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SARS-CoV-2 Omicron variants are highly resistant to vaccine-induced immunity and human monoclonal antibodies. Here, we demonstrate that a novel nanobody TP86 potently neutralized both BA.1 and BA.2 Omicron variants, and that the TP17 and TP86 nanobody cocktail broadly neutralized in vitro all VOCs as well as D614G. Furthermore, this cocktail showed therapeutic efficacy in vivo on VOCs using human ACE2 transgenic mice.

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted—most successfully, vaccination1–3—imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)—namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

SummarySARS-CoV-2 Lambda, a new variant of interest, is now spreading in some South American countries; however, its virological features and evolutionary trait remain unknown. Here we reveal that the spike protein of the Lambda variant is more infectious and it is attributed to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino-acid deletion mutation in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the insertion of the RSYLTPGD246-253N mutation is closely associated with the massive infection spread of the Lambda variant in South America.HighlightsLambda S is highly infectious and T76I and L452Q are responsible for this propertyLambda S is more susceptible to an infection-enhancing antibodyRSYLTPGD246-253N, L452Q and F490S confer resistance to antiviral immunityGraphical Abstract

SummaryDuring the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity.HighlightsP681R mutation is highly conserved in the B.1.617 lineagesP681R mutation accelerates and enhances SARS-CoV-2 S-mediated fusionPromotion of viral fusion by P681R mutation is augmented by TMPRSS2

Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used cap analysis of gene expression (CAGE) to compare the transcriptional landscape of latently infected cells with that of noninfected or productively infected cells., The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus-producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a green fluorescent protein (GFP) reporter under the HIV-1 promoter and a monomeric Kusabira orange 2 (mKO2) reporter under the internal elongation factor alpha (EF1α) promoter. This viral construct enables direct identification of both productively and latently HIV-1-infected cells. In this study, we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using cap analysis of gene expression (CAGE). We deep sequenced CAGE tags in non-infected and latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus-producing cells had differentially expressed TSSs related to T-cell activation and apoptosis compared to those of non-infected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to those of non-infected cells. Among these, SPP1 and APOE were downregulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have antiviral properties. Components of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway, MLST8, 4EBP, and RPS6, were significant TSSs in productively infected cells, and S6 kinase (S6K) phosphorylation was increased compared to that in latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent. IMPORTANCE Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used cap analysis of gene expression (CAGE) to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T cells cannot recognize incoming viral components or the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1-infected cells.

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86, 493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

Interdigitating dendritic cell sarcoma (IDCS) is a rare neoplasm considered to derive from a dendritic cell. Recent studies have shown that B- or T-lymphoblastic leukemia/lymphomas can develop clonally related histiocytic/dendritic cell (H/DC) neoplasms, such as histiocytic sarcoma, Langerhans cell

Neurolymphomatosis is a rare entity defined as nerve infiltration by neurotropic abnormal lymphocytes which can lead to the development of neuropathy, with typical presentations including pain, hypoesthesia, paresthesis and palsy. We herein report two cases where critical bilateral vocal cord paralysis due to neurolymphomatosis in recurrent nerves occurred in refractory Burkitt lymphoma and adult T-cell lymphoma patients. High-dose methotrexate and intrathecal chemotherapy injection for the nervous lesions were ineffective, and the patients died. Neurolymphomatosis of the recurrent nerve is an emergent and difficult complication and should be suspected when sudden onset of aphasia, hoarseness or shortness of breath is found in refractory lymphoma patients.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of A3B alone in cancer cells. To investigate A3B function in myeloma cells easily and comprehensively, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced under known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeting drugs, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed the EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage response triggers A3B expression through ATM, ATR and DNA-PK signaling.

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors., がんに遺伝子変異を導入する酵素の分子スイッチを発見 --リン酸化によるDNAシトシン脱アミノ化酵素の活性制御機構--. 京都大学プレスリリース. 2019-06-07.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminases have emerged as potential genomic mutators in various cancers. Multiple myeloma accumulates APOBEC signature mutations as it progresses; however, the mechanisms underlying APOBEC signature acquisition and its consequences remain elusive. In this study, we examined the significance and clinical impact of APOBEC3B (A3B) activity in multiple myeloma. Among APOBECs, only highly expressed A3B was associated with poor prognosis in myeloma patients, independent of other known poor prognostic factors. Quantitative PCR revealed that CD138-positive primary myeloma cells and myeloma cell lines exhibited remarkably high A3B expression levels. Interestingly, lentiviral A3B knockdown prevented the generation of deletion and loss-of-function mutations in exogenous DNA, whereas in control cells, these mutations accumulated with time. A3B knockdown also decreased the basal levels of γ-H2AX foci, suggesting that A3B promotes constitutive DNA double-strand breaks in myeloma cells. Importantly, among control shRNA-transduced cells, we observed the generation of clones that harboured diverse mutations in exogenous genes and several endogenous genes frequently mutated in myeloma, including TP53. Taken together, the results suggest that A3B constitutively mutates the tumour genome beyond the protection of the DNA repair system, which may lead to clonal evolution and genomic instability in myeloma., 多発性骨髄腫における遺伝子変異蓄積の分子メカニズムの一端を解明 --新たな治療標的としてのDNAシトシン脱アミノ化酵素の可能性--. 京都大学プレスリリース. 2019-05-17.

Few studies have examined the prognostic impact of blood markers [other than the five factors in the enhanced International Prognostic Index (NCCN-IPI)] in elderly patients with diffuse large B cell lymphoma (DLBCL). We retrospectively analyzed 391 DLBCL patients receiving rituximab plus anthracycline-containing chemotherapy to examine the prognostic impact of simple blood markers. The NCCN-IPI was more accurate for discriminating prognoses than the original IPI. Multivariate analysis identified platelet count (

APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family proteins restrict retroviruses and retrotransposons by inducing hypermutation or degradation of the replication intermediates through their DNA cytidine deaminase activity. APOBECs can also act as endogenous sources of DNA damage that mutate many human cancers. Accumulation of APOBEC signature mutations is associated with disease progression and poor overall survival in multiple myeloma (Walker et al. Nat Commun, 2015). Among APOBEC3 enzymes, APOBEC3B (A3B) is the only family member that is predominantly located in the nucleus throughout the cell cycle. We previously reported that A3B knockdown decreased cytidine deaminase activity in myeloma cells, suggesting that among APOBECs, A3B plays a major role in cytidine deamination-related mutagenesis in myeloma cells (Yamazaki et al., Sci Rep, 2019). Recent studies showed that cofactors of A3B could affect the functions of A3B: heterogeneous nuclear ribonucleoproteins (hnRNPs) interact with surface hydrophobic residues of the N-terminal domain in order to bind to A3B (Xiao et al., Nuc. Acids Res, 2017; Zhang al., Cell Microbiol, 2008); BORF2, an Epstein-Barr viral protein, interacts with the A3B catalytic domain and inhibits A3B DNA cytidine deaminase activity (Cheng et al., Nat Microbiol, 2018). However, the biological mechanisms of how endogenous A3B induces mutations in genomic DNA are still unclear. In this study, we aim to ascertain the cofactors for nucleic acid binding and elucidate the regulatory mechanisms that prevent APOBEC-mediated genomic mutagenesis. Because of the high homology between APOBEC3 proteins, a specific antibody against A3B is not available, and it is difficult to analyze A3B-interaction at the endogenous expression level. To overcome this technical impediment, we used a lentiCRISPR system to insert a FLAG-tag sequence at the C-terminus of the A3B gene in A3B highly expressing myeloma cell lines (AMO1 and RPMI8226). We then conducted A3B-immunoprecipitation with the anti-FLAG M2 antibody, followed by mass spectrometry (MS) analysis to identify potential A3B interacting proteins. MS analysis identified 40 putative interacting proteins and these proteins were clustered largely into two interaction networks: ribonucleoprotein complex and ribosomal-associated proteins. We also performed Gene Ontology (GO) enrichment analysis and revealed that spliceosome, ribosome, and RNA transport were significantly enriched terms. We confirmed the binding between A3B and selected A3B interacting proteins: hnRNPs, interleukin enhancer-binding factor 2 and 3 (ILF2, ILF3) in myeloma cell lines by co-immunoprecipitation assays. Next, we tested the intracellular colocalization of overexpressed A3B and interacting proteins in Hela cells by immunofluorescence microscopy. We found that ILF2 presents strong colocalization with A3B in the nuclei of cells. We also employed density-gradient sedimentation analysis to test if these proteins form high molecular mass (HMM) complexes with A3B in the nucleus using HEK293T cells expressing FLAG-tagged A3B. We detected that ILF2 is one of the components of HMM A3B complexes. To check whether these putative interacting proteins affect A3B cytidine deaminase activity, we next performed an in vitro luminescence-based screening assay (AlphaScreen)using a FLAG-GST protein library which was produced by the wheat cell-free protein production system. We found that hnRNP A1 and ILF2 decreased A3B cytidine deaminase activity. This study provides for the first time a proteomic characterization of A3B interactome in a myeloma cell context. Our findings reveal putative A3B cofactors in myeloma cell lines which may regulate the catalytic activity of A3B. We discuss how these proteins bind A3B and affect its activity in myeloma cells. Disclosures Takaori-Kondo: Pfizer: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding.

A 19-year-old woman complaining of fever and a sore throat was diagnosed with very severe aplastic anemia (AA) by bone marrow examination at a local hospital. Despite administration of antibiotics and granulocyte-colony stimulating factor to treat the soft tissue infection in her neck, her neutrophil count showed no increase. Because emergent allogeneic stem cell transplantation (SCT) was necessary, she was referred to our hospital. On admission, computed tomography revealed right-sided severe pharyngitis and lymphadenitis causing tracheal stenosis, and emergent intubation was required the next day. Granulocyte transfusion therapy (GTX) from related donors coupled with broad-spectrum antibiotic administration controlled the otherwise overwhelming infection. The patient received allogeneic peripheral blood SCT using a reduced-intensity conditioning regimen. After allogeneic SCT, successful engraftment was obtained. She was discharged from the hospital 59 days after allogeneic SCT. She remains alive and well, as of the latest follow up. This case clearly demonstrates that GTX is useful for controlling severe infection and enables patients with severe AA to receive allogeneic SCT safely.

Acetylation is a reversible process under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetyl proteome analysis revealed that acetylation regulates various cellular processes through both histone and non-histone proteins (Choudhary et al., Science, 2008). Subsets of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) have inactivating mutations of HATs, CBP and p300 (Pasqualucci et al., Nature, 2011). In addition, p300 mutation is a poor prognostic factor in FL patients (Pastore et al., Lancet Oncol, 2015). In a mouse model, CBP deficiency promotes B cell lymphomagenesis through H3K27 deacetylation of enhancer mediated by the HDAC3, which forms repressor complex with Bcl-6, SMRT and NCoR (Jiang et al., Cancer Discov, 2016). Human HDACs consist of 18 isoenzymes that are classified into 4 classes (I-IV). Among the 4 classes, class I includes 4 HDACs (HDAC1, 2, 3 and 8) that are ubiquitous. Four HDAC inhibitors are FDA approved for the treatment of cutaneous T cell lymphoma (romidepsin, vorinostat), peripheral T cell lymphoma (belinostat) and multiple myeloma (panobinostat). These inhibitors mainly target class I and class II HDACs, each with a different specificity. This non-specific nature of current HDAC inhibitors limits their efficacy and causes adverse effects, therefore, several selective HDAC inhibitors has been developed. Selective HDAC3 inhibition restricts myeloma cell growth in vitro and in vivo more efficiently than selective HDAC1 or HDAC2 inhibition, allowing for DNMT1 acetylation, accelerating its degradation by the proteasome (Harada et al., Leukemia, 2017). However, the efficacy of selective HDAC3 inhibition against B cell lymphoma remains unclear. We first knocked down HDAC3 by shRNA in a B-cell lymphoma cell line (KIS1). We found that selective HDAC3 knock down suppressed cell growth and induced apoptosis. We next tested selective HDAC3 inhibitors (RGFP966 and AA-1) using five B cell lymphoma cell lines (Raji, Ramos, KIS1, SUDHL-6 and Granta519). We confirmed that these inhibitors reduced cell viability in all treated cell lines, significantly increased the number of apoptotic cells in Ramos, KIS1 and SUDHL-6 and induced cell cycle arrest in Raji and Granta519, rather than apoptosis. Lentiviral shRNA against HDAC3 or the inhibitors also led to the cleavage and subsequent activation of caspase9 and caspase3. These results show that HDAC3 inhibition induces apoptosis by activating the intrinsic apoptotic pathway. To clarify how HDAC3 inhibitors affect global protein acetylation and how they induce apoptosis in B cell lymphoma, we conducted acetyl proteome analysis using LC-MS/MS. Briefly, we immunoprecipitated acetyl lysine peptides from whole cell lysates of SUDHL-6 with or without RGFP966 treatment, and analyzed them by mass spectrometry. We identified 673 and 1,328 acetylation sites in RGFP966 treated and untreated samples, respectively, and 1,425 acetylation sites in total. Among these 1,425 sites, 153, including histones and HATs, were more than two fold upregulated in the RGFP966 treated sample. To identify which cellular processes are affected by RGFP966 treatment, we performed Gene Ontology (GO) enrichment analysis of the proteins with upregulated acetylation. GO enrichment analysis revealed that bromodomain, glycolysis and unfolded protein binding were significantly enriched terms upon RGFP966 treatment. Our data show that selective HDAC3 inhibition is also effective against B cell lymphoma and that protein folding and metabolic processes as well as epigenetic mechanisms might be involved in activation of the intrinsic apoptosis pathway upon HDAC3 inhibition. We discuss how acetylation of these proteins leads to cell growth inhibition and apoptosis in B cell lymphoma. Disclosures Takaori-Kondo: Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.

Multiple myeloma (MM) is an incurable plasma cell malignancy and the reason for this is not yet completely understood. It might be partially attributable to heterogeneity and clonal evolution through DNA mutation accumulation. Recent next generation sequencing (NGS) studies have revealed that apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) 3B (A3B) is one of the cause of clonal evolution in MM. APOBEC protein family consists of 11 proteins and most of them, including A3B, have cytidine deaminase (CDA) activity on single-strand DNA, which catalyzes deamination of cytosine to uridine, resulting in C-to-T or C-to-G mutations. We have previously shown in human cell line models that A3B induces genomic mutations (Shinohara et al., Sci Rep, 2012). Therefore, controlling A3B CDA activity is an attractive therapeutic strategy to overcome the unfavorable prognosis of patients with these malignancies. Enzymatic activity of other APBEC family proteins, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G) is regulated by protein kinase A (PKA)-mediated phosphorylation. A3B also has two consensus PKA phosphorylation motifs conserved upstream of each CDA domain. Thus, we hypothesized that PKA phosphorylates A3B which may alter A3B CDA activity. Using NetPhosK1.0 and ScanSite programs, two A3B residues (serine at the 46th and threonine at the 214th position) are predicted to be PKA phosphorylation sites. Firstly, we examined the binding between A3B and PKA. We transfected HEK293T cells with expression vectors for C-terminal HA-tagged A3B and N-terminal FLAG-tagged PKA catalytic subunit a (PKACA) and used CoIP assays with anti-HA or anti-FLAG antibodies to demonstrate physical binding between A3B and PKACA. Secondly, to determine whether the putative residues are phosphorylated, we overexpressed PKACA and A3B alanine mutants in HEK293T cells and performed immunoblotting analysis with anti-phospho PKA motif antibodies and found that threonine-214 is phosphorylated by PKA. To confirm that PKA phosphorylates A3B directly, we employed in vitro phosphorylation assays using purified C-terminal A3B and PKACA and found that PKACA phosphorylated A3B, but not the A3B T214A mutant, thus we concluded that PKACA directly phosphorylates threonine-214 in A3B. Next, to investigate the effect of this phosphorylation on the CDA activity of A3B, we constructed phosphomimetic mutants of these residues, T214D and T214E. We carried out in vitro CDA assays using cell lysates from HEK293T cells overexpressing A3B or mutants (T214A, T214D, T214E), and found that T214D and T214E almost completely lost their CDA activity. We also performed in vitro CDA assays using purified C-terminal A3B and mutant proteins. The purified A3B T214D mutant also lost CDA activity, suggesting that PKA-mediated phosphorylation of A3B might also lead to loss of its CDA activity. To confirm this result in cell lines, we used differential DNA denaturation PCR (3D-PCR) of DNA from HEK293T cells transfected with vectors for uracil DNA glycosylase inhibitor, EGFP and A3B wild type or mutants. 3D-PCR products of the EGFP sequence were amplified at a lower denaturation temperature in cells expressing the wild type A3B compared to those in cells expressing phosphomimetic A3B or catalytically inactivated A3B. These results indicate that phosphomimetic A3B no longer has exogenous DNA editing activity. We further plan to evaluate the PKA phosphorylation of A3B and the consequences on its mutagenic potential in myeloma cell lines. In conclusion, PKA induces phosphorylation at threonine-214 in A3B, and this phosphorylation suppresses its CDA activity. PKA-mediated phosphorylation of A3B may be an attractive therapeutic target for inhibiting A3B-induced clonal evolution in lymphoid malignancies, especially in MM. Disclosures Takaori-Kondo: Kyowa Kirin: Research Funding; Alexion Pharmaceuticals: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) has an aggressive clinical course and, even in the R-CHOP era, about 10-20% of DLBCL patients (pts) show primary refractory disease. For pts with primary refractory disease, salvage chemotherapy with or without stem cell transplantation (SCT) is generally performed, but even with this treatment, less than half of the pts survive. To elucidate the pts population who require innovative therapy, we retrospectively analyzed the risk factors that lead to an unsuccessful outcome of primary refractory DLBCL. DLBCL pts who received R-CHOP with curative intent were included. Pts with primary mediastinal large B-cell lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma and iatrogenic immunodeficiency-associated lymphoproliferative disorders were excluded. In this study, primary refractory disease was defined as disease progression during first-line immuno-chemotherapy or recurrence within 3 months after completion of the first-line therapy. The primary endpoint was overall survival (OS) after treatment failure, which was assessed using the Kaplan-Meier method. The log-rank test and Cox regression analysis were used to assess the prognostic values of each clinical variable. From January, 2004 to December, 2013, 440 DLBCL pts were referred to our institute. Among them, 60 pts (20 females and 40 males) were considered to have primary refractory disease (55 with progressive disease and 5 with early relapse). At the time of treatment failure, the median age was 72 (range, 31–90) and 44 pts (73%) showed advanced stage (stage IIIor more) disease. Nineteen (32%) had an ECOG performance status (PS) of 2 or more, and 23 (38%) had a high IPI score. In 31 pts, disease progression was observed at novel sites that had not been detected during the initial evaluation; patients with these sites were designated as "new lesion pts". The remaining 29 pts displayed regrowth of known lesions and were designated as "regrowth pts". Among new lesion pts, 29 displayed extranodal disease. After disease progression, 52 pts received salvage chemotherapy or radiotherapy, and 21 pts achieved a clinical response. Among the 24 patients less than 70 years old, 11 received autologous or allogeneic stem cell transplantation (SCT). After a median follow-up time of 17 months (range, 1–81 months), 39 pts had died of lymphoma. The 1-year OS rate (1y-OS) was 38.8% [95% confidence interval (CI), 25.5-51.8%].Univariate analysis using clinical variables at the time of treatment failure revealed that new lesion pts or patients with an IPI score >3 had a poor 1y-OS rate (p Primary refractory DLBCL pts with new lesions, most of which were extranodal sites, have an extremely poor outcome. Disease progression at novel extranodal sites may indicate that the disease has chemotherapy-resistant characteristics. Routine approaches using platinum- or cytarabine-based salvage chemotherapy followed by SCT rarely overcome the progressive nature of the disease, and a novel treatment strategy is required. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The prognostic impact of interim positron emission tomography scans (I-PET) for patients with diffuse large B-cell lymphoma (DLBCL) is a matter of debate because its positive predictive value (20–80%) is low. Here, we aimed to improve the prognostic impact of I-PET by combining it with interim analysis of serum soluble interleukin-2 receptor (sIL2R) levels, the levels of which at the time of diagnosis are associated with the prognosis of DLBCL. We retrospectively examined data from DLBCL patients diagnosed at our institution between January 2006 and October 2013. Patients were included in the analysis if they met all of the following criteria: six or more cycles of rituximab plus CHOP regimen (R-CHOP); I-PET performed after 2-4 cycles of chemotherapy; and sIL2R levels measured after each cycle. Patients with primary central nervous system lymphoma, primary mediastinal large B-cell lymphoma, or transformed DLBCL from indolent lymphoma were excluded. I-PET was assessed visually according to the International Harmonization Project criteria. The interim sIL2R (I-IL2) level was defined as the value measured just before the fourth R-CHOP cycle. I-IL2 levels > 800 U/ml, or 2000 U/ml if serum creatinine was > 2.0 mg/dl (sIL2R is influenced by renal function), were regarded as positive. The primary endpoint of the study was progression-free survival (PFS). The unadjusted probabilities of PFS were estimated using the Kaplan-Meier method. The log-lank test and multivariate Cox regression analysis were used to assess the prognostic values of each clinical variable. In total, 135 patients were enrolled. The median age was 66 years (range, 34–89) and 66 patients (48.9%) were male, 27 (20%) had an ECOG performance status >1, 18 (13.3%) had bulky disease, 81 (60%) had advanced disease, and 61 (45.2%) had a high or high-intermediate International Prognostic Index (IPI) score. The median follow-up time was 25.6 months (range, 6.3–88.7) and the 2 year progression-free survival rate (2-y PFS) of the entire cohort was 72.9% (95% confidence interval (CI), 63.8–80). I-PET and I-IL2 were positive in 47 (34.8%) and 15 (11.1%) patients, respectively. Univariate analysis revealed that a high IPI score, a positive I-PET, and a positive I-IL2 had statistically significant poor prognostic effects on PFS, although gender and bulky disease did not. The three significant variables were entered into multivariate analysis, which identified positive I-PET and I-IL2 values (but not IPI) as independently associated with a poor prognosis. The 2-y PFS was 81.8% (95% CI, 70.4–89.1) for I-PET-negative and 56.3% (95% CI, 40.7–69.3; p DLBCL patients that were both I-PET- and I-IL2-positive suffered a high rate of progression; therefore, such patients should be targeted by novel therapeutic approaches. Because the study was based on a retrospective analysis and a limited follow-up period, further studies are needed to confirm the prognostic impact of the combined use of I-PET and I-IL2. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background: High-dose cytarabine (HD-AraC) is a promising therapy for acute myeloid leukemia (AML). However, it causes substantial toxicity, such as severe cytopenia and cytarabine-encephalopathy. The efficacy of HD-AraC for the treatment of core-binding-factor (CBF) AML has been well established; however, whether or not HD-AraC is suitable for the treatment of non-CBF AML is unclear. Moreover, it remains controversial whether HD-AraC is effective before allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the treatment of non-CBF AML. In this study, we investigated the efficacy of HD-AraC in terms of optimal numbers of HD-AraC cycles required to treat non-CBF AML patients who did and did not receive allo-HSCT. Methods: We retrospectively collected the data of consecutive AML patients who were aged 15-70 years, treated in our hospital and diagnosed between Jan. 2000 and Apr. 2014. Patients with acute promyelocytic leukemia (APL) and CBF AML were excluded. Only patients who achieved complete remission (CR) and received post-remission therapies with a curative intent were included; namely, only those who received allo-HSCT or completed more than one cycle of consolidation chemotherapy. Patients who exclusively received azacytidine-based or low-dose AraC-based therapies were excluded. HD-AraC therapy was defined as a chemotherapeutic regimen utilizing 12 g/m2/cycle or more of AraC. Patients who were not suitable for HD-AraC because of comorbidities were also excluded. Overall survival (OS) was separately analyzed in patients who underwent allo-HSCT in the 1st CR (group A) and in those who did not (group B). The unadjusted probabilities of OS were estimated using the Kaplan-Meier method, and univariate analysis was done using the log-rank test. Multivariate analysis was performed with Cox proportional hazard regression models, and 95% confidence intervals (CIs) were calculated. In multivariate analysis, the number of cycles of HD-AraC, age, cytogenetic data, and whether or not the patient achieved CR after the 1st cycle of chemotherapy were used as covariates. In group A, donor source and conditioning were also considered as covariates. Results: A total of 166 non-APL, non-CBF AML patients were identified. Seventy-one patients were excluded for the following reasons: 50 did not achieve CR, 13 underwent incomplete consolidation therapies because of complications or early relapse, 7 had missing data, and 1 had chronic renal disease. For HD-AraC, toxicity was not a consideration for the discontinuation of treatment, but the dose of AraC was reduced to an intermediate dose (AraC2 cycles of HD-AraC were 50.6%, 85.7%, 75.0%, and 68.6% (n=22, 9, 8, 11) in group A, and 44.3%, 16.0%, 65.6%, and 50.0% (n=19, 10, 10, 6) in group B, respectively. Univariate analysis showed that patients in group A who received one or more cycles of HD-AraC had a longer 3y-OS (75.4% vs 50.6%, p=0.024) (figure 1), whereas patients in group B who received two or more cycles of HD-AraC had a longer 3y-OS (57.1% vs 36.2%, p=0.078) (figure 2). Multivariate analysis of patients in group A showed that one or more cycles of HD-AraC resulted in a survival benefit (Hazard ratio=0.26, 95%CI: 0.070-0.98, p=0.046), whereas multivariate analysis of patients in group B showed that two or more cycles of HD-AraC (Hazard ratio=0.36, 95%CI: 0.13-0.97, p=0.044) and intermediate-risk of cytogenetics resulted in a survival benefit. Conclusion: For consolidation chemotherapy, at least one cycle of HD-AraC is recommended for patients who plan to receive allo-HSCT, and at least two cycles is required for other patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Title Risk stratification of DLBCL patients according to NCCN-IPI in our hospital Background Recently, a robust prognostic tool termed enhanced International Prognostic Index (NCCN-IPI) for the rituximab era was reported. The aim of this study was to assess the usefulness of NCCN-IPI in risk discrimination and its suitability in clinical applications. Patients and Methods We retrospectively analyzed consecutive patients with de novo diffuse large B cell lymphoma (DLBCL) who were diagnosed and treated with R-CHOP or CHOP-like regimens between January 2004 and December 2013. Patients were required to be cancer-free for 5 years before diagnosis and had to have no prior documented history of indolent lymphoma. We stratified DLBCL patients using IPI and NCCN-IPI, and estimated overall survival (OS) in each risk group. The unadjusted probabilities of OS were estimated using the Kaplan-Meier(K-M) method. The log-lank test and multivariate Cox regression analysis were used to assess the prognostic values of each clinical variable. Results Three-hundred and seventy one patients were identified. The median age was 69 (20–93) years. The median follow-up time was 41 months. The numbers of patients with NCCN-IPI-defined low (L), low-intermediate (L-I), high-intermediate (H-I), and high (H) risk were 35 (9.4%), 125 (33%), 139 (37%), and 72(19%), respectively. The 3-year OS in each risk group was 93%, 89%, 70%, and 52%, respectively. NCCN-IPI was better for the discrimination of low- and high-risk groups (3-year OS, 93% vs 52%) than IPI (3-year OS, 87% vs 56%), and NCCN-IPI gave a better concordance index than IPI (c-Harrel = 0.678 vs 0.665). However, the discriminating power of NCCN-IPI was not as good as previously reported. In the multivariate analysis using the five independent variables of NCCN-IPI as covariates, age was not a significant factor in the 1-3 groups. Ann Arbor stage was not a significant factor either. Next, we examined extranodal involvement, which was a significant prognostic factor (HR, 2.3; p75, 1pt), LDH ratio (>3, 1pt), extranodal disease (bone marrow, lung, CNS, or GI tract, but not liver, 1pt), and ECOG PS (≥2, 1pt) and not using Ann Arbor stage as a prognostic factor. Four distinct groups were formed based on K-M curves for OS: low (L, 0 pt), low-intermediate (L-I, 1pt), high-intermediate (H-I, 2pt), and high (H, 3-4 pt). Simplified NCCN-IPI better discriminated low- and high-risk groups (3-year OS, 93% vs 34%; c-Harrel = 0.705) than NCCN-IPI. When patient age was compared in each scoring system, high-risk patients were older in the NCCN-IPI (median age, 79 years old) and its simplified version (median age, 79 years old) than in IPI (median age, 72 years old). Conclusion NCCN-IPI showed better discrimination in our cohort than IPI. However, we found that the enhancement in predicting outcomes by including age and LDH was not as useful as previously reported, and stage and liver involvement was not an independent prognostic factor in our cohort. In the rituximab era, Ann Arbor stage is not a useful prognostic factor. Further validation and optimization of cut-off in each variable could improve the NCCN-IPI. The majority of the high risk patients evaluated by both NCCN-IPI and simplified NCCN-IPI were elderly, so innovative therapeutic approaches adjusted for age are required to improve the outcome of the high-risk group. To identify high-risk groups especially among younger patients, a refined scoring system including not only conventional clinical factors but also other factors such as biological markers will be required. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Background Recent reports have suggested that allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-matched related donors improves the outcome of elderly acute myeloid leukemia (AML) patients in first complete remission (CR1) (Kurosawa et al BBMT 2011). However, the efficacy of allo-HSCT from alternative donors for the management of elderly AML patients in CR1 remains to be clarified. In this study, we examined the efficacy of allo-HCST for the management of elderly AML patients. Methods We retrospectively collected the data of consecutive AML patients who were aged 50-70 years and diagnosed in our hospital between January 1, 2000 and May 31, 2013. Patients with acute promyelocytic leukemia (APL) and patients who could not receive intensive therapy or died within 60 days after diagnosis were excluded. The primary endpoint of this study was overall survival (OS), which was defined as the interval between diagnosis and the last follow up. The unadjusted probabilities of OS were estimated using the Kaplan-Meier method. To compare OS between the treatment groups, the log-rank test was used. Multivariate analysis were performed with Cox proportional hazard regression models and 95% confidence intervals (CIs) were calculated. Results A total of 131 elderly AML (non-APL) patients were identified. Except for 14 patents, all received chemotherapy. 12 patients who died within 60 days were excluded from the study. Among the remaining 105 patients, 41 could not be induced into complete remission (CR) and showed a dismal outcome; the probabilities of 2 years (2y)-OS were 0% and 4.4% for 5 patients who received allo-HSCT and 36 patients who did not, respectively. In contrast, the probability of 2y-OS of 64 patients who achieved CR1 was 48.5%. Of these 64 patients, 20 proceeded to allo-HSCT. 13 patients were transplanted in CR1, 3 in second CR (CR2), and 4 in relapse. The probabilities of 2y-OS were 56.1% and 45.0% for patients who received and who did not receive allo-HSCT, respectively (p=0.27). When the disease status at allo-HSCT was considered, the 2y-OS of patients who received allo-HSCT in CR1 reached 83.3%, whereas that of patients transplanted in CR2 or relapse reached only 14.3%. Next, we performed univariate and multivariate analysis of OS of patients who achieved CR1. In addition to whether allo-HSCT was performed in CR1 or not, the following factors were also considered as covariates: age and leukocyte counts at diagnosis, cytogenetic classification according to National Comprehensive Cancer Network guidelines, presence or absence of preceding hematological diseases, and the number of induction regimens required for achieving CR1. Univariate analysis showed that allo-HSCT in CR1 was associated with improved OS (2y-OS: 83.3% vs 40.6%, p=0.013) (figure1). Multivariate analysis also showed that allo-HSCT in CR1 was the only favorable prognostic factor for OS (Hazard ratio=0.25, 95%CI: 0.077-0.82, p=0.022). Stem cell sources of allo-HSCT done in CR1 were 1 HLA-matched related donor and 12 unrelated donors including umbilical cord blood from 1 donor. Conclusion Allo-HSCT, even from an unrelated donor, improves the outcome of elderly AML patients in CR1. However, allo-HSCT for those with relapsed or refractory disease could hardly extend their survival. Disclosures: No relevant conflicts of interest to declare.

Introduction Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) has an indolent clinical course, with overall patient survival at 5 years after diagnosis of more than 85%. Although the cause of death in a significant proportion of MALT lymphoma patients is not the lymphoma itself, some patients experience early relapse or refractory disease. However, the risk factors capable of predicting aggressive disease in MALT lymphoma have not been clearly defined. Patients and Methods This is a retrospective pooled analysis of pathologically confirmed extranodal MALT lymphoma patients treated at our institute. Patients with nodal or splenic marginal zone lymphoma were excluded. The primary endpoint was progression-free survival (PFS), which was assessed using the Kaplan-Meier method. The log-rank test and multivariate Cox regression analysis were used to assess the prognostic value of each clinical variable. Results From January 2000 to June 2013, 52 extranodal MALT lymphoma patients (26 females and 26 males) were referred to our institution. The median age of the patients was 68 years (range, 43–89 years). Thirty-three (63%) patients had limited stage disease (stage I/II) and 48 (92%) patients had an ECOG performance status of less than 2. The cumulative numbers of patients who were diagnosed with orbit, thyroid, salivary gland, stomach, lung, and intestine disease were 7 (13%), 5 (10%), 9 (17%), 16 (31%), 16 (31%), and 4 (8%), respectively. Fifteen (29%) patients had a prior history of autoimmune disease, and a serum electrophoresis study detected M-protein in 9 (17%) patients (5 patients had IgG, 3 patients had IgM, and 1 patient had IgA). There was no significant correlation between the presence of M-protein and prior history of autoimmune disease (Fisher's exact test, p=0.46). Of the 32 patients who underwent cytogenetic studies, 9 (28%) had cytogenetic aberrations involving MALT1. Histological transformation to diffuse large B-cell lymphoma (DLBCL) was confirmed in 3 patients. After a median follow-up time of 53 months (range, 1–142 months), 16 patients had relapsed (MALT lymphoma relapse or transformation to DLBCL), and 1 patient had died due to lymphoma. The 4-year PFS and overall survival rate were 76.3% (95% confidence interval (CI), 60.0–86.7%) and 98.0% (95% CI, 86.6–99.7%), respectively. Among the clinical variables measured at diagnosis, univariate analysis found that advanced stage, three or more extranodal sites, serum soluble IL-2 receptor concentration higher than 700 IU/L, lymphocyte count higher than 1x10^9/l, hemoglobin concentration lower than 12.5 g/dl, and the presence of M-protein adversely affected PFS (p Conclusion Extranodal MALT lymphoma patients with M-protein had an increased risk of early disease progression. Patients with M-protein were more likely to had advanced stage disease (p=0.005) and more than three extranodal sites (p=0.02) compared to patients without M-protein. However, multivariate analysis showed that the prognostic impact of M-protein was better than that of advanced stage disease and the number of extranodal sites. Further studies are required to determine differences between the biological backgrounds of patients with and without M-protein, and a novel treatment strategy to obtain a durable disease control. Disclosures: No relevant conflicts of interest to declare.

Background About 70-80% of adult patients (pts) with acute myeloid leukemia (AML) achieve complete remission (CR); however, around a half of them experience a relapse. For the purpose of creating accurate decision-making process of post-consolidation strategy, stratification system using karyotype and recurrent gene mutations have been widely utilized. As is confirmed in childhood acute lymphocytic leukemia, however, the information of minimal residual disease (MRD) status would substantially improve the reliance of decision-making process of adult AML pts. Unfortunately, approximately a half of AML patients lack molecular targets suitable for MRD monitoring. The aims of this study are to evaluate the applicability of MRD detection using multiparametric flow cytometry (MPFC) and to estimate the impact of MRD measured with MPFC at the end of consolidation therapy in improving decision-making process. Patients and Methods We retrospectively studied 81 consecutive pts with newly diagnosed AML who received induction therapies and achieved CR in our institute between January 2007 and March 2013. Pts with acute promyelocytic leukemia were excluded. We routinely analyzed the bone marrow specimens with MPFC for the detection of leukemia-associated immunophenotypes (LAIPs) at diagnosis. Since April 2010, RT-PCR assay examined FLT3-ITD mutation in the same specimens. In pts who had traceable LAIPs, the relationships of the levels of MRD at the end of consolidation therapy with relapse free survival were analyzed. Positive MRD was defined as the detection of 0.2% and more LAIPs-positive cells with MPFC. We compared two patient groups: those with MRD at the end of consolidation (MRDp group) and those without (MRDn group). Relapse-free survival (RFS) was analyzed using the Kaplan-Meier method and the log-rank test was used for comparison between each group. A multivariate Cox regression analysis for RFS was fit to assess the effect of the followings: age at diagnosis (≥ vs. < 65 years old), the number of induction regimens required for achieving CR (≥ vs. < 2 times), cytogenetic risk groups of SWOG (unfavorable vs. favorable/intermediate). Results In 57 / 81 pts, MPFC could detect LAIPs in the bone marrow specimens at diagnosis (70.4% of all subjects; 15-82 years-old; follow-up time [median] 98-2211[517] days). FLT-ITD mutations were found in 13 pts, but not in 39 pts (the remaining 5 pts were not examined). The rate of detection of LAIPs with 6-color MPFC was significantly superior to 3-color MPFC (82.1% vs. 61.0%, p Conclusion Our retrospective study confirmed that LAIPs as MRD targets were applicable to the majority of pts with AML; MRD positivity measured with LAIPs was a promising predictor for early relapses at the end of consolidation, as was previously reported. When combined with FLT-ITD status, it might become a more sensitive prognostic factor. Disclosures: Takahashi: celgene: Research Funding.

Objectives Prospective clinical trials show that 5-Azicitidine (5-Aza) is superior to conventional care regimens in prolonging the survival of patients with high-risk myelodysplastic syndromes (MDS) (pts). To confirm the survival benefit of 5-Aza in daily clinical practice, we compared the survival of high-risk MDS patients who received parental treatment before 5-Aza approval to those who could be initially treated with 5-Aza. Methods In Japan, 5-Aza was approved in January 2011. We collected data of consecutive adult patients (pts) with high-risk MDS, namely refractory anemia with excess blasts-1 (RAEB-1), RAEB-2, and acute myeloid leukemia with MDS related features (AML/MRF), who received the first parental chemotherapy between January 2000 and April 2013. Survival was not calculated from the day on which the first chemotherapy was delivered, but from the day on which the diagnosis of high-risk MDS was established. Survival analysis was done by the Kaplan-Meier method. The Cox regression model was used for the analyses. A total of 107 pts with high-risk MDS were identified. They were divided into two groups (grps) on the basis of whether or not they could be initially treated with 5-Aza. In brief, pts who received the first parental treatment before January 2011 were categorized into the conventional care (CC) grp (N=75), and pts for whom treatment began after January 2011 were categorized in the 5-Aza grp (N=32). The following variables were also considered to affect survival in multivariate analysis: sex, age (>=65 years (yrs) old or Results Median follow-up times of CC and 5-Aza grp were 43 and 11 months, respectively. As shown in Table 1, the distributions of sex, age, WHO classification and IPSS were nearly the same in both grps. As for initial treatment, pts in the CC group mainly received low-dose cytarabine (ld-AraC), whereas most pts in the 5-Aza grp were treated with 5-Aza. Unadjusted survival curves of the two grps are shown in Figure 1. The unadjusted 2-yr survival rate was 33% in the CC grp and 52% in 5-Aza era grp. Median survival times were 11 and 24 months in CC and 5-Aza grps, respectively. Uni-variate analysis of overall survival revealed a trend for improved survival in younger pts, but this was contradicted by the results of multi-variate analysis. In uni- and multi-variate analyses, the 5-Aza group showed an overall, and statistically significant higher survival benefit (Table 2). Removing the 26 patients allografted after parental treatment (10 in 5-Aza grp and 16 in CC grp) at the time of alloSCT from the analyses did not modify the survival advantage of the 5-Aza grp. Conclusion This retrospective analysis confirms that the introduction of 5-Aza has certainly improved the outcome of high-risk MDS in daily clinical practice. Table 1 . Patients characteristics at diagnosis of high-risk MDS Patients characteristics >=2011 (N=32) Total (N=107) Sex(male/female) 57(76)/18(24) 28(88)/4(12) 85(79)/22(21) Age (median) >=65 68 29(39) 46(61) 68.5 14(44) 18(56) 68 43(40) 64(60) Initial treatment low-dose AraC 5-Aza intensive chemotherapy 63(84) 0(0) 12(16) 3(9) 26(81) 3(9) 66(62) 26(24) 15 (14) WHO Classification RAEB1, RAEB2, AML/MRF Missing/Unevaluable 19(25) 31(42) 15(20) 10(13) 7(22) 13(41) 8(25) 4(13) 26(24) 44(41) 23(21) 14(13) IPSS low Int-1 Int-2 high Missing/Unevaluable 1(1) 15(20) 21(28) 24(32) 14(19) 0(0) 7(22) 8(25) 12(38) 5(16) 1(1) 22(21) 29(27) 36(34) 19(18) Download : Download high-res image (84KB) Download : Download full-size image Figure 1 . Unadjusted survival curves of the two grps Table 2 . Uni- and multi-variate analysis of overall survival Variables Univariate Multivariate RR (95%CI) P RR (95%CI) P Age (vs. >=65y 1.5(0.98-2.41) 0.061 1.5(0.94-2.5) 0.84 Sex male 0.92(0.53-1.60) 0.76 0.73(0.39-1.4) 0.33 Disease status (vs.RAEB1) RAEB2 AML/MRFMissing/Unevaluable 0.73(0.42-1.3) 0.78(0.41-1.5) 0.69(0.34-1.4) 0.27 0.450.32 0.72(0.37-1.4) 1.3(0.51-3.4) 0.76(0.25-2.3) 0.33 0.580.62 Treatment Era (vs. Conventional Care Era) 5-Aza 0.54(0.32-1.0) 0.027 0.39(0.18-0.83) 0.043 IPSS (vs. Low) Intermediate-1 Intermediate-2 High Missing/Unevaluable 2.6(0.35-20) 2.3(0.31-17) 2.0(0.26-15) 1.9(0.24-14.) 0.35 0.420.51 0.55 5.0(0.53-48) 2.5(0.22-28) 1.3(0.099-17) 3.0(0.21-44) 0.16 0.470.84 0.42 Disclosures: No relevant conflicts of interest to declare.