Rikio Suzuki

Preferential hypermethylation of the Dickkopf-1 promoter in core-binding factor leukaemia.

The Dickkopf‐1 (DKK1) gene product is an extracellular Wnt inhibitor. Hypermethylation of the DKK1 promoter results in transcriptional silencing and may play an important role in cancer development. Here, we investigated hypermethylation of the DKK1 promoter in patients with acute myeloid leukaemia (AML), especially core‐binding factor (CBF) leukaemia. The methylation status of DKK1 was analysed using methylation‐specific polymerase chain reaction in 47 patients with AML. DKK1 methylation was found in 14 (29·8%) patients, and more frequently in those with CBF leukaemia (6 of 12 patients), than in those with acute promyelocytic leukaemia (APL) (0 of 6 patients) (P = 0·03). In contrast, Wnt inhibitory factor‐1 methylation was found in APL (4 of 6 patients) but not in CBF leukaemia (0 of 12 patients) (P = 0·001). Multivariate analyses suggested that DKK1 methylation was a risk factor for poorer overall survival. Sequential analysis using four paired samples obtained at diagnosis and relapse suggested that DKK1 methylation was involved in the progression of leukaemia. Therefore, DKK1 methylation may be involved in leukaemogenesis, especially in CBF leukaemia, and may be a useful prognostic marker in AML.

Association of autoimmune disease-related gene polymorphisms with chronic graft-versus-host disease

Chronic graft‐versus‐host disease (GVHD) is the most common cause of poor outcomes after haematopoietic stem cell transplantation (HSCT), while the pathophysiology of chronic GVHD remains poorly understood. As both chronic GVHD and autoimmune disease share clinical features, we speculated that autoimmune disease‐related genes might be candidate chronic GVHD‐related genes. Recent large‐scale cohort studies showed that Fc receptor‐like 3 gene (FCRL3) single nucleotide polymorphism (SNP) and peptidylarginine deiminases citrullinating enzymes 4 gene (PADI4) haplotype were associated with autoimmune disease. The present study investigated the association between polymorphisms of these two genes and the incidence of chronic GVHD. We analysed 123 cases of Japanese human leucocyte antigen‐matched sibling recipients and their donors who underwent HSCT. Although PADI4, which is the rheumatoid arthritis‐specific related gene, was not associated with the occurrence of chronic GVHD, the recipient FCRL3‐169C/C genotype was significantly less frequent in chronic GVHD patients than in those without chronic GVHD (P = 0·0086). There was no relationship between FCRL3 polymorphism and acute GVHD. As FCRL3 is expressed by B cells and might have an important role in immunoregulation, this significant protective genetic effect raises the question of whether FCRL3 might also be involved in the pathogenesis of chronic GVHD.

Prognostic Significance of FLT3 Internal Tandem Duplication and NPM1 Mutations in Acute Myeloid Leukemia in an Unselected Patient Population

Mutations in the fms-like tyrosine kinase 3(FLT3) gene containing an internal tandem duplication(@#@ FLT3/ITD@#@) or mutations in the nucleophosmin 1 gene(NPM1) are thought to be prognostic indicators in acute myeloid leukemia (AML). Previous studies suggested that FLT3/ITD mutation indicates a poor prognosis and thatNPM1 mutation indicates a more favorable one, but these studies were often performed with selected patient populations. We investigated the clinical significance of these mutations at our institution with an unselected group of patients with newly diagnosed AML. This group included patients ≥60 years old and those with a poor performance status. Using polymerase chain reaction and sequencing analyses, we detected FLT3/ITD mutations in 12 patients (20.0%) andNPM1 mutations in 7 patients (11.7%) among a group of 60 patients. There was a nonsignificant trend for FLT3/ITD mutation to be associated with a poorer predicted overall survival (OS) probability in this population. In contrast, OS was significantly higher in patients with wild-typeNPM1 than in patients withNPM1 mutation, both for all AML patients and for AML patients with a normal karyotype. In this general and unselected AML patient population,NPM1 mutation was not a prognostic indicator of a favorable outcome.

Functional analysis of the SEPT9-ABL1 chimeric fusion gene derived from T-prolymphocytic leukemia

We analyzed the function of a SEPT9-ABL1 fusion identified in a case of T-prolymphocytic leukemia with tyrosine kinase inhibitor (TKI) resistance. Five isoforms with different N-termini, including SEPT9a-ABL1, SEPT9b-ABL1, SEPT9d-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1, were detected in the leukemic cells. All isoforms except SEPT9d-ABL1 are localized in the cytoplasm, undergo autophosphorylation and phosphorylate the downstream targets, STAT-5 and Crkl, and provided IL-3-independence and in vivo invasiveness to 32D cells. Additionally, these SEPT9-ABL1 isoforms were resistant to TKIs in vitro and in vivo, in comparison to BCR-ABL1. These findings demonstrated that SEPT9-ABL1 had oncogenic activity and conferred resistance to TKIs.

Identification of a novel SEPT9-ABL1 fusion gene in a patient with T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

Jagged1-induced Notch activation contributes to the acquisition of bortezomib resistance in myeloma cells

Multiple myeloma (MM) is a hematologic malignancy characterized by the proliferation of malignant plasma cells in the bone marrow (BM). Despite recent advances in therapy, MM remains incurable, largely due to the emergence and maintenance of drug-resistant myeloma cells, whose interactions within the BM microenvironment, the BM myeloma niche, are critical. Because Notch signaling, a pathway activated only via cell-cell contacts, has been implicated as an integral part of the onset and progression of MM, we investigated how Notch activation in myeloma cells, specifically through interactions with ligand-expressing niche cells, affects the pathophysiology of MM. We first demonstrated the presence of at least one Notch receptor in 10 arbitrarily selected MM cell lines (Supplementary Figure 1a and b). The presence of these receptors indicates that Notch signaling can be activated and is functional in human myeloma cells. Of the two Notch ligands examined, Jagged1, but not Jagged2, is known to be abundantly expressed in many types of cells in the BM myeloma niche . Therefore, we investigated the effects of niche-induced Jagged1-Notch activation on myeloma cells. To distinguish niche-induced Notch activation from the homotypic activation of Notch in myeloma cells, in which Notch and its ligands can be simultaneously expressed, we selected five MM cell lines that expressed Jagged1 weakly or not at all for analyses. Contrary to previous reports, the presence of immobilized human Jagged1 did not significantly alter the proliferation of any of the five MM cell lines in vitro (Supplementary Figure 1c). To further investigate the roles of niche-induced Notch signaling, we established a clinically relevant animal model of human MM that would allow us to examine myeloma cell interactions within the BM niche (Supplementary Figure 1d), where malignant plasma cells primarily proliferate in patients with MM. The engraftment of human MM cells in the BM did not increase even when U266 cells were transplanted into non-obese diabetic/severe combined immunodeficient/IL2Rγnull (NOG) mice expressing human Jagged1 in osteoblasts (NOGJ), a cell type that constitutes the BM myeloma niche (Supplementary Figure 1e). The results indicate that niche-induced Jagged1-Notch signaling is not specifically associated with the proliferation of myeloma cells. We then assessed whether niche-induced Jagged1Notch activation affects the sensitivity of myeloma cells to drugs used clinically to treat MM patients. In co-culture experiments, transgenic expression of human Jagged1 in stromal cells (ST2J) increased the survival of U266 cells only when the cells were exposed to bortezomib (BTZ), which coincided with the upregulation of human Hey1 and Hes1 expression in culture (Supplementary Figure 2a-c), indicating a role of Notch signaling in BTZ resistance. Five other MM cell lines similarly demonstrated resistance to BTZ in the presence of ST2J cells (Supplementary Figure 2d). As expected, cells cultured on immobilized human Jagged1 demonstrated significant resistance to BTZ treatment (Fig. 1a) but not to melphalan treatment (Supplementary Figure 2e). In addition, a marked upregulation in the expression of Hey1 and Hes1 was detected in cells that survived BTZ treatment (Fig. 1b), confirming that the observed resistance to BTZ

Tamibarotene maintenance improved relapse-free survival of acute promyelocytic leukemia: a final result of prospective, randomized, JALSG-APL204 study

Between April 2004 and December 2010, we conducted a prospective randomized controlled study comparing tamibarotene with all-trans retinoic acid (ATRA) in the maintenance therapy of newly diagnosed acute promyelocytic leukemia (APL), and here report the final results of this study with a median follow-up of 7.3 years. Of 344 eligible patients who had received ATRA and chemotherapy, 319 (93%) achieved complete remission (CR). After completion of three courses of consolidation chemotherapy, 269 patients in molecular remission underwent maintenance randomization, 135 to ATRA (45 mg/m2 daily), and 134 to tamibarotene (6 mg/m2 daily) for 14 days every 3 months for 2 years. The primary endpoint was relapse-free survival (RFS). The 7-year RFS was 84% in the ATRA arm and 93% in the tamibarotene arm (p = 0.027, HR = 0.44, 95% CI, 0.21 to 0.93). The difference was prominent in high-risk patients with initial leukocytes ≥ 10.0 × 109/L (62% vs. 89%; p = 0.034). Tamibarotene was significantly superior to ATRA by decreasing relapse in high-risk patients. Overall survival after randomization did not differ (96% vs. 97%; p = 0.520). Secondary hematopoietic disorders developed in nine patients, secondary malignancies in 11, and grade 3 or more late cardiac comorbidities in three. These late complications did not differ between the two arms.

Refractory acute promyelocytic leukemia successfully treated with combination therapy of arsenic trioxide and tamibarotene: A case report

A 40-year-old male developed refractory acute promyelocytic leukemia (APL) after various treatments including all-trans retinoic acid, tamibarotene, arsenic trioxide (As2O3), conventional chemotherapy, and autologous peripheral blood stem cell transplantation. We attempted to use both tamibarotene and As2O3 as a combination therapy, and he achieved molecular complete remission. Grade 2 prolongation of the QTc interval on the electrocardiogram was observed during the therapy. The combination therapy of As2O3 and tamibarotene may be effective and tolerable for treating refractory APL cases who have no treatment options, even when they have previously been treated with tamibarotene and As2O3as a single agent.