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Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established three ANKL-patient-derived xenograft mice (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a non-canonical hematopoietic organ in adults, serves as a principal niche for ANKL, and that inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

The characteristics of adult patients with chronic active Epstein-Barr virus infection are poorly recognized, hindering early diagnosis and an improved prognosis. We studied 54 patients with adult-onset chronic active Epstein-Barr virus infection diagnosed between 2005 and 2015. Adult onset was defined as an estimated age of onset of 15 years or older. To characterize the clinical features of these adults, we compared them to those of 75 pediatric cases (estimated age of onset

// Fumito Abe 1, * , Akihiro Kitadate 1, * , Sho Ikeda 1 , Junsuke Yamashita 2 , Hiroki Nakanishi 3 , Naoto Takahashi 1 , Chikara Asaka 4 , Kazuaki Teshima 5 , Tomomitsu Miyagaki 6 , Makoto Sugaya 6 , Hiroyuki Tagawa 1 1 Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine, Akita, Japan 2 Division of Bioscience Center, Radioisotope, Akita University, Akita, Japan 3 Research Center for Biosignal, Akita University, Akita, Japan 4 Department of Otolaryngology, Noshiro Kousei Medical Center, Noshiro, Japan 5 Department of Hematology, Hiraka General Hospital, Yokote, Japan 6 Department of Dermatology, University of Tokyo, Tokyo, Japan * These authors share first authorship Correspondence to: Hiroyuki Tagawa, email: htagawa0279jp@yahoo.co.jp Keywords: HDACI, miR-150, CCR6, CTCL, metastasis Received: September 24, 2016 Accepted: November 24, 2016 Published: December 07, 2016 ABSTRACT Tumor suppressive microRNA (miR)-150 inhibits metastasis by combining with the C-C chemokine receptor 6 (CCR6) “seed sequence” mRNA of the 3′-untranslated region (3′-UTR) in advanced cutaneous T-cell lymphoma (CTCL). Because the histone deacetylase inhibitor (HDACI) vorinostat showed excellent outcomes for treating advanced CTCL, HDACIs may reduce the metastasis of CTCL by targeting miR-150 and/ or CCR6. To examine whether these candidate molecules are essential HDACI targets in advanced CTCL, we used the My-La, HH, and HUT78 CTCL cell lines for functional analysis because we previously demonstrated that their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) induced multiple metastases. We found that pan- HDACIs (vorinostat and panobinostat) inhibited the migration of CTCL cells and downregulated CCR6. The miRNA microarray analysis against CTCL cell lines demonstrated that these pan-HDACIs commonly upregulated 161 miRNAs, including 34 known tumor suppressive miRNAs such as miR-150. Although 35 miRNAs possessing the CCR6 “seed sequence” were included in these 161 miRNAs, miR-150 and miR-185-5p were downregulated in CTCL cells compared to in normal CD4+ T-cells. The transduction of 12 candidate miRNAs against CTCL cells revealed that miR-150 most efficiently inhibited their migration capabilities and downregulated CCR6. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that miR-150 was downregulated in advanced but not early CTCL primary cases. Finally, we injected miR-150 or siCCR6 into CTCL mice and found that mouse survival was significantly prolonged. These results indicate that miR-150 and its target, CCR6, are essential therapeutic targets of pan-HDACIs in advanced CTCL with metastatic potential.

Hemophagocytic syndrome (HPS) is frequently associated with hematopoietic stem cell transplantation and is treated with some benefit derived from TNF-α inhibitors. However, the mechanisms of how HPS occurs and how a TNF-α inhibitor exerts some benefit to HPS management have remained unclear. We evaluated the effect of toll-like receptor (TLR) ligands, especially focusing on cytosine-phosphorothionate-guanine oligodeoxynucleotide (CpG), a TLR9 ligand, on HPS in mice that underwent transplantation with syngeneic or allogeneic bone marrow (BM) cells (Syn-BMT, Allo-BMT), or with allogeneic BM cells plus splenocytes to promote graft-versus-host disease (GVHD mice). Hemophagocytosis was a common feature early after all BMT, but it subsided in Syn-BMT and Allo-BMT mice. In GVHD mice, however, hemophagocytosis persisted and was accompanied by upregulated production of IFN-γ but not TNF-α, and it was suppressed by blockade of IFN-γ but not TNF-α. A single injection of the TLR9 ligand CpG promoted HPS in all BMT mice and was lethal in GVHD mice, accompanied by greatly upregulated production of TNF-α, IL-6, and IFN-γ. Blocking of TNF-α, but not IL-6 or IFN-γ, suppressed CpG-induced HPS in all BMT mice and rescued GVHD mice from CpG-induced mortality. Thus, TLR9 signaling mediates TNF-α–driven HPS in BMT mice and is effectively treated through TNF-α inhibition.

Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice. Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6). Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat. Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. Disclosures Kitadate: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa: TaNeDS (Daiichi Sankyo): Research Funding.

In multiple myeloma (MM), the bone marrow (BM) microenvironment may contain a myeloma cell fraction that has acquired treatment resistance by undergoing an epigenetic gene expression change. Hypoxic stress is an important factor in the BM microenvironment. Recently, we demonstrated that miR-210 was upregulated in hypoxia and downregulated IRF4, which is known as an essential factor in myeloma oncogenesis in normoxia. In the study, we demonstrated that myeloma cells still showed a strong antiapoptotic phenotype despite IRF4 downregulation, suggesting that another antiapoptotic factor might be involved under hypoxic stress. To determine the factor or factors, we conducted gene expression analysis on myeloma cells (primary samples and cell lines) that were exposed to chronic hypoxia and observed upregulation of glycolytic genes and genes encoding H3K9 demethylases in myeloma cells with hypoxia. Among these, KDM3A was most significantly upregulated in all examined cells, and its knockdown induced apoptosis of myeloma cells in chronic hypoxia. Expression of KDM3A was dependent on HIF-1α, which is a transcription factor specifically upregulated in hypoxia. We further demonstrated that an essential target of KDM3A was a noncoding gene, MALAT1, whose upregulation contributed to acquisition of an antiapoptotic phenotype by accumulation of HIF-1α, leading to upregulation of glycolytic genes under hypoxia. This process was independent from IRF4. These results led us to conclude that the hypoxia-inducible HIF-1α-KDM3A-MALAT1 axis also contributes to acquisition of the antiapoptotic phenotype via upregulation of glycolysis-promoting genes. Thus, this axis is a promising therapeutic target against myeloma cells in the BM microenvironment.

MicroRNAs (miRNAs) are non-coding regulatory RNAs consisting of 20-24 nucleotides. Over 4,500 miRNAs have been identified in humans, and it is known that nearly all human protein-encoding genes can be controlled by miRNAs in both healthy and malignant cells. Abnormal miRNA expression is known to occur in many cancers, including in malignant lymphomas (MLs). Detailed genome-wide miRNA expression analysis has been performed in various ML subtypes, and these analyses have led to the discovery of subtype-specific miRNA alterations. Actually, in B-cell lymphomas, several miRNAs have been used as prognostic markers, and their targets are for new agents for ML therapy. Successful studies for delineating miRNA functions in B-cell lymphomas lead us to hypothesize that miRNA dysregulation may also be deeply associated with the pathogenesis of T-cell lymphomas. Indeed, studies for delineating essential miRNAs have been conduced against comparatively well-defined T-cell lymphoma entities. In this review, we describe several key miRNAs and their targets in distinct T-cell lymphoma subsets and their roles in their pathogenesis, studies of which will lead to new therapeutic strategies against T-cell lymphomas.

(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.

Liposomal amphotericin B (L-AmB) is recommended as an empirical antifungal treatment for patients at increased risk of fungal infections although renal toxicity remains a clinical problem. We therefore conducted a pilot study to evaluate the safety and efficacy of low-dose L-AmB as an empirical antifungal therapy for patients with prolonged neutropenia. High-risk patients with hematological malignancies were eligible to enroll in this study provided they had: exhibited neutropenia for at least 1 week; suffered from high-grade fever for 4 days despite treatment with a broad-spectrum antibacterial; and no identified fever-causing pathogen. Low-dose L-AmB (1 mg/kg) was administrated as empirical antifungal therapy. Sixteen patients were registered and, of these, data from the13 patients who did not receive allogeneic stem cell transplantation were analyzed. The median duration of low-dose L-AmB treatment was 8 days. Hypokalemia was seen in one patient: administration of potassium supplements for 10 days restored potassium levels to the normal range. A two-fold increase in creatinine levels was not found in any patients even those taking concomitant nephrotoxic drugs (e.g., amynoglycoside) during the study. One patient stopped receiving the drug due to an infusion-related adverse event. No patients showed breakthrough fungal infections or died during therapy or within 7 days after the end of the study. Increase in the L-AmB dose was necessary due to persistent fever in three patients who withdrew from the study. The satisfactory response rate for low-dose L-AmB was 69 %. This study suggests that low-dose L-AmB may be an effective option as empirical antifungal therapy for high-risk patients with febrile neutropenia.

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Recent studies have shown that anemia is commonly observed after exposure to pathogens or pathogen-derived products, which are recognized via Toll-like receptor 9 (TLR9). In the current study, we demonstrate that CpG oligodeoxynucleotide-2006, a TLR9 ligand with phosphodiester (PO; 2006-PO) but not with the phosphorothioate backbone, selectively inhibits the erythroid growth derived from human CD34+ cells. The 2006-PO was internalized by the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA was not detected in these cells. The 2006-PO directly inhibited burst-forming unit-erythroid growth, resulted in the accumulation of cells in S and G2/M phases, and increased cell size and frequency of apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19 that causes pure red cell aplasia. The consensus sequence of 2006-PO was defined as 5′-GTTTTGT-3′, which was located in the P6-promoter region of B19 and inhibited erythroid growth in a sequence-specific manner and down-regulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19 genome extracted from serum also inhibited erythroid growth and down-regulated expression of EPOR on glycophorin A+ cells. These results provide a possible insight into our understanding of the mechanisms of human parvovirus B19-mediated inhibition of erythropoiesis.

Intravascular large B cell lymphoma (IVLBCL) is a rare type of non-Hodgkin lymphoma characterized by a disseminated intravascular proliferation of tumor cells in the lumina of small vessels. Although the kidney is one of the target organs of IVLBCL, it is extremely rare that lymphoma cells are localized only in the kidney. We report here a Japanese patient with kidney-limited IVLBCL. The patient presented with mild proteinuria and a good performance status without B symptoms at presentation. A renal biopsy showed large B cell neoplastic lymphocytes in the glomerular capillary lumina. Extensive systemic examinations showed no other organ involvement. The patient responded well to rituximab and anthracycline-based chemotherapy. A follow-up renal biopsy showed the disappearance of intraglomerular lymphoma cells with restoration of glomerular architecture. Within 20 months past the discontinuation of chemotherapy, no evidence of recurrence was observed. Although IVLBCL is commonly a fatal disease, favorable clinical courses were reported in some cases of IVLBCL, such as the cutaneous variant. To our knowledge, there are 8 reported cases of kidney-limited IVLBCL in the English literature. All 4 patients treated with intensive chemotherapy responded well to the treatment as our patient. We suggest that kidney-limited IVLBCL might be a distinct variant of IVLBCL.

Aberrant overexpression of the miR-17-92 polycistron is strongly associated with B-cell lymphomagenesis. Recent studies have shown that miR-17-92 down-regulates the proapoptotic protein Bim, leading to overexpression of Bcl2, which likely plays a key role in lymphomagenesis. However, the fact that Jeko-1 cells derived from mantle cell lymphoma exhibit both homozygous deletion of BIM and overexpression of miR-17-92 suggests other targets are also involved in B-cell lymphomagenesis. To identify essential target(s) of miR-17-92 in lymphomagenesis, we first transfected miR-17-92 into 2 genetically distinct B-cell lymphoma cell lines: Raji, which overexpress c-Myc, and SUDHL4, which overexpress Bcl2. Raji transfected with miR-17-19b-1 exhibited down-regulated expression of Bim and a slight up-regulation in Bcl2 expression. On the other hand, SUDHL4 transfectants showed aggressive cell growth reflecting facilitated cell cycle progression at the G1 to S transition and decreased expression of CDKN1A mRNA and p21 protein (CDKN1A/p21) that was independent of p53 expression. Conversely, transfection of antisense oligonucleotides against miR-17 and miR-20a into Jeko-1 led to up-regulation of CDKN1A/p21, resulting in decreased cell growth with G1 to S arrest. Thus, CDKN1A/p21 appears to be an essential target of miR-17-92 during B-cell lymphomagenesis, which suggests the miR-17-92 polycistron has distinct targets in different B-cell lymphoma subtypes.

Background The differentiation of biologically and clinically different malignant lymphoma diseases or subtypes is crucial because it leads to better prognostication and therapeutic decision-making. Attempts have been made at subtype classification for diagnosing lymphomas on the basis of gene-expression profiling. Although array-based comparative genomic hybridization (array CGH) has identified a characteristic genomic alteration pattern for each disease entity, it has not been clear whether each patient with certain genomic alterations can be classified by array CGH data. Design and Methods Data on copy number gains and losses for 46 diffuse large B-cell lymphomas and 29 mantle cell lymphomas were used. The gene expressions of the diffuse large B-cell lymphomas cases were profiled and hierarchical clustering revealed that 28 of them were of the activated B-cell type and 18 were of the germinal center-B-cell type. Using these data, we developed a computer algorithm to classify lymphoma diseases or subtypes on the basis of copy number gains and losses. Results The method correctly classified 88% of the diffuse large B-cell lymphomas and mantle cell lymphomas, and 83% of the activated B-cell and germinal center-B-cell subtypes. These results demonstrate that copy number gains and losses detected by array CGH can be used for classifying lymphomas into biologically and clinically distinct diseases or subtypes. Conclusions Our computer algorithm based on array CGH data successfully classified diffuse large B-cell lymphomas and mantle cell lymphomas and activated B-cell and germinal center-B-cell subtypes with high accuracy. An important finding is that the regions automatically identified by the computer algorithm were located in the critical regions that are likely to be involved in the development of lymphoma.

Diffuse large B-cell lymphoma (DLBCL) comprises molecularly distinct subgroups such as activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCLs. We previously reported that CD5(+) and CD5(-)CD10(+) DLBCL constitute clinically relevant subgroups. To determine whether these 2 subgroups are related to ABC and GCB DLBCLs, we analyzed the genomic imbalance of 99 cases (36 CD5(+), 19 CD5(-)CD10(+), and 44 CD5(-)CD10(-)) using array-based comparative genomic hybridization (CGH). Forty-six of these cases (22 CD5(+), 7 CD5(-)CD10(+), and 17 CD5(-)CD10(-)) were subsequently subjected to gene-expression profiling, resulting in their division into 28 ABC (19 CD5(+) and 9 CD5(-)CD10(-)) and 18 GCB (3 CD5(+), 7 CD5(-)CD10(+), and 8 CD5(-)CD10(-)) types. A comparison of genome profiles of distinct subgroups of DLBCL demonstrated that (1) ABC DLBCL is characterized by gain of 3q, 18q, and 19q and loss of 6q and 9p21, and GCB DLBCL is characterized by gain of 1q, 2p, 7q, and 12q; (2) the genomic imbalances characteristic of the CD5(+) and CD5(-)CD10(+) groups were similar to those of the ABC and GCB types, respectively. These findings suggest that CD5(+) and CD5(-)CD10(+) subgroups are included, respectively, in the ABC and GCB types. Finally, when searching for genomic imbalances that affect patients' prognosis, we found that 9p21 loss (p16(INK4a) locus) marks the most aggressive type of DLBCL.

Angioimmunoblastic T-cell lymphoma (AITL) is a distinct peripheral T-cell lymphoma entity exhibiting peculiar clinical features and poor prognosis. Its clinical characteristics and prognostic factors are not well established. To clarify the clinical characteristics and prognostic features of AITL, we conducted a multicenter, retrospective study. Fifty-six patients were enrolled. The median patient age was 68 years. Immunohistochemical examinations of tumor cells showed positivity for CD10 and T-cell markers, and chromosomal examination detected several types of abnormalities. More than 80 % of patients show advanced disease at diagnosis and poor prognostic scores. A high proportion of patients showed accompanying B symptoms, splenomegaly, and hepatomegaly at diagnosis. The 5-year overall survival (OS) rate was 48 % and progression-free survival was 25 %. Univariate analysis revealed higher age, fever, poor performance status, anemia, and low albumin level to be poor prognostic factors for OS. In addition to these factors, both IPI and PIT were also predictive of OS. Multivariate analysis indicated only a low level of serum albumin to be a significant prognostic factor for OS. Serum albumin may be one of the important prognostic factors for AITL. Further investigation is needed to confirm these results.

Background: It is known that gene expression patterns in tumor cells are heterogeneous even in the same individual, not only in solid tumors but also in hematological malignancies, including multiple myeloma (MM). MM is characterized by the accumulation of a population of malignant plasma cells within the bone marrow (BM) and microenvironment (endosteal and vascular niches). Hypoxic oxygen stress occurs in the microenvironment, leading to various epigenetic gene alterations. For instance, hypoxia lead to cell cycle arrest, glycolysis, epithelial-mesenchymal transition (EMT)-related machinery, drug resistance and immature phenotype decreasing expression of CD138 and IRF4. Recently, it is reported that a microRNA (miRNA) in exosomes of myeloma treated in hypoxia induces vessel formation and promotes survival of myeloma cells via upregulation of HIF-1. However, to the best of our knowledge, there have been no reports describing expression changes of miRNAs in myeloma cells during hypoxia. The aim of this study was to detect epigenetically regulated miRNA and essential targets that could be affected by hypoxia in MM. Methods: To identify hypoxia-induced miRNA(s) and their target gene(s), we conducted whole miRNA and mRNA microarray screening using myeloma cell lines (MM.1S, RPMI-8226, KMS12BM, KMS11, and U266) and primary samples (n=4) that were subjected to chronic hypoxia conditions (1% O2 for 48 hr). We purified CD38++ cells (CD38: a marker for activated B-cells and plasma cells) from primary samples, and then conducted functional analysis of target gene/proteins of identified hypoxia inducible miRNA. Results: We recognized a significant upregulation of microRNA-210 (miR-210) under chronic hypoxia. We explored target genes of miR-210 as well as downregulated genes by hypoxia comprehensively, and we found that five candidate genes including DIMT1, CIAPIN1, TTC13, ARMC1, and NOL12 by TargetScan program. Among these, 18S rRNA base methyltransferase DIMT1, which is necessary for the production of ribosome, was the most likely candidate target of miR-210 in myeloma cells because the product was only directly reduced by miR-210 transduction of myeloma cells. We confirmed significant upregulation of miR-210 and downregulation of DIMT1 in primary samples (n=15) cultured in hypoxia by q-RT-PCR. In the examination of the myeloma patient samples on several GEO data sets, we found that expression of DIMT1 increased significantly with disease progression, and that DIMT1 had a positive correlation of IRF4. Notably, immunohistochemical analysis revealed that DIMT1 was strongly stained at myeloma cells but not other BM cells including various lymphocytes. The knock down of DIMT1 for MM.1S, H929, and KMS11 cell lines lead to apoptosis and downregulation of IRF4. Interestingly, miR-210 transduction also reduces expression of IRF4. Furthermore, we found that pan-HDAC inhibitors (panobinostat and vorinostat) inhibit cell survival via downregulation of the DIMT1-IRF4 axis in myeloma cells. Conclusion: By screening for targets of hypoxia inducible miR-210, we identified DIMT1 as a novel diagnostic marker and therapeutic target for all molecular subtypes of MM. Our results suggest that the master antiapoptosis regulator might be different between normoxia and hypoxia. In hypoxia, HIFs and/or the HIF-miR-210 axis may convert myeloma cells to an antiapoptotic phenotype. However, in normoxia, DIMT1 was highly expressed in myeloma cells and, herein, activation might convert myeloma cells to the antiapoptotic phenotype. Figure Figure. Disclosures Ikeda: Novartis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding. Kitadate:Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Abe:Novartis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding. Takahashi:Pfizer: Honoraria, Research Funding; BMS: Honoraria; Novartis: Honoraria, Research Funding. Tagawa:Kyowa Kirin: Research Funding; Otsuka: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

Background: Various types of histone deacetylase inhibitor (HDACi) are currently under development or being used for non-Hodgkin's lymphomas including cutaneous T-cell lymphoma (CTCL). However, there is little molecular-based evidence to support the proposed efficacy of HDACi against advanced CTCL or information on the possible pivotal target genes of HDACi. We have shown that a tumor suppressive microRNA (miRNA), miR-150 inhibits metastasis via combining "seed sequence" of messenger RNA of a chemokine receptor CCR6 in advanced CTCL (Blood 2014). Because HDACi yielded excellent outcome for treating advanced CTCL, HDACi might have a potential to reduce metastasis capability of CTCL via targeting miR-150 and CCR6. Indeed we previously showed that a tumor suppressive miRNA, miR-16 was epigenetically downregulated and HDACi restored its expression leading to induce cellular senescence or apoptosis in various T-cell lymphomas (Oncogene 2015). This led us to consider that HDACis might upregulate variety of tumor suppressive miRNAs and there might be deep association of restoration for some critical gene(s) or/and miRNA(s) expression with HDACis in CTCL oncogenesis. In this study, we focused on to investigate relationship between miRNAs and HDACis, especially focused on to detect CCR6 associated miRNA(s). Methods: My-La, HH, and HUT78 (CTCL cell lines) were used for functional analysis because their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) shortened survival, owing to multiple metastases. To investigate changes of miRNA expression by HDACis, we conducted miRNA microarray analysis. To examine the expression of miR-150 in primary CTCL, we conducted a qRT-PCR analysis of samples from early (patch and/or plaque phase, n = 26) and advanced cases (tumor phase, n = 14). The control samples were obtained from patients with atopic dermatitis (AD, n = 18). Results: We demonstrated that pan-HDACis (vorinostat and panobinostat) inhibited migration of CTCL cells with downregulating CCR6. When we examined miRNA array against CTCL cells treated with and without respective HDACi, 161 miRNAs were commonly upregulated by pan-HDACis in My-La, HH and HUT78. Among these 161 miRNAs, 35 miRNAs that possesses seed sequence of CCR6 showed upregulation by HDACis treatment (e.g., miR-150, miR-96, miR-183, miR-185, miR-194, miR-320a, miR-371a, miR-3135b). To examine the miRNAs that has a functional role in CTCL metastasis, we conducted northern blot analysis of these candidate miRNAs in the CTCL cell lines and primary tissue samples. We found that the miR-150 was strongly expressed in normal CD4+ cells with downregulation of advanced CTCL samples, but the others showed very low or no expressions. In addition, the transduction of candidate miRNAs against the CTCL cells revealed that miR-150 downregulated CCR6 and inhibited their migration most efficiently. These data strongly suggest that the most likely target miRNA of the pan-HDACis in metastasis inhibition was the miR-150 in the metastatic CTCL. To examine whether there was a change in miR-150 expression during disease progression of primary CTCL, we conducted a qRT-PCR analysis of samples from early and advanced cases. The qRT-PCR demonstrated that the expression of miR-150 was significantly lower in the advanced specimens than it was in samples from patients with AD. The miR-150 levels did not significantly differ between the early and the AD specimens, but a significant difference was detected between the early and advanced specimens. These results suggest that miR-150 expression declines with disease progression in CTCL. Finally, we conducted in vivo experiment to examine whether miR-150 indeed inhibit tumor metastasis. We demonstrated that administration of miR-150 against CTCL model mice led to prolong their survivals. Conclusion: miR-150 and CCR6 are essential targets of pan-HDACi in advanced CTCL. Our results provide rational reasons for using pan-HDACi against metastatic CTCL. Furthermore, these results suggest that miR-150 could be not only powerful biomarker for molecular diagnosis, and predictive of metastasis, but also novel therapeutic molecule in advanced CTCL. Disclosures Kitadate: Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Abe:AsahiKasei: Research Funding; Chugai: Research Funding; Otsuka: Research Funding; Kyowa Kirin: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding. Ikeda:AsahiKasei: Research Funding; Chugai: Research Funding; Otsuka: Research Funding; Kyowa Kirin: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding. Takahashi:BMS: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Tagawa:Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

MicroRNA (miRNA; miR) is a class of small regulatory RNA molecules, the aberrant expression of which can lead to the development of cancer. We recently reported that overexpression of miR-21 and/or miR-155 leads to activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway in malignant lymphomas expressing CD3(-)CD56(+) natural killer (NK) cell antigen. Through expression analysis, we show in this study that in both NK/T-cell lymphoma lines and samples of primary lymphoma, levels of miR-150 expression are significantly lower than in normal NK cells. To examine its role in lymphomagenesis, we transduced miR-150 into NK/T-cell lymphoma cells, which increased the incidence of apoptosis and reduced cell proliferation. Moreover, the miR-150 transductants appeared senescent and showed lower telomerase activity, resulting in shortened telomeric DNA. We also found that miR-150 directly downregulated expression of DKC1 and AKT2, reduced levels of phosphorylated AKT(ser473/4) and increased levels of tumor suppressors such as Bim and p53. Collectively, these results suggest that miR-150 functions as a tumor suppressor, and that its aberrant downregulation induces continuous activation of the PI3K-AKT pathway, leading to telomerase activation and immortalization of cancer cells. These findings provide new insight into the pathogenesis of malignant lymphoma.

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = -0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.

We have developed a method that cells exhibiting typical dendritic cell (DC) characteristics are generated from human CD34(+) cells and phagocytose cogenerating erythroid progenitor cells in the presence of tumor necrosis factor-alpha (TNF-alpha), interleukin-3, stem cell factor and erythropoietin. Using this system, we titrated the effects of TNF antagonists, etanercept and infliximab, on TNF-alpha activity. We found that 1 microg/ml etanercept dramatically inhibited the generation of CD11c(+) cells accompanying with a complete recovery of the generation of erythroid progenitors. Infliximab at 200 microg/ml exhibited a similar effect to that observed for etanercept. The delayed addition of etanercept to this culture system at day five resulted in significant inhibitory effects on the generation of CD11c(+), CD4(+) and CD86(+) cells. These results indicate that TNF antagonists administered at a concentration that is achievable in vivo, neutralize the biologic effects of TNF-alpha in generating CD11c(+) cells and that a delay in the administration of these antagonists for as long as 5 days partially inhibits the biologic activity of TNF-alpha. These findings may contribute to a great understanding of anti-TNF therapy in patients with an overproduction of cytokines such as hemophagocytic syndromes.

The gene(s) responsible for natural killer (NK)–cell lymphoma/leukemia have not been identified. In the present study, we found that in NK-cell lymphoma lines (n = 10) and specimens of primary lymphoma (n = 10), levels of miR-21 and miR-155 expression were inversely related and were significantly greater than those found in normal natural killer (CD3−CD56+) cells (n = 8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides (ASOs) targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced by the use of lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to up-regulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21– and ASO-155–treated cell lines all showed down-regulation of phosphorylated AKTser473. Moreover, transduction with either miR-21 or miR-155 led to down-regulation of PTEN and PDCD4 or SHIP1 with up-regulation of phosphorylated AKTser473. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia.

The constitutive activation of nuclear factor-κB (NF-κB) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-κB pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus–infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-κB activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-κB pathways may be advantageous for treatment.

Multiple sequential genetic and epigenetic alterations, including alterations in microRNAs (miRNAs), underlie the development and progression of cancers. For example, overcoming cellular senescence is an early step in cancer pathogenesis. We show here that a tumor-suppressive miRNA, microRNA-16/miR-16 has a potential to induce cellular senescence in cutaneous T-cell lymphoma (CTCL). Firstly, to examine the role of miR-16 in the progression of primary CTCL, we performed q-PCR analysis against early (n=26) and advanced (n=14) Mycosis Fungoides samples. As for control (CD4+ T-cells), we used samples from patients with atopic dermatitis (AD; n=18). We found that expression of miR-16 in both early and advanced stages were significantly lower than AD, with the expression of advanced showing significantly more reduced than early stage. These results suggest that miR-16 is associated with lymphomagenesis from early to advanced stages of CTCL. To examine in vivo tumor-suppressive effects of miR-16, we used NOD/Shi-scid IL2Rgnull (NOG) mouse xenograft model, which would die around day 30-40 after CTCL cells (My-La, MJ, HH or HUT78) injection by invasion and metastasis (Ito et al, Blood 2014). By use of the xenograft model, we found that miR-16 transduced CTCL cells transplanted NOG mice showed significant prolonged survivals than control xenograft mice. We previously demonstrated that miR-16 did not inhibit migration capability of CTCL cells. When we considered that miR-16 successfully contribute to extend survival of mice models despite of lacking migration-inhibition capability, the result suggested that other tumor suppressive mechanisms might exist. Therefore we conducted functional analysis of miR-16, and found that miR-16 could induce cellular senescence against CTCL cells. In CTCL cells that expressed wild-type p53 (My-La and MJ), forced expression of miR-16 enhanced p21, which is a key mediator of senescence, in a p53 dependent manner. Because miR-16 family members are also able to directly suppress the polycomb group protein Bmi1, which is known to negatively regulate p21, we assessed Bmi1 expression in miR-16-transduced CTCL cells and found it to be diminished in all four CTCL cell lines tested. When we knocked down BMI1, expression of p21 was elevated in cells expressing wild-type p53 and the incidence of senescence was increased. We further demonstrated that Bmi1 could directly interact with the promoter lesion of CDKN1A/p21 by cross-linking/chromatin immunoprecipitation assays. From these results, miR-16 has potential to induce cellular senescence via targeting Bmi1-p21 pathway in p53-wild type CTCL cells. Although miR-16 could not induce senescence in p53 non-functional CTCL cells (HH and HUT78), in that case, we found that miR-16 can compensatory induce apoptosis by negative regulation of Bmi1-Survivin pathway. To elucidate this senescence-apoptosis switch mechanism, we examined co-transfection of miR-16 and siCDKN1A/p21 against p53 wild-type CTCL cells. We found that co-transfection of miR-16 and siCDKN1A significantly decreased senescent cells and increased apoptotic cells, with downregulation of Bmi1 and Survivin. In addition, miR-16 negatively regulated cyclinD1-Rb pathway regardless of p53 status, leading to cell growth inhibition against CTCL cells. Together, these results strongly support our idea that presence or absence of functional p53-p21 is an essential factor in miR-16-mediated senescence-to-apoptosis switch in CTCL. Finally, because a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA, vorinostat) has been recently applied and marks good responses in advanced CTCL, we tried to examine whether SAHA might share essential targets with miR-16 in CTCL lymphomagenesis. We found that SAHA restored miR-16 and its essential targets (p21, Bmi1, Survivin), and induced senescence in CTCL cells with wild-type p53 and apoptosis in cells with non-functional p53. Moreover, we found that T or NK/T-cell lymphoma cells (n=8) derived from various subtypes also showed similar tumor-suppressive effects by miR-16 transduction or SAHA treatment. In conclusion, through detailed study of miR-16 function, we have shed light on the mechanism by which lymphoma cells avoid senescence. Furthermore, identification of essential SAHA targets may provide supporting evidence of its clinical application for various non-Hodgkin T/NK-cell lymphomas. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

CD10 and MUM1 are representative B cell differentiation markers. Follicular lymphoma (FL) is usually positive for CD10 and negative for MUM1. In this study, however, we compared 22 FLs with peculiar phenotype CD10−MUM1+ with 119 typical CD10+MUM1− FLs. All CD10−MUM1+ FL patients exhibited follicular structure with follicular dendritic meshwork, and a high rate of somatic hypermutation and ongoing mutation, similar to typical FL. However, CD10−MUM1+ FLs were encountered frequently in the elderly compared with CD10+MUM1− typical FLs (67.0 versus 58.7 years, P < .01), showed high grade (grade 3A or 3B) morphology (91% versus 17%, P < .001), diffuse proliferation (59% vs 19%, P < .001), and lacked BCL2/IGH translocation (5% versus 92.5%, P < .001), which is the most characteristic aberration in FL, and 88% showed BCL6 gene abnormalities (translocation or amplification). Our results indicate that CD10−MUM1+ FL is different from typical FL with respect to biologic and clinical features.

Aggressive adult T-cell leukemia/lymphoma (ATLL) such as acute and lymphoma types are fatal diseases with poor prognosis. Although these 2 subtypes feature different clinicopathologic characteristics, no detailed comparative analyses of genomic/genetic alterations have been reported. We performed array-based comparative genomic hybridization for 17 acute and 49 lymphoma cases as well as real-time quantitative polymerase chain reaction (PCR) to identify the target genes of recurrently amplified regions. Comparison of the genome profiles of acute and lymphoma types revealed that the lymphoma type had significantly more frequent gains at 1q, 2p, 4q, 7p, and 7q, and losses of 10p, 13q, 16q, and 18p, whereas the acute type showed a gain of 3/3p. Of the recurrent high-level amplifications found at 1p36, 6p25, 7p22, 7q, and 14q32 in the lymphoma type, we were able to demonstrate that CARMA1 is a possible target gene of the 7p22 amplification for the lymphoma type but not for the acute type. Furthermore, we found BCL11B overexpression in the acute type regardless of the 14q32 gain/amplification, but no or low expression of the gene in the lymphoma type. These results suggest that acute and lymphoma types are genomically distinct subtypes, and thus may develop tumors via distinct genetic pathways.

MicroRNA (miRNA)s are a class of small regulatory RNA molecules that play important roles in tumor development by pairing with the 3' untranslated region (UTR) of target messenger RNAs to repress their productive translation. Thus although the genes responsible for T-cell lymphomas remain largely unknown, in some lymphoma subtypes, miRNA dysregulation may contribute to tumorigenesis. For example, altered expression of various oncRogenic or tumor suppressive miRNAs has been identified in lymphomas/leukemias and solid tumors We show here that a tumor suppressive miRNA, miR-150, is significantly downregulated in advanced cutaneous T-cell lymphoma (CTCL), and that the downregulation is strongly associated with tumor invasion and metastasis. Inoculation of cutaneous T-cell lymphoma cell lines into NOD/Shi-scid IL-2γnul mice led to CTCL cell migration to multiple organs, but this invasion/metastasis was substantially reduced in miR-150-transfected CTCL cells due to direct downregulation of CCR6, a specific receptor for the chemokine CCL20. MiR-150 thus appears to negatively regulate the interaction between CCL20 and CCR6 in CTCL cells, thereby inhibiting autocrine CTCL cell metastasis. CD4+ T helper cells are divided into TH1, TH2, TH17, and TH22 subsets. Among these, the TH22 subset produces only IL-22, while TH17 cells produce both IL-17 and IL-22. Normally, IL-22 activates CCL20 transcription by binding to the IL-22RA1/IL-10RB receptor, which is not expressed in lymphoid organs or lymphocytes. In the present study, we further found that both IL-22 and its receptor subunit, IL-22RA1, are aberrantly overexpressed in advanced CTCL, but IL-17 is not expressed, suggesting CTCL is derived from the TH22 subset. In the presence of continuous upregulation of CCR6 accompanied by downregulation of miR-150, IL-22 activation could lead to continuous CCL20-CCR6 interaction in CTCL cells and, in turn, autocrine metastasis to distal organs. This is the first report demonstrating an invasion/metastasis mechanism in advanced CTCL. Disclosures: No relevant conflicts of interest to declare.

Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2 gamma nul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e. g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e. g., EZH2, EPC1) and ubiquitin-proteasome (e. g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.

Abstract 4921 Introduction: Lenalidomide, a novel therapeutic agent, has been found to be effective for the treatment of relapsed or refractory myeloma. However, the ideal lenalidomide dose to obtain sufficient therapeutic efficacy without causing hematologic toxicity is yet to be determined. Although Dimopoulos et al. reported that the initial lenalidomide dose can determined on the basis of renal function (i. e., creatinine clearance) and peripheral blood cell counts (namely, neutrophil and platelet counts), dose adjustments, particularly after drug administration, were necessary in some cases because of interindividual differences in pharmacokinetics. We believe that it might be possible to determine the ideal dose by measuring the plasma concentration of lenalidomide. Purpose: We aimed to develop an equation for the predicted total area under the observed plasma concentration–time curve (AUC) of lenalidomide and to set the ideal lenalidomide dose in patients with multiple myelomas by using only 1 or 2 sampling points. Methods: Twenty-one myeloma patients treated with lenalidomide according to the dose recommended by Dimopoulos et al. were enrolled in this study after obtaining written informed consent. Plasma concentrations of lenalidomide from samples obtained just prior to and 1, 2, 4, 8, and 12 h after oral administration of lenalidomide were analyzed using high-performance liquid chromatography (HPLC) (Figure A). Pharmacokinetic analysis of lenalidomide was carried out with a standard non-compartmental method using WinNonlin (Pharsight Co., CA, version 4. 0. 1). The AUC0–24 was calculated using the linear trapezoidal rule. Results: The plasma concentrations of lenalidomide at 2 and 4 h (C2h and C4h, respectively) after its administration were highly correlated with the AUC0–24 value for lenalidomide. The correlation observed between the measured and predicted AUC0–24 values for lenalidomide by using only the C2h and C4h sampling points in the equation (predicted AUC0–24 = 2. 0·C2h + 6. 8 ·C4h +55. 3) is shown in Figure B. The coefficient of determination between the predicted and measured AUC0–24 values was 0. 9213 (P < 0. 0001). In 4 patients for whom the drug was discontinued or administered at a lower dose because of the development of hematologic toxicity in a subsequent course of therapy, the measured AUC0–24 values were significantly higher than the corresponding values in 17 patients who continued to receive lenalidomide (1162 ng·h/ml vs. 646 ng·h/ml, P = 0. 004). Moreover, the results of receiver-operating characteristic curve (ROC) analysis of best sensitivity (100%) and specificity (94. 1%) showed that the ideal AUC0–24 of lenalidomide could be set below 940 ng·h/ml to avoid hematological toxicity. The area under the ROC curve was 0. 985 +-0. 023. Conclusion: The predicted AUC0–24 value might be a new indicator for the management of lenalidomide therapy. In this study, the threshold predicted AUC0–24 values suggested that 4 of 21 patients (19%) should have received a lower initial dose than that recommended by Dimopoulos, et al. It is possible to adjust the ideal dose by using the equation for AUC0–24 with the C2h and C4h values in a test before lenalidomide therapy is actually initiated. Therapeutic drug management (TDM) of lenalidomide can be performed by establishing the minimum effective concentration and the minimum toxic concentration in a large prospective study in the future. These findings should provide economic benefits and facilitate personalized medicine in myeloma therapy with lenalidomide. Disclosures: No relevant conflicts of interest to declare.

Abstract 3939 Background: Multiple myeloma (MM) is characterized by the accumulation of a population of malignant plasma cells within the bone marrow. Cytotoxic chemotherapy-based treatment is not curative, and the disease eventually recurs. Although currently available anti-MM strategies are effective at targeting the bulk of tumor cells, it is not clear that these agents are targeting the tumor-initiating subpopulation, or cancer stem cells. Side Population (SP) cells are an enriched source of cancer-initiating cells with stem cell properties, which have been identified in solid tumors, as well as in hematopoietic malignancies. SP cells express high levels of various members of the ABC transporter family, which are responsible for their drug resistance. A recent our work demonstrated that SP cells in MM have shown to exhibit stem cell like characteristics as well as high tumorigenicity. Therefore, it is worthy to identify gene/proteins specifically expressed in MM SP cells, which could be essential therapeutic targets. Purpose: The aim of this study was to identify genes and transcripts that could serve as molecular markers for targeting the MM SP cells, and to identify candidate agents for the MM SP cells. Experimental design: We used Hoechst 33342 dye to detect the MM SP in five MM cell lines (RPMI 8226, AMO1, KMS-12BM, KMS-11 and JJN3) and eight primary samples. We then tested whether the MM SP cells have stem-like characteristics and performed gene expression analysis to detect genes specifically expressed in the MM SP. On that basis, we tested candidate agents such as an aurora kinase inhibitor (VX-680), a histone methyltransferase inhibitor (DZNep), lenalidomide, thalidomide and a proteasome inhibitor (bortezomib) for their ability to target MM SP cells. Results: We found that clonogenic MM SP cells exhibit “stem cell-like” properties, including self renewal, differentiation and repopulation. Gene expression analysis of MM cell lines and primary samples revealed that, in SP cells, expression of genes related to G2/M phase (e.g. CDC2, CCNB1)-, microtubule attachment (e.g. BIRC5, CENPE, SKA1)-, mitosis or centrosomes (e.g. AURKB, KIF2C, KIF11, KIF15)-, proliferation (e.g. TOP2A, ASPM)-, polycomb (e.g. EZH2, EPC1)- and proteasomes(e.g. UBE2D3, UBE3C, PSMA5)- was significantly stronger in SP than non-SP cells. On that basis, we used VX-680, DZNep, lenalidomide, thalidomide and bortezomib against MM cells. Of these, bortezomib reduced the SP fraction most effectively due to its ability to reduce levels of target gene transcripts including phospho-histone H3, aurora kinase B and EZH2. Finally we tried to examine effects of those candidate agents to “clonogenic ability of SP”, and found that bortezomib possessed the most powerful effects for reduction of SP colonies. These results suggest that bortezomib has a broader range of targets than other agents and could include cell cycle, centrosome, polycomb and proteasome genes/proteins. Conclusion: Our findings are i) the first to identify genes specifically expressed in the MM SP, ii) the first to provide a rationale for treating MM using agents against genes and encoded proteins that are specifically expressed in MM SP cells. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria.

Abstract 861 Background: Mantle cell lymphoma (MCL) is categorized as an indolent CD5+ B-cell lymphoma and is associated with numerous genomic copy number alterations, including 9p21 deletion (CDKN2A) and 10p12 amplification (BMI1). The target gene of the 10p12 amplification has been identified as BMI1, whose overexpression is frequently observed in the blastoid variant of MCL. CDKN2A is also well-known target of BMI1 in solid tumor. So, it has been hypothesized that BMI1 regulates CDKN2A in MCL. However there are the MCL cases with both 10p12 amplification and 9p21 homozygous deletion, suggesting that BMI1 might regulate the other target gene(s). The proto-oncogene, BMI1 is crucially involved in cancer stem cell maintenance and the upregulation has been demonstrated in aggressive or relapsed cases of solid tumors. Cancer stem cells are often identified in the side population (SP) of cancer cells, which is detected based on the cell's ability to export Hoechst 33342 dye via an ATP-binding cassette (ABC) membrane transporter, which gives the SP a distinct low-staining pattern. Aim of the study: The aim of this study is to determine the role of BMI1 in MCL initiating cells, especially in the relapsed cases. In this presentation, we show that the SP fraction has stem cell-like characteristics and high tumorigenic potential, and that BMI1 expression is upregulated in the SP in both relapsed MCL cases and MCL cell lines. Further we show that miR-16 is upstream regulator of the BMI1 in MCL. Results: To determine the role of BMI1 in the pathogenesis of MCL-initiating cells, we firstly examined BMI1 expression at primary MCL cases and found that its expression is stronger in cases of recurrent MCL than at initial diagnosis. We next characterized the MCL SP and found that the SP cells exhibit cancer stem cell-like features and upregulated BMI1 expression, which appears to enhance anti-apoptosis activity. Knocking down of BMI1 increases apoptosis and reduces tumorigenicity in CDKN2A−/− MCL cell lines (REC1 and Z138c). Subcutaneous inoculation of NOD/Shi-scid IL-2γnul (NOG) mice with CDKN2A−/− MCL cell lines, siBMI1-expressing cells were significantly smaller than those in mice receiving control siRNA in vivo. Chip assay showed that BMI1 interacts with BCL2L11/Bim and PMAIP3/Noxa, which were recently shown to be Bmi-1 target. These results suggest that BMI1/Bmi-1 may regulate Bim and/or Noxa to inhibit apoptosis in MCL cells. Furthermore, upon screening for upstream regulator of BMI1, we found that expression of a non-cording regulatory RNA, microRNA-16 (miR-16) is weaker in MCL SP cells than in non-SP cells. To investigate relationship between BMI1 and miR-16, we transfected miR-16 into MCL cell lines, and found that it directly downregulated BMI1, leading to reductions in tumor size following in vivo lymphoma xenograft (NOG mice). Finally, we find that bortezomib, which is known to be a proteasome inhibitor, led to dose-and time- dependent reductions in Bmi-1 expression with re-upregulation of miR-16 in both cell lines and a primary sample. Conclusion: We conclude that dysregulation of miR-16 and BMI1 plays a key role in lymphomagenesis by reducing MCL cell apoptosis, especially in refractory/recurrent cases via enhancement of anti apoptotic function. Disclosures: No relevant conflicts of interest to declare.

Abstract 3429 Background: Idiopathic PRCA and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are major subtypes of adult-onset chronic PRCA. We have previously shown that these types of PRCA are responsive to immunosuppressive therapy but most patients require long-term maintenance immunosuppressive treatment. These results suggest that acquired chronic PRCA is an autoimmune disorder mediated by T lymphocytes and pathogenic T cell clones may be persistently present during remission. We have previously made an interesting observation that a thymoma-associated PRCA patient had an increase of Vd1 gd T cells in blood. We have also reported that recipients of allogeneic hematopoietic stem cell grafts had an oligoclonal expansion of Vd1 gd T cells and that Vd1 gd T clones had cytotoxicity against autologous EBV-transformed B cell line. Thus, gd T cell repertoires may be altered in PRCA patients in response to certain antigens. Objective: In order to clarify the role for gd T cells in the pathogenesis of chronic acquired PRCA, we have examined the gd T cell receptor repertoire in acquired chronic PRCA patients. Materials and Methods: Nineteen PRCA (8 idiopathic, 6 thymoma, 3 LGL-leukemia and 2 SLE) and 107 healthy volunteer donors were included in the study. This study was approved by the Institutional Review Board at Akita University and conducted in accordance with the Declaration of Helsinki. Blood lymphocyte subsets were analyzed by flow cytometry. Clonality of T cells was determined by complementarity-determining region 3 (CDR3) size distribution analysis and junctional sequence was determined by subcloning of PCR products and DNA sequencing. In some experiments, purified gd T cells from PRCA patients were co-cultured with allogeneic erythroid progenitor cells derived from CD34-positive cells in vitro in order to learn whether patient's gd T cells would exert cytotoxic or growth-inhibitory effect on erythroid progenitor cells. Results: The absolute numbers of ab T cells and gd T cells were normal in patients with PRCA, but there were an increase of Vd1 gd T cells and a decrease of Vd2 T cells (Table 1). More than 50% of Vd1 T cells from PRCA patients expressed HLA-DR, while 20 to 30% of those from healthy individuals expressed HLA-DR (Fig. 1). CDR3 size spectratyping revealed that CDR3 size distribution patterns were skewed in 9 out of 13 PRCA patients examined, although skewed CDR3 size distribution patterns were also observed in 7 out of 10 healthy individuals. In order to determine whether a particular Vd1-Jd rearrangement size was selected in PRCA patients, we performed statistical analysis comparing the CDR3 size distribution of 115 Vd1 TCR clones obtained by subcloning of PCR products in 7 PRCA patients versus 7 controls. No significant difference was found between the two groups (p=0.795 by Mann-Whitney test). Moreover, no apparent consensus amino acid motifs were identified in PRCA patients. Although the T cell clone carrying the -YWGIR- sequence in the CDR3d region was detected in 3 PRCA patients, the T cell clone carrying the -YWGIR- sequence was also detected in one healthy donor. Purified gdT lymphocytes from idiopathic PRCA neither showed an inhibitory effect on proliferation nor cytotoxicity against erythroid progenitor cells in vitro. Adjusted p value was calculated by Kruskal-Wallis ANOVA test. Conclusions: Expansion of Vd1 T cells and depletion of Vd2 T cells are unique features for chronic acquired PRCA. Expansion of Vd1 T cells does not seem to be the consequence of CDR3-dependent selection. Depletion of Vd2 T cells may be the result of chronic stimulation, because our previous study has revealed that the numbers of Vd2 T cells show an age-dependent decrease and Vd2 T cells are susceptible to activation-induced cell death (Int J Hematol, in press). Failure to demonstrate the cytotoxicity of gd T cells from a PRCA patient against erythroid progenitor cells suggests that expanded gd T cells are not effector T cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 1807 Dasatinib (DA) is approved for use in imatinib-resistant or intolerant chronic myeloid leukemia (CML)/Philadelphia-positive acute lymphoid leukemia (Ph+ALL) and may also be useful for central nervous system (CNS) leukemia accompanied with CML/Ph+ALL; however, little is known about the relationship between DA pharmacokinetics and its ability to penetrate the blood-brain barrier. Consequently, we measured DA plasma and cerebrospinal fluid (CSF) levels by high-performance liquid chromatography in 20 samples obtained from 11 DA-treated patients (seven Ph+ALL and four lymphoid crisis CML). DA was detected in 10 CSF samples from five patients who were treated with 100 mg QD of DA (CSF C4h of detectable group; 3.526±2.604 ng/mL, 1.11–7.95 ng/mL), which was above the IC50 level for wild type BCR-ABL positive leukemia cells in vitro (0.8 nM = 0.39 ng/mL). However, DA was not detected in 10 CSF samples from 7 patients (CSF C4h of non-detectable group; Disclosures: No relevant conflicts of interest to declare.

Abstract 465 Disease specific genetic alteration or translocation have not been identified in NK/T cell lymphoma. We recently demonstrated that over expressions of miR-21 and/or miR-155 are frequently occurring in natural killer (NK) cell lymphoma/leukemia and deeply associated with their lymphomagenesis by deregulation of AKT signaling. In this study, we tried to identify tumor suppressor miRNA(s) in malignant lymphomas including B-cell, NK-cell and CD4+T-cell lymphoma, by using quantitative PCR and/or Northern blot analysis. We found that miR-150 in both cell lines and primary samples of NK and T-cell lymphoma showed significantly lower expression than those of normal natural killer cells and CD4+T cells. To examine the role for lymphomagenesis, we stably transduced miR-150 into lymphoma cell lines. Enforced expression of miR-150 in NK/T cell lymphoma cell lines showed increased levels of susceptibility of apoptosis, and showed senescence with reduced levels of telomerase activity and telomere DNA shortening. We further demonstrated that miR-150 directly down regulate AKT2, leading to reduced expression of phosphorylated AKTser473/474 with upregulation of tumor suppressors, Bim, p27 and p53. Since it has been proven that AKT kinase phosphorylate hTERT (human telomerase reverse transcript), downregulation of miR-150 in lymphomas lead to activate telomerase activity, resulting immortalization of the cancer cells. These results suggest that miR-150 play as a role of tumor suppressor in NK/T-cell lymphoma. Our recent and previous report demonstrate that downregulation of miR-150 and upregulation of miR-21/miR-155 collaborately contribute to NK/T-cell lymphomagenesis by deregulating AKT signaling. These findings will give a new insight into the pathogenesis of NK/T cell lymphoma and the miR-150 itself and/or AKT targeting therapy can be a useful against aggressive lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 1917 Poster Board I-940 Background: Natural Killer (NK) cell lymphomas/leukemias are characterized groups of highly aggressive lymphoid malignancies, which are comprised of “extranodal NK/T cell lymphoma, nasal type” and “aggressive NK-cell leukemia”. Notably, these two subtypes show many similarities in their morphologic features, immmunophenotypes and genotypes, and are invariably associated with Epstein-Barr virus (EBV), which suggests they may share the same genetic alterations. The gene(s) responsible for natural killer (NK)-cell lymphoma/leukemia have not been identified. Materials and Methods: In the present study, we used Northern and quantitative PCR analyses to screen for and quantitatively assess miRNA expression in NK-cell lymphomas/leukemias using 66 probe sets of miRNAs. For function analysis, we conducted antisense oligonucleotides and lentivirus transfection assays using NK-cell lymphoma cell lines. Results: We found that in NK-cell lymphoma lines (n=10) and specimens of primary lymphoma (n=10), levels of miR-21 and miR-155 expression were inversely related, and were significantly higher than those seen in normal natural killer (CD3-CD56+) cells (n=8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced using lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to upregulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21- and ASO-155-treated cell lines all showed downregulation of phosphorylated AKTser473 (pAKT). Moreover, transduction with either miR-21 or miR-155 led to downregulation of PTEN and PDCD4, or SHIP1 with upregulation of pAKT. Conclusions: Because recent reports suggest that EBV infection can lead directly to upregulation of miR-21 and miR-155, aberrant overexpressions of these miRNAs via EBV infection could be the first-hit genetic alterations during NK-cell lymphomagenesis. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia. Disclosures: No relevant conflicts of interest to declare.

Aberrant overexpression of the miR-17-92 polycistron is strongly associated with B-cell lymphomagenesis. Recent studies have shown that miR-17-92 downregulates the pro-apoptotic protein Bim, leading to overexpression of Bcl2, which likely plays a key role in lymphomagenesis. However, the fact that Jeko-1 cells derived from mantle cell lymphoma exhibit both homozygous deletion of BIM and overexpression of miR-17-92 suggests other targets are also involved in B-cell lymphomagenesis. To identify essential target(s) of miR-17-92 in lymphomagenesis, we first transfected miR-17-92 into genetically distinct B-cell lymphoma cell lines, including Raji cells, which overexpress c-Myc, and SUDHL4 cells, which overexpress Bcl2. Raji cells transfected with miR- 17-19b exhibited downregulation of Bim and reversible upregulation of Bcl2. On the other hand, SUDHL4 cells transfected with miR-17-19b showed aggressive cell growth reflecting facilitated cell cycle progression at the G1-S transition, and decreased expression of CDKN1A mRNA and p21 protein (CDKN1A/p21) that was independent of p53 expression. Conversely, transfection of antisense oligonucleotides against miR-17 and miR-20a into Jeko-1 cells led to upregulation of CDKN1A/p21, resulting in decreased cell growth with G1-S arrest. Thus, CDKN1A/p21 appears to be an essential target of miR-17-92 during B-cell lymphomagenesis, which suggests the polycistron has distinct targets in different B-cell lymphoma subtypes.

Peripheral T-cell lymphoma, unspecified (PTCL-U) is one of the most commonly encountered group among peripheral T-cell lymphomas (PTCLs). This category consists of the cases which do not belong to any of the recognized subtypes of PTCLs in the World Health Organization classification. PTCL-U comprises histologically and clinically heterogeneous groups, which makes it difficult to assess conventional therapies accurately and to develop new therapeutic targets. The first priority is thus to identify subgroups of PTCL-U. However, the molecular basis of their clinical heterogeneity is still poorly understood. Here we analyzed 51 cases of PTCL-U by means of array CGH consisting of 2304 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. DNA gains and losses were detected in 29 cases (total regions: average 15.6, range 1–31; gains: average 8.0 regions, range 0–24; losses: average 7.6 regions, range 0–21). Regions of frequent gain(>15%; ≥5cases) were detected on 1q24.2, 1q25.1–25.2, 3q25–26.1, 3q26.2–27.1, 3q27.3–29, 4p16.2–15.2, 4q21.1–21.21, 4q21.23–28.1, 4q28.3–31.23, 4q32.1, 4q32.3–35.1, 7p, 7q, 9q31.3–32, 9q33.2–34.2, 11q14.1, 11q14.3-tel, and 16p13.2–12.2, and regions of frequent loss on 1p31.2–13.1, 2q37.3, 6q14.3, 9p21.3, 10p14–13, 10p12.31–11.22, 10q11.21–21.1, 10q23.2–23.31, 10q24.2–26.3, 13q14.11–14.2, 13q21.1–34, 16q12.1–21, 17p13.3–11.2, and 18p11.31d–11.22b. Three consecutive BAC clones at 14q32.2 and at 9p21.3 were identified as regions of recurrent high-level amplification and homozygous loss, respectively. Our array CGH analysis could reveal a distinct subgroup consisted of 29 cases which have genomic alterations. Regarding to tumor burden, histopathology, the expression of chemokine receptors, and prognosis, this subgroup displayed significantly different features compared with 22 cases showing no genomic alteration. Our findings showed that PTCL-U with genomic alteration has pathological and clinical relevance distinct from those without, implying that distinct mechanisms underlay in molecular pathogenesis of PTCL-U.

Malignant lymphoma frequently shows genomic amplification of 13q31-32, which harbor c13orf25, a host of mature microRNA-17, 18, 19a, 19b, 20a, and 92–1 (Ota et al., Cancer Res 2004). It has been reported that Eμ-myc mice enforced by over-expression of the miR-17-19b, could develop tumor significantly earlier than those without the expression (He et al., Nature 2005), and that overexpression of a proto-oncogene, c-MYC can up-regulate miR-17 cluster via binding directory upstream of miR-17–92 locus (O’ Donnell et al., Nature 2005). These findings suggest that the miR-17 cluster and c-MYC synergistically contribute to cancer development. However, it is still unclear how c-myc acts synergistically in tumorigenesis with the miR-17 cluster, and which genes are essentially regulated by the miR-17 cluster. In this study, we inserted 674 or 755 bp fragments spanning a given miRNA-genomic region in a modified PMXs vector such that they are placed under the control of a LTR promoter, and thus established Rat-1 and NIH3T3 cells which stably expressed miR-17–18-19a–20a-19b-92 or miR-17–18-19a-20-19b (miR-17–92/miR-17-19b). We demonstrated that the miR-17 cluster is highly upregulated under the constitutive expression of c-myc, and that a rat fibroblast (Rat-1) stably expressing both the miR-17 cluster and c-myc (miRs+myc) shows a full-blown neoplastic transformation phenotype. Because the transformation of Rat-1 cells could be due to the suppression of tumor suppressor gene(s), we next conducted screening for predicted tumor related gene(s) by a Western blot analysis with lysate from Rat-1 transfectants. Our examined predicted tumor suppressor genes of miR-17 cluster were p130 (RBR2), PTEN, SOSC-3, Smad2, MeCp2, and TGF-β type II receptor (TβRII). Among these, we could detect significant reduced expression of TβRII protein in the miRs+myc transfectant, although no other predicted targets showed such a significant difference. The expression levels of TβRII gene in Rat-1 and NIH3T3 transfected with miRs+myc were also repressed in comparison to those of the other transfectants. These results demonstrate that a combined effect of RNA destruction and translation inhibition is used by the miR-17 cluster to silence TβRII. To further substantiate TβRII as a direct target of miR-17 cluster, we cloned its 3′UTR sequence downstream of the firefly luciferase gene (pGL3-TβRII). As a control, a construct with the mutated target sequence was also made. Transfection experiments showed a significant repression of the luciferase activity in the wild type construct in comparison to mutated TβRII, thus suggesting that TβRII is direct downstream target of the miR-17 cluster. Up-regulation of miR-17 cluster and c-myc might contribute to tumorigenesis via the down regulation of both the TβRII-Smads and the c-myc-Miz-1-p15INK4b pathways, respectively. Furthermore, we demonstrated that aggressive B-cell lymphoma frequently represents both c-MYC rearrangement and the upregulation of the miR-17 cluster via 13q31 amplification. These results suggest that the deregulation of the miR-17 cluster and c-MYC synergistically contribute to the aggressive clinicopathological features of cancers.

Peripheral T-cell lymphoma, unspecified (PTCL-U) is the most common group among Peripheral T-cell lymphomas (PTCLs). This category consists of the cases which do not belong to any of the recognizable subtypes of PTCLs in WHO classification. PTCL-U comprises heterogeneous groups in morphology and phenotype. Molecular basis of clinical heterogeneity is needed to identify distinct subgroups with clnical relevance. Several reports of conventional cytogenetic studies including comparative genomic hybridization (CGH) showed some recurrent aberrations, but failed to identify the genetic hallmarks to categorize distinct subgroups. So far, no array-based comparative genomic hybridization (array CGH) study for PTCL-U has been reported. Here we analyzed 29 cases of PTCL-U by means of array CGH consisting of 2265 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. The analysis clearly divided these cases into two distinct subgroups on the basis of frequency of genomic alterations. One group consists of 17 cases which showed significant lower copy number changes (average copy number gains: 0.5 regions, average copy number losses: 0.1 regions). The other group had average copy number gains of 15.7 regions and losses of 15.0 regions in 12 cases. We designate the former as “simple type” and the latter as “complex type”. In the complex type, regions of recurrent (>20%) gain are detected on chromosome 1q23.3-24.2, 3q25.31-tel, 4p15.1-16.1, 4q28.3-31.23, 5q34, 6p24.1-25.1, 7p21.3-tel, 7p21.1, 7q, 8q24.23, 11q13.4-tel, 12p11.21-11.22, 16p12.3-13.3, 17q11.2-22. Regions of recurrent (>20%) losses are detected on chromosome 1p13.1-13.3, 2q37.3, 4q21.21-21.23, 4q34.3-35.1, 5q21.2-23.1, 6p12.1-q14.3, 6q23.2-24.1, 6q25.1-26, 7p14.3-22.1, 9p21.3, 10p14-qtel, 12p13.1-13.2, 13, 14q12, 16q, 17p, 18p, 20q13-2, 22q11.21-12.2. Median age is 62 years in the simple type and 73 years in the complex type, respectively. Median survival is 27 months in the simple type and 11 months in the complex type. Log-rank test for overall survival between the simple type and the complex type showed inferior survival for the complex type but significance was marginal (p=0.21). Our findings showed that PTCL-U comprised two genetically distinct subgroups, implying that distinct mechanisms underlay in molecular pathogenesis of PTCL-U. Furthermore cilinicopathological features of each group are also being studied.

Background: Approximately 70% of low grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma cases are successfully treated by H. pylori eradication. The MALT lymphoma resistant to this therapy needs an additional therapy. A part of the tumors transform to an aggressive lymphoma including diffuse large B-cell lymphoma (DLBCL). The t(11;18)(q21;q21) characteristic to MALT lymphoma is recognized as a marker for H. pylori independency, but this marker is found in only a half of the MALT lymphomas resistant to H. pylori eradication. Therefore the detailed genomic features of eradication resistant group as well as dependent group are needed to understand molecular basis. Patients and Methods: We performed array-based comparative genomic hybridization for 30 gastric MALT lymphomas treated with H. pylori eradication. These included 10 cases of t(11;18)(q21;q21)-positive MALT group, 10 cases of t(11;18)(q21;q21)-negative MALT with H. pylori dependent group and 10 cases of t(11;18)(q21;q21)-negative MALT with H. pylori independent group. The presence of t(11;18)(q21;q21) was identified by a multiplex reverse transcriptase polymerase chain reaction of the API2-MALT1 chimeric transcript. Results: Almost no significant genetic alterations were found in t(11;18)(q21;q21)-positive MALT lymphoma, whereas numerous genomic alterations were found in those without t(11;18)(q21;q21). These aberrations were mostly similar to those found in DLBCL (Tagawa et al., Blood 106:5, 2005). Trisomy 3 is most recurrent and 7q loss specific to SMZL is also recurrent. Within the t(11;18)(q21;q21)-negative MALT lymphoma group, presence of genomic imbalances is more frequent in H. pylori independent group (one of ten (10%) vs seven of ten (70%); p =0.019, two-sided fisher’s exact test). Neither locally advanced disease nor including large cell component is correlated with H. pylori independency, respectively. Conclusions: Genomic imbalances are associated with H. pylori independency in t(11;18)(q21;q21)-negative gastric MALT lymphomas. Thus, the genomic imbalances may have a role in the acquisition of H. pylori-independent growth.

Genomic amplification of the 2p arm has been identified as a recurrent alteration in classical Hodgkin lymphoma, follicular lymphoma, primary mediastinal large B cell lymphoma and diffuse large B-cell lymphoma (DLBCL). We previously reported that 2p15 was gained in 25 out of 100 DLBCL patients by use of a genome-wide array-comparative genomic hybridization (array-CGH). In DLBCL with 2p amplification, genomic co-amplification of REL and BCL11A has been observed. Recent studies suggest that REL amplification is infrequently associated with nuclear REL expression and NFkB activation. In an attempt to identify the target gene at 2p15 amplification, we made BAC contig array CGH glasses for 2p15 region with 33 BAC clones covering 4.5Mb, and found that seven samples of the DLBCL with 2p amplification displayed alterations. REL and BCL11A were located within majority of the gained regions. The minimal common region of amplification was mapped to 0.5 Mb and we found that this region did not include BCL11A. To investigate the relationship between genomic gains and gene expression, we performed real-time quantitative polymerase chain reaction (RQ-PCR) analysis. The results indicated that REL, rather than BCL11A, is the target of 2p15 alterations in DLBCL.

Background: Genomic gain/amplification of 13q31-q32 is frequently observed in malignant lymphomas. C13orf25, recently established as a candidate gene in malignant lymphoma via 13q31-q32 genomic amplification, encodes two variant transcripts by alternative splicing (Ota et al, Cancer Res 2004). Seven microRNA genes (miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20 and miR-92) are clustered in C13orf25 transcript variant 2 (C13orf25 v2). Because microRNAs display dynamic temporal and spatial expression patterns, disruption of these microRNAs may be associated with tumorigenesis. Purpose: The purposes of this study are i) to reveal frequencies of the 13q gain/amplification in various lymphoma types, and ii) to examine the expression of C13orf25 v2 and seven microRNAs using various lymphoma cell lines and tumors with and without 13q gain/amplification. Experimental Design: We analyzed genomic alterations of chromosome 13 for 12 malignant lymphoma cell lines (eight B-cell and four T-cell lymphomas), and 214 cases of B-cell lymphomas (136 cases of diffuse large B-cell lymphoma (DLBCL), 27 cases of sporadic Burkitt’s lymphoma (sBL), 29 of mantle cell lymphoma (MCL), 22 of follicular lymphoma (FCL)) and 20 cases of T-cell lymphoma by using array-based comparative genomic hybridization. The expression levels of seven microRNAs using 12 lymphoma cell lines with (four) and without (eight) 13q gain/amplification were examined by Northern-blot and quantitative real-time PCR (RQ-PCR) analyses. RQ-PCR for C13orf25 v2 (microRNA cluster) was also conducted for 21 cases of DLBCL (eight cases with 13q gain/13 cases without), 10 cases of sBL (four cases with 13q gain/amp/six cases without) and 10 cases of mantle cell lymphoma. Results: Frequent (> 20%) gain/amplification of 13q were detected in DLBCL (31 cases, 23%) and Burkitt’s lymphoma (8 cases, 30%) but no gain/amplification at 13q was found in MCL, FCL and T-cell lymphomas. Genomic amplification of 13q31-q32 was observed in four cases of DLBCL and two cases of sBL, four of which were c-MYC rearranged (two cases of DLBCL and two cases of sBL). RQ-PCR and Northern blot analyses revealed that five of the seven mature microRNAs displayed overexpression in lymphoma cell lines with 13q31 genomic gain/amplification but not in those without. RQ-PCR analysis for 21 cases of DLBCL demonstrated that the cases with 13q gain/amplification (8 cases) showed significantly higher expression of C13orf25 v2 than those without (13 cases) (Mann Whitney U test, P < 0.05). Significant higher levels of the five microRNAs in sBL with 13q gain/amplification were also confirmed by Northern blot analysis. Lower expression levels of the microRNAs were found in T cell lymphoma cell lines and tumors. Conclusion: These results suggest that the microRNA cluster (C13orf25 v2) is a target of 13q/13q31 genomic gain/amplification in DLBCL and sBL.

Background: Adult T cell leukemia/lymphoma (ATLL) is a human T-lymphotropic virus type-I mediated neoplasm and four major clinical subtypes are recognized; i.e., smoldering, chronic, lymphoma and acute types. Among these subtypes, acute and lymphoma types are fetal disease with poor prognosis. Although these two subtypes represent different clinical features, detailed analysis for genomic/genetic differences has not been sufficiently investigated. Purpose: To investigate genomic aberrations of these subtypes, we performed array-based comparative genomic hybridization (array CGH: 2308 BAC clones spotted in duplicate) for 66 cases of ATLL (17 cases of acute and 49 cases of lymphoma subtypes). To determine target genes in recurrently amplified regions, quantitative real-time PCR (RQ-PCR) was conducted. Results: Array CGH: Comparison of genome profiles revealed that lymphoma type shows more frequent gain and loss than those of acute type. The numbers of respective genomic gain and loss regions of lymphoma type were 9.7 and 9.8 on average. The numbers of genomic gain and loss regions of acute type were 4.2 and 4.5 on average. Gain of 1q, 2p, 4q, 7p and 7q, and loss of 10p, 13q, 16q and 18p were characteristic to lymphoma type whereas gain of 3p and 3q was specific to acute type. Recurrent high-level amplifications were found at 1p35, 6p25, 7p22.2, 7q21–q22, 7q31–q36 and 14q32 in lymphoma type. However, such genomic amplifications were not found in acute type. RQ-PCR: CARMA1 (7p22 locus), which encodes signaling proteins associated with development of B and T cells, was a possible target of the 7p22 amplification in lymphoma type. The extremely higher expression of BCL11B (14q32 locus) which is a key regulator of both differentiation and survival during thymocyte development, was found in acute type irrespective of the 14q32 amplification. However, no overexpression of BCL11B was found in lymphoma type with high copy number gain, indicating that BCL11B is not the target gene for 14q32 amplification in the lymphoma type. Conclusion: Array-CGH revealed that patterns of genomic aberrations between acute and lymphoma types were different. Quantitative gene expression analysis also implicated distinct mechanism of aberrant gene expressions between acute and lymphoma types. Combination of array-CGH and quantitative gene expression analysis provided evidence that the acute and lymphoma types are developed via distinct genomic/genetic pathways.