Morito Kurata

Adult Xp11 Translocation Renal Cell Carcinoma Diagnosed by Cytogenetics and Immunohistochemistry

Purpose: To determine the incidence of Xp11 translocation renal cell carcinoma (RCC) in adult patients using cytogenetics and immunohistochemstry. Experimental Design: Cytogenetic studies were prospectively done using tumor samples from 443 consecutive adult Japanese patients (ages 15-89 years) who underwent nephrectomy for RCC. TFE3 immunohistochemistry was done for cases in which cytogenetic results were not obtained. Clinicopathologic characteristics of Xp11 translocation RCC were examined. Results: Mitotic cells suitable for cytogenetic analysis were obtained in 244 tumor samples (55%); among these, we identified 4 cases (1.6%) of Xp11 translocation RCC. TFE3 immunohistochemistry identified 3 positive cases (1.5%) among the remaining 199 cases. The median age of the 7 patients was 41 years (range, 15-59 years), and 15% of RCC patients (4 of 26) who were younger than ages 45 years had this type of RCC. Of the four Xp11 translocation RCC patients whose karyotypes were determined, two had an ASPL-TFE3 gene fusion. Of these 2, 1 had pulmonary metastasis at presentation, and the other developed liver metastasis 12 months after nephrectomy and died of the disease. The remaining two patients had PRCC-TFE3 and PSF-TFE3 gene fusions, respectively. Both had nodal involvement but remained disease free for 3 and 5 years, respectively, after surgical resection of lymph node metastases. Of the 3 immunohistochemically diagnosed patients, 1 had nodal metastases at presentation and died 9 months after surgery. Conclusions: This is the first report to determine the incidence of Xp11 translocation RCC in adult patients. We found that this disease is relatively common in young adults.

DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells

Dysregulation of the G(1)/S transition in the cell cycle contributes to tumor development. The oncogenic transcription factors c-Jun and c-Myc are indispensable regulators at this transition, and their aberrant expression is associated with many malignancies. Degradation of c-Jun/c-Myc is a critical process for the G(1)/S transition, which is initiated upon phosphorylation by glycogen synthase kinase 3 β (GSK3β). However, a specific kinase or kinases responsible for priming phosphorylation events that precede this GSK3β modification has not been definitively identified. Here, we found that the dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 functions as a priming kinase of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G(1) phase and accelerated cell proliferation due to escape of c-Jun and c-Myc from ubiquitination-mediated degradation. In concert with these results, silencing DYRK2 increased cell proliferation in human cancer cells, and this promotion was completely impeded by codeprivation of c-Jun or c-Myc in vivo. We also found marked attenuation of DYRK2 expression in multiple human tumor samples. Downregulation of DYRK2 correlated with high levels of unphosphorylated c-Jun and c-Myc and, importantly, with invasiveness of human breast cancers. These results reveal that DYRK2 regulates tumor progression through modulation of c-Jun and c-Myc.

Regulation of angiogenesis in the bone marrow of myelodysplastic syndromes transforming to overt leukaemia

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS → OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor α (TNFα), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang‐1 and Ang‐2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti‐angiogenic mediator, transforming growth factor‐β (TGFβ) exhibited significantly higher expression in the bone marrow of MDS → OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS → OL and de novo AML differs remarkably, suggesting the different efficacy of anti‐angiogenic therapy between de novo AML and leukaemia secondary to MDS.

Anti-apoptotic function of Xbp1 as an IL-3 signaling molecule in hematopoietic cells

Cytokine signaling is critical for proliferation, survival and differentiation of hematopoietic cell, and interleukin-3 (IL-3) is required for maintenance of many hematopoietic cell lines, such as BaF3. We have isolated apoptosis-resistant clones of BaF3 using retroviral insertional mutagenesis and the Xbp1 locus was identified as a retroviral integration site. Expression and splicing of the Xbp1 transcript was conserved in the resistant clone but was promptly disappeared on IL-3 withdrawal in parental BaF3. IL-3 stimulation of BaF3 cells enhanced Xbp1 promoter activity and induced phosphorylation of the endoplasmic reticulum stress sensor protein IRE1, resulting in the increase in Xbp1S that activates unfolded protein response. When downstream signaling from IL-3 was blocked by LY294002 and/or dn-Stat5, Xbp1 expression was downregulated and IRE1 phosphorylation was suppressed. Inhibition of IL-3 signaling as well as knockdown of Xbp1-induced apoptosis in BaF3 cells. In contrast, constitutive expression of Xbp1S protected BaF3 from apoptosis during IL-3 depletion. However, cell cycle arrest at the G1 stage was observed in BaF3 and myeloid differentiation was induced in IL-3-dependent 32Dcl3 cells. Expression of apoptosis-, cell cycle- and differentiation-related genes was modulated by Xbp1S expression. These results indicate that the proper transcriptional and splicing regulation of Xbp1 by IL-3 signaling is important in homeostasis of hematopoietic cells.

Expression of multidrug resistance 1 gene in B-cell lymphomas: association with follicular dendritic cells

Aims:  Multidrug resistance (MDR) in B‐cell lymphomas still constitutes a major obstacle to the effectiveness of chemotherapy even in the anti‐CD20 antibody therapy era. The aim of this study was to investigate the expression of MDR‐associated molecules in reactive lymphadenopathy (RL), follicular lymphoma (FL), and diffuse large B‐cell lymphoma (DLBCL).

Differential expression of Toll-like receptors in follicular lymphoma, diffuse large B-cell lymphoma and peripheral T-cell lymphoma

Although Toll-like receptors (TLRs) in mammals are well-known to play important roles in innate immunity, newer roles for the TLRs have suggested that cells with aberrant TLR expression may have a survival advantage over normal cells. Lymphocytes are one of a small number of cell types that express many of the TLRs, suggesting that abnormal TLR levels/signaling may potentially influence the progression of malignant lymphomas. Thus, frozen samples of 51 lymph nodes from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) were analyzed for the expression of TLR1 to 9 using quantitative real-time PCR, and compared to those in reactive lymphadenopathy (RL) samples. TLR2 was over-expressed in both DLBCL and PTCL but not in FL when compared to RL. TLR1 and TLR4 expression was up-regulated in PTCL, while TLR8 was highly expressed in DLBCL. Although TLR5 showed lower expression in FL, expression of TLR3, TLR6, TLR7 and TLR9 did not vary significantly between different lymphoma subtypes. Double immunostaining revealed an increase in the number of TLR2 and/or TLR8 expressing lymphoma cells in DLBCL. In PTCL, TLR2 and TLR4 expression was localized to neoplastic T cells. TLR expression is highly variable among lymphoma subtypes. However, despite this some significant differences exist that may prove useful in the development of novel therapeutic strategies.

CXCL12 + stromal cells as bone marrow niche for CD34 + hematopoietic cells and their association with disease progression in myelodysplastic syndromes

The bone marrow microenvironment, known as ‘hematopoietic stem cell niche,’ is essential for the survival and maintenance of hematopoietic stem cells. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell diseases, which eventually result in leukemic transformation (acute myelogenous leukemia with myelodysplasia-related changes, AML-MRC). However, the precise components and functions of the MDS niche remain unclear. Recently, CXCL12-abundant reticular cells were shown to act as a hematopoietic stem cell niche in the murine bone marrow. Using immunohistochemistry, we show here that CXCL12+ cells were located in the cellular marrow or perivascular area, and were in contact with CD34+ hematopoietic cells in control and MDS/AML-MRC bone marrow. MDS bone marrow exhibited higher CXCL12+ cell density than control or AML, not otherwise specified (AML-NOS) bone marrow. Moreover, AML-MRC bone marrow also exhibited higher CXCL12+ cell density than control bone marrow. CXCL12+ cell density correlated positively with bone marrow blast ratio in MDS cases. CXCL12 mRNA level was also higher in MDS bone marrow than in control or AML-NOS bone marrow. In vitro coculture analysis revealed that overexpression of CXCL12 in stromal cells upregulated BCL-2 expression of leukemia cell lines. Triple immunostaining revealed that the CD34+ hematopoietic cells of MDS bone marrow in contact with CXCL12+ cells were BCL-2-positive and TUNEL-negative. In the bone marrow of MDS cases, CXCL12-high group showed significantly higher Bcl-2+/CD34+ cell ratio and lower apoptotic cell ratio than CXCL12-low group. Moreover, CXCL12-high refractory cytopenia with multilineage dysplasia (RCMD) cases had a greater tendency to progress to refractory anemia with excess blasts (RAEBs) or AML-MRC than CXCL12-low RCMD cases. These results suggest that CXCL12+ cells constitute the niche for CD34+ hematopoietic cells, and may be associated with the survival/antiapoptosis of CD34+ hematopoietic cells and disease progression in MDS. Thus, CXCL12+ cells may represent a novel MDS therapeutic target.

Expression of Toll-like receptor 9 in bone marrow cells of myelodysplastic syndromes is down-regulated during transformation to overt leukemia.

Toll-like receptors (TLRs) play a crucial role in the host defense against invading microorganisms by recognizing pathogen-associated molecular patterns. Recently, a number of endogenous molecules have been reported to be ligands of TLRs. Some of these molecules are known to be expressed in cancer tissue and activate intracellular signal pathways via TLRs during cancer progression. Thus, in the present study, we analyzed the expression dynamics of TLRs in the bone marrow of myelodysplastic syndromes (MDS) during the course of transformation to overt leukemia (OL) using real-time RT-PCR. MDS bone marrow cells at the time of initial diagnosis tended to express higher levels of TLR2, TLR4 and TLR9 than control bone marrow cells. Among these TLRs, TLR9 exhibited a significant decrease of expression at the time of transformation to OL. The expression of TLR9 and TNF-alpha showed significant correlation in bone marrow cells from patients with MDS and OL. Immunohistochemically, TLR2 was mostly localized to neutrophils of the control and MDS bone marrow. TLR4 was observed in a subset of neutrophils and a few mononuclear cells in control and MDS bone marrow. In addition, TLR4 was weakly expressed in nearly half of immature myeloid cells of MDS cases. TLR9 was mainly localized to neutrophils in the control and RA bone marrow and strongly expressed in the immature myeloid cells of RAEB cases, although the blastic cells of OL cases did not express TLR9. Bone marrow cells in MDS exhibit frequent apoptosis, while OL cells are prone to be immortal. Thus, TLR9 might be associated with regulation of apoptotic/proliferative signals via TNF-alpha in the MDS bone marrow.

Angiogenic mediators of the angiopoietin system are highly expressed by CD10‐positive lymphoma cells in angioimmunoblastic T‐cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AILT) is a malignant disease of peripheral T‐cell origin that is characterized by a prominent proliferation of high endothelial venules in the lymph node. To investigate angiogenic mechanisms in AILT we measured the angiogenic mediator gene expression levels in the lymph nodes of 54 non‐Hodgkin lymphoma patients, by immunostaining and quantitative reverse transcription polymerase chain reaction. Angiogenic mediators angiopoietin (Ang) 1 (ANGPT1), Ang2 (ANGPT2) and their receptor, Tie2 (TEK), vascular endothelial growth factor (VEGF; VEGFA) and its receptor, VEGFR2 (KDR), and hepatocyte growth factor (HGF) and its receptor, c‐Met (MET) were all more highly expressed in AILT lymph nodes (16 cases) than in B‐cell lymphomas (24 cases). Moreover, significantly higher Ang1 and Tie2 expression was detected in AILT cases with CD10‐positive neoplastic T‐cells by comparison with unspecified peripheral T‐cell lymphoma (14 cases). Immunostaining confirmed the expression of Ang1 and VEGF by both neoplastic T‐cells and follicular dendritic cells. These results suggest that the angiopoietin system may play an important role in the development of high vascularity in AILT lymph nodes. Consequently, as neoplastic T‐cells and follicular dendritic cells are both increased in AILT and may represent an important source of angiogenic mediators, targeting these cells with anti‐angiogenic strategies might represent a novel therapy for AILT.

Overexpression of MCM2 in myelodysplastic syndromes: Association with bone marrow cell apoptosis and peripheral cytopenia

Myelodysplastic syndromes (MDS) are characterized by proliferation and apoptosis of bone marrow cells. Minichromosome maintenance protein (MCM) 2, which is known to be essential for regulating DNA replication, has proven to have a pro-apoptotic effect in our recent study. Thus, to determine the role of MCM2 in MDS, real-time PCR, immunohistochemistry and in vitro analysis were performed. Our results showed higher MCM2 expression in MDS than in control and AML. Notably, there was no correlation between MCM2 and Ki67-labeling indices (LIs) in MDS, while MCM2 LIs were significantly correlated with cleaved caspase 3 LIs in MDS. In vitro analysis revealed that MCM2 overexpression induced apoptosis in HL60 cells. Furthermore, MDS bone marrow exhibited higher ratio of MCM2 and cleaved caspase 3 double-positive cells and the ratio was correlated with the degree of leukocytopenia. These results suggest that the up-regulated expression of MCM2 is associated with frequent apoptosis in MDS and may have an important role in the pathogenesis of MDS.