Norimichi Hattori

Rituximab with chemotherapy improves survival of non-germinal center type untreated diffuse large B-cell lymphoma

Rituximab with chemotherapy improves survival of non-germinal center type untreated diffuse large B-cell lymphoma

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

KPT-330 has antitumour activity against non-small cell lung cancer.

Background:We investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo.Methods:The in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed.Results:KPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation.Conclusions:Our results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC.

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway.

We investigated the oncogenic role of SETDB1, focusing on non‐small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT–β‐catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1. Copyright

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD)

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

LNK (SH2B3): paradoxical effects in ovarian cancer.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography–mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

Corrigendum: Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia (Leukemia (2016) 30 (1672-1681) DOI: 10.1038/leu.2016.69)

V Madan, P Shyamsunder, L Han, A Mayakonda, Y Nagata, J Sundaresan, D Kanojia, K Yoshida, S Ganesan, N Hattori, N Fulton, KT Tan, T Alpermann, MC Kuo, S Rostami, J Matthews, M Sanada, L-Z Liu, Y Shiraishi, S Miyano, E Chendamarai, HA Hou, G Malnassy, T Ma, M Garg, LW Ding, QY Sun, W Chien, T Ikezoe, M Lill, A Biondi, RA Larson, BL Powell, M Lübbert, WJ Chng, HF Tien, M Heuser, A Ganser, M Koren-Michowitz, SM Kornblau, HM Kantarjian, D Nowak, WK Hofmann, H Yang, W Stock, A Ghavamzadeh, K Alimoghaddam, T Haferlach, S Ogawa, LY Shih, V Mathews and HP Koeffler

Over-expression of CCL3 MIP-1α in a blastoid mantle cell lymphoma with hypercalcemia

We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL‐BV), a rare subtype of B‐cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme‐linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), macrophage inflammatory protein‐1α (MIP‐1α), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone‐related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT‐PCR) analysis, we found predominant expression of mRNA for MIP‐1α in addition to those for receptor‐activator of nuclear‐factor kappa B ligand (RANKL), TNF‐α, and IL‐6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP‐1α significantly stimulated 3H‐thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP‐1α. In the present case, MIP‐1α, an osteoclast‐activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T‐cell leukemia/lymphoma (ATLL). Furthermore, MIP‐1α is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP‐1α is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B‐cell lymphoma.

Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.

Improvement of the thermal amplitude after rituximab treatment for cold agglutinin disease with Waldenström’s macroglobulinemia

Dear Editor, Cold agglutinin disease (CAD) is an uncommon autoimmune hemolytic anemia characterized by the production of cold agglutinins (CA) [1]. CA exhibits the strongest affinity for the antigen at 0–4°C but binding may occur at temperatures approaching the normal body temperature of mammals, depending on the thermal amplitude of the antibody. Rituximab has been demonstrated to be useful in treating CAD that is refractory to conventional therapies [2]. However, CA titers did not concur with the observed clinical and hematologic responses, and it has become apparent that the CA titer is not an important index for the clinical features of CAD. The detailed effect(s) of rituximab on CAD have not been reported. We present a case of a 68-year-old man who was diagnosed with Waldenstrom’s macroglobulinemia with CAD in 1999, in whom the thermal amplitude was improved after rituximab treatment. His Hb level was 4.9 g/dL, and an immunoglobulin M (IgM) kappa monoclonal protein was evident at diagnosis (IgM, 58.2 g/L). His bone marrow was diffusely infiltrated with CD20+ lymphoplasmacytoid lymphocytes. The direct antiglobulin test was positive for complement (C3d). CAwas positive with a titer of 8,192 at 4°C. He underwent six courses of cyclophosphamide, adriamycin, vinctistine, and prednisolone and low-dose prednisolone up to July 2007. Due to worsening anemia with acrocyanosis, he was treated with rituximab alone at 375 mg/m four times every 8 weeks. Although the CAD-related symptoms were improved, the high-CA titers at 4°C remained. He relapsed 42 weeks after the initial treatment, and the duration to response was 10 months. After informed consent was obtained, we determined the agglutination titer and tested the thermal amplitude of the patient’s plasma and serum, as described previously [3]. As shown in Table 1, 4 weeks after the initial injection of rituximab, increases of hemoglobin and improvement of the thermal amplitude of the antibody were observed simultaneously. Up to 24 weeks later, the improvement of the thermal amplitude remained. Hemoglobin increased progressively up to 28 weeks. Based on the changes of the thermal range in reactivity of the antibody, the clinical sign(s), such as acrocyanosis, improved along with amelioration of the symptoms related to anemia. Temperatures of 30°C and lower normally occur in the superficial skin vessels of the parts of the body exposed to cold air or water. The thermal range of the antibody is more important than the agglutination titer for clinical symptoms [4]. The present findings suggested that both elevation of hemoglobin and resolution of clinical sign(s) are closely associated with improvement of thermal amplitude of the CA after rituximab treatment. The present case suggested that hemoglobin was elevated as a consequence of the improvement of thermal amplitude after rituximab treatment. Schoellkopf et al. observed a significant and persistent reduction of serum IgM concentration after rituximab Ann Hematol (2010) 89:103–104 DOI 10.1007/s00277-009-0773-z