (P = .83), respectively. CONCLUSION The incidence of febrile neutropenia was not affected by footwear exchange. In hematology units, changing shoes does not appear to affect the rate of infections during neutropenic periods.

Standardized uptake value on FDG-PET as a marker for disease activity in patients with non-Hodgkin’s lymphoma: comparison with serum soluble interleukin-2 receptor values

BackgroundWe compared standardized uptake values (SUVs) on positron emission tomography with a glucose analog, 2-[F-18] fluoro-2-deoxy-D-glucose (FDG-PET) and serum soluble interleukin-2 receptor (sIL-2R) values in patients with non-Hodgkin’s lymphoma (NHL) in the pre-, mid- (after three or four cycles), and post-treatment periods of chemotherapy (pre, mid, and post, respectively), and we examined whether the SUV was a useful tumor marker for NHL.MethodsThe SUVs on PET and sIL-2R values were retrospectively evaluated based on all the clinical information available in 40 patients (31 in pre, 24 in mid, and 24 in the post periods). Patients in complete remission status were classified as group A and those with active residual disease in the mid and post periods were classified as group B.ResultsIn pre, the SUV and sIL-2R values exhibited sensitivity of 100% and 84%, respectively. In mid, the SUV was lower in group A than in group B, while sIL-2R was not different. The SUV yielded better specificity than sIL-2R (88 % vs 25 %, respectively), though the difference was not significant. In mid, the SUV in patients later assigned to group A in post was lower than than the SUV in group B, whereas sIL-2R was not different. In post, the specificity and accuracy of SUV were better than those of sIL-2R (95 vs 47 %, and 96 vs 58 %, respectively). Both the SUV and sIL-2R were lower in group A than in group B.ConclusionThe SUV on PET was better than the serum sIL-2R as a marker to evaluate the disease status of NHL, and was considered to be a useful tumor marker for NHL.

Successful treatment with rituximab for angioimmunoblastic T-cell lymphoma

We experienced a patient with angioimmunoblastic T-cell lymphoma (AITL) without Epstein-Barr virus-positive B (EBV-B) cells at initial presentation who progressed to AITL with expansion of EBV-B cells at relapse. Based on the results of repeated biopsy, the patient was successfully treated with rituximab in combination with chemotherapy at relapse. A repeat biopsy may be necessary to determine the optimum therapeutic strategy at relapse, particularly for patients with suspected expansion of B cell and/or EBV-B cells. Although a recent report found no significant prognostic advantage of rituximab, it is one of the active drugs for selected patients with AITL.

Reversible posterior leukoencephalopathy syndrome of bilateral thalamus in acute lymphoblastic leukemia

Numerous cases of reversible posterior leukoencephalopathy syndrome (RPLS) are reported in patients having risk factors such as malignant hypertension, eclampsia, receiving solid organ/hematopoietic stem cell transplant, and exposure to chemotherapeutic agents and immunosuppressive drugs. We experienced a very interesting case of atypical RPLS in the thalamus bilaterally accompanied by brain hemorrhage in acute lymphoblastic leukemia (ALL), and described here. A 62-year-old Japanese female was referred to our hospital with fever, general fatigue and leukocytosis in May 2011. Her white blood cell count was 139.6 10 9 /L, of which lymphoblasts constituted 96%, hemoglobin level was 8.2 g/ dL and platelet count was 15.0 10 9 /L. Leukemic lymphoblasts were peroxidase negative and their surface markers were positive for CD10, CD19, CD34 and human leukocyte antigen (HLA)-DR. Th e karyotype was 46, XY; the BCR/ABL fusion gene was not detected. Th e patient was diagnosed as having ALL and treated with cyclophosphamide, vincristine, adriamycin and prednisolone. Although leukemia cells were not present in peripheral blood at 9 days after starting chemotherapy, febrile neutropenia occurred, and antibiotic treatment was started immediately. After the administration of antibiotic and antifungal agents, erythroderma developed on her trunk, extremities and back. Stevens – Johnson syndrome (SJS) was diagnosed, and high-dose methylprednisolone was given. Hyponatremia (124 mEq/L) also occurred at this time, as an eff ect of the chemotherapeutic agents. Although the erythroderma and hyponatremia improved slowly over the next 4 days, her level of consciousness decreased gradually before falling suddenly, accompanied by high fever. Blood culture was positive for Pseudomonas aeruginosa while cerebrospinal fl uid (CSF) culture was negative. Th e patient ’ s level of consciousness improved after antibiotic treatment for sepsis; however, she lost consciousness once more, accompanied by high blood pressure (180/118 mmHg). Emergency computed tomography (CT) of the brain revealed multiple low-density areas in the thalamus, midbrain and pons bilaterally. Magnetic resonance imaging (MRI) of the brain performed 2 days after the CT scan showed multiple high signal intensity lesions with small hemorrhagic areas on T2-weighted fl uid attenuated inversion recovery imaging, T2 star-weighted imaging and diff usion-weighted imaging (Figure 1). No thrombosis of the bilateral internal vein and Galen vein was detected in brain CT and MRI. At 10 days after completion of treatment for sepsis and control of blood pressure, the patient was fully conscious. She was diagnosed with hemorrhage in atypical RPLS. MRI obtained 2 months after treatment showed no RPLS lesions