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Extended post-therapy long-term survival of patients with diffuse large B cell lymphoma (DLBCL) may also lead to an increase of late adverse events. We retrospectively investigated the frequency and clinical manifestation of second primary malignancy (SPM) after rituximab-containing immunochemotherapy in patients with DLBCL treated at seven institutes belonging to the Kyoto Clinical Hematology Study Group (KOTOSG) from the perspective of the existence of past or synchronous cancer history. In a median follow-up period of 899 days, 69 SPMs were observed in 58 of 809 patients. The most frequent SPM was gastric cancer, followed by lung cancer and colorectal cancer. The cumulative incidence of SPM increased steadily over time and was not significantly influenced by the presence or absence of past or synchronous cancer history. Our study suggests the need for careful attention to SPM in patients with DLBCL in daily practice.

An increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) is associated with disease progression and treatment resistance in multiple myeloma (MM). We investigated the mechanisms underlying MDSC induction, and sought to discover a strategy for prevention of MDSC induction in MM. Using a transwell co-culture system, four of nine examined human myeloma-derived cell lines (HMCLs) were potent in inducing monocytic (M)-MDSCs from normal peripheral blood mononuclear cells (PBMCs). As the results, we identified that secretion of C-C motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF) by myeloma cells is a prerequisite for induction of MDSCs in MM. The immunomodulatory drug (IMiD) compounds, such as lenalidomide (LEN) and pomalidomide (POM), were identified as potent inhibitors of MDSC induction through bidirectional molecular effects of cereblon (CRBN)-dependent and -independent downregulation of CCL5 and MIF in myeloma cells; and downregulation of C-C motif chemokine receptor 5, a receptor for CCL5, and induction of interferon regulatory factor 8, a critical transcription factor for monocytic differentiation, in PBMCs. In the present study of the molecular mechanisms underlying MDSC induction, we identified a novel effect of LEN and POM of inhibiting MDSC induction via overlapping regulatory effects in myeloma cells and normal PBMCs.

Carfilzomib (CFZ) constitutes powerful combinatory therapy for relapsed/refractory multiple myeloma (RRMM); however, cardiovascular adverse events (CVAEs) have been shown as major treatment obstacles with the use of CFZ. Along with our multi-institutional prospective observational study by the Kyoto Clinical Hematology Study Group on the efficacy and safety of CFZ-based treatments (UMIN000025108), we here performed an ad hoc analysis of CFZ-related CVAEs in 50 patients with RRMM. We analyzed the association between CFZ-related CVAEs and pre-planned examinations, including patients' background, electrocardiographic findings, echocardiographic findings, and serum/plasma levels of 18 potential candidate biomarkers. The common CVAEs were hypertension (42%), arrhythmia (14%), and prolongation of QT corrected interval (10%), whereas no serious CVAEs occurred. The pretreatment serum level of interleukin-6 was identified as a significant risk factor for CFZ-related hypertension. This study revealed hypertension as the most frequent CFZ-related CVAE and suggested that baseline serum interleukin-6 is a useful predictor for CFZ-induced hypertension.

Summary Background BCR-ABL1 tyrosine kinase inhibitors (TKIs) are commonly initiated in older patients with chronic myeloid leukaemia in the chronic phase at standard doses. However, because of their safety profile in this population, appropriate therapy has not been established. We aimed to investigate whether a lower than standard dose of dasatinib was an appropriate therapy for older patients with chronic myeloid leukaemia in the chronic phase. Methods DAsatinib, Very Low-dose, for Elderly CML-CP patients (DAVLEC) was a multicentre, single-arm, phase 2 trial done in 25 Japanese hospitals. We enrolled patients older than 70 years with newly diagnosed chronic myeloid leukaemia in the chronic phase, ECOG performance status 0–2, and no previous treatment for CML other than hydroxyurea within 4 weeks. Second-generation TKI dasatinib was given orally at a starting dose of 20% of the standard dose (20 mg/day). If the treatment was assessed as optimal response at 3 months, 6 months, and 9 months and adverse events were grade 2 or better (according to the NCI Common Toxicity Criteria v 4.0), the same dose was continued. If response was suboptimal and adverse events were grade 2 or better, the dose was increased by 20 mg/day. Once a dose reduction had been made because of a grade 3 or worse adverse event, there were no further dose increases. Treatment was discontinued if assessed as failure (disease progression to the accelerated phase or acute phase). The primary endpoint was the achievement of major molecular response at 12 months, assessed using a per-protocol analysis. This trial is registered at with the UMIN clinical trial registry, UMIN000024548, and has completed its planned observation period. Findings Between Nov 1, 2016, and Oct 30, 2019, 52 patients received first-line dasatinib therapy at 20 mg/day. The median age at diagnosis was 77·5 years (73·5–83·0). 35 (67%) patients were male and 17 (33%) were female. 31 (60%) of 52 patients reached major molecular response at 12 months (one-sided 95% CI 48–71), with a median follow-up of 366 days (IQR 353–372). Grade 3–4 adverse events were reported in 12 (23%) patients. Neutropenia was the most frequent grade 3–4 adverse event, occurring in three (6%) patients. No treatment-related deaths were observed. Interpretation Low-dose dasatinib at 20mg/day is worthy of consideration as a starting dose for older patients with newly diagnosed chronic myeloid leukaemia in the chronic phase. However, this dose needs to be further studied in a larger cohort and with a more ethnically diverse population. Funding Bristol-Myers Squibb.

This multicenter phase II study (UMIN000008145) aims to investigate the efficacy and safety of six cycles of combination therapy (RBD) comprising rituximab, bendamustine, and dexamethasone (DEX) for relapsed or refractory (RR) indolent B-cell non-Hodgkin lymphoma (B-NHL) and mantle cell lymphoma (MCL). Although the initial study protocol comprised 20 mg/body DEX on days 1 and 2, and 10 mg/body on days 3-5 [high-dose (HD-) DEX group], the dose of DEX was later decreased to 8 mg/body on days 1 and 2 [low-dose (LD-) DEX group] due to frequent cytomegalovirus (CMV) antigenemia and recurrent retinitis. We enrolled 33 patients, and LD-DEX and HD-DEX were administered in 15 and 18 patients, respectively. The overall response and the 3-year progression-free survival rates were 88% and 75.5%, respectively. The leading adverse event was myelosuppression. Incidence of grade 3-4 leukocytopenia, neutropenia, and lymphocytopenia was 55%, 67%, and 91%, respectively. The most frequent nonhematological adverse events were CMV antigenemia and rash (33% and 30%, respectively). Incidence of CMV antigenemia over 10/100,000 white blood cells was significantly lower with LD-DEX than that with HD-DEX (P = 0.0127). In conclusion, RBD showed significant effectiveness for RR indolent B-NHL and MCL.

Background Combinatory strategies with carfilzomib (CFZ), a second‐generation proteasome inhibitor, plus dexamethasone (DEX) with or without lenalidomide (LEN) have shown promising efficacy for patients with relapsed/refractory multiple myeloma (RRMM) in pivotal clinical trials. However, their effects on patients who were resistance to bortezomib (BTZ) and/or LEN have not been fully evaluated in a daily practice setting. Aims To evaluate the real‐world efficacy and safety of CFZ‐based treatments; that is, CFZ with LEN plus DEX (KRD therapy) and CFZ with DEX (KD therapy), in Asian patients, we conducted a multicenter pilot prospective observational study in the Kyoto Clinical Hematology Study Group. Methods and Results All 50 patients with RRMM enrolled in this study were treated with CFZ‐based treatments between 2017 and 2019. KRD and KD were administered to 31 and 19 patients, respectively. The overall response rates (ORRs) were 80.6% with KRD and 73.7% with KD. Two‐year progression‐free survival (PFS) and overall survival (OS) were 58.5% and 79.7% with KRD, and 23.1% and 52.6% with KD. By multivariate analysis, refractoriness to BTZ and to LEN were identified as independent unfavorable factors for both PFS and OS. The common non‐hematologic AEs included hypertension (42.0%), fever (24.0%), fatigue (24.0%), and infection (16.0%). No serious heart failure was observed. This study is registered as UMIN000025108. Conclusion This study suggests the need of the development of novel CFZ‐containing strategy which can overcome the refractoriness to BTZ and/or LEN, while both KRD and KD were shown to be mostly feasible in Asian patients in a daily practice setting.

Multiple myeloma (MM) is cytogenetically, genetically and molecularly heterogenous even among subclones in one patient, therefore, it is essential to identify both frequent and patient-specific drivers of molecular abnormality. Following previous molecular investigations, we in this study investigated the expression patterns and function of the Ewing sarcoma breakpoint region 1 (EWSR1) gene in MM. The EWSR1 transcriptional level in CD138-positive myeloma cells was higher in 36.4% of monoclonal gammopathy of undetermined significance, in 67.4% of MM patients compared with normal plasma cells, and significantly higher in ten human myeloma-derived cell lines (HMCLs) examined. EWSR1 gene knockdown caused growth inhibition with an increase of apoptotic cells in NCI-H929 and KMS-12-BM cells. Gene expression profiling using microarray analysis suggested EWSR1 gene knockdown caused transcriptional modulation of several genes associated with processes such as cell proliferation, cell motility, cell metabolism, and gene expression. Of particular, EWSR1 gene knockdown caused upregulation of let-7c and downregulation of its known targets K-RAS and AKT. Finally, our analysis using community database suggested that high EWSR1 expression positively associates with poor prognosis and advanced disease stage in MM. These findings suggest that EWSR1 overexpression is a pro-oncogenic molecular abnormality that may participate in MM progression.

Introduction: Tyrosine kinase inhibitors (TKIs) dramatically improved the prognosis of chronic myelogenous leukemia (CML), as nearly half of CML patients presenting with a deep molecular response (DMR) over several years could achieve treatment-free remission. DASISION study reported dasatinib (DAS), a second-generation TKI was superior to imatinib to achieve faster and deeper response in treating newly-diagnosed chronic CML. However, standard initial dosage of 100 mg DAS/day might be too potent for patients aged >70 years with CML, possibly owing to the presence of other health conditions that threaten the life spans (e.g., lung and cardiovascular disease), thereby preventing TKI continuation. In this open-label, multicenter, single-arm, phase 2 study (DAVLEC), we evaluated the efficacy and safety of a reduced initial dose of DAS (20 mg/day) in the elderly with chronic CML. Patients and Methods: We included CML patients aged >70 years with an ECOG performance status of 0-2 and adequate organ function. After diagnosis, they began orally taking 20 mg of DAS per day. BCR-ABL mRNA values were measured and aligned to an international scale at a central laboratory to evaluate the drug's efficacy at 3, 6, 9, and 12 mo of therapy, per recommendations by European LeukemiaNet. The dosage was maintained in case of the optimal, increased by 20 mg/day when the warning appeared and could be decreased when an adverse event (AE) > grade 3 appeared. Patients were excluded if the respond was the failure or disease progression was detected. Primary endpoint was the cumulative major molecular response (MMR) rate at 12 mo. Secondary endpoints included the rates of the cumulative DMR, treatment discontinuation due to AEs, and failure to DAS treatment or disease progression. The non-inferiority of primary endpoint MMR (non-inferiority limit of 38%) was evaluated by binomial test with normal approximation. Other differences were investigated by Mann-Whitney tests, with p < 0.05 indicating significance. Results: Among the 56 patients enrolled from 25 centers, 4 declined participation, and so 52 (median age, 77.5 years) began taking 20 mg/day of DAS. In total, 73.1% of the patients had comorbidities. Median BCR-ABL values at 3, 6, and 12 mo were 1.47%, 0.23%, and 0.03%, respectively; cumulative MMR rate at 3, 6, 9, and 12 mo were 11.5%, 36.5%, 40.4%, and 59.6%, respectively. Calculated MMR at 12 mo was higher than 38%, a value selected based upon findings from the DASISION trial, accounting for a non-inferiority margin of 10% (p=0.047). At 12 mo, among the 31 patients achieving MMR, 23 were only given 20 mg DAS/day, and 14 and 7 respectively achieved MR4.0 and 4.5. Patients discontinued the study due to treatment failure (n=3), content withdrawal (n=2), drug-related AEs (Long QT Syndrome; n=1), or others (n=2). No patient discontinued it due to disease progression. Treatment-related AEs of all grades were noted in 96.2%and that of grade 3 or 4 events in 23.1% of the patients. Median dose interruptions for a median of 7 days (range: 5-36) were respectively noted in 3 and 2 patients due to hematological and non-hematological AEs. Furthermore, 4 patients experienced pleural effusion < grade 2, and 1 had lymphocytosis. Patients who achieved MMR at 12 mo (n=31) had a significantly lower BCR-ABL value at 3 mo (0.41% vs. 4.26%, p=0.0020) and a lower halving time from diagnosis to 3 mo (11.35 vs. 18.34 days, p=0.0050) than those who did not (n=21). Patients who achieved MMR by 12 mo with only 20 mg/day of DAS (n=23) had lower BCR-ABL values at 1 and 3 mo (25.51 vs. 63.63 %, p=0.00028; 0.33 vs. 5.89%, p Patients who achieved MMR at 12 mo (n=31) had higher plasma DAS concentration (23.5 vs. 8.0 ng/mL, p Conclusion: This study discovered that prescribing a starting very-low dose DAS (20 mg/day) while monitoring IS and AEs was a successful initial therapeutic strategy for the elderly with chronic CML comparable to previous studies using standard-dose. Notably, rapid BCR-ABL downregulation, early molecular responses at 1 and 3 mo, a shortened halving time from diagnosis to 3 mo, and sufficient increase in plasma DAS concentrations 2 h post-intake after 12 mo of treatment were key indicators for successful low-dose therapy. Disclosures Kumagai: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria; Otsuka Pharmaceuticals: Honoraria, Speakers Bureau. Murai: Bristol Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; CHUGAI Pharmaceutical Co., Ltd.: Honoraria; TAKEDA Pharmaceutical Co., Ltd.: Honoraria; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Asahi Kasei Pharma Corporation.: Honoraria. Tanaka: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Otsuka Pharmaceuticals: Honoraria. Nishiwaki: Kyowa-Kirin: Research Funding; Alexion: Honoraria. Inokuchi: Bristol-Myers Squibb: Research Funding. Yoshida: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis KK,: Honoraria; Pfizer Japan Inc.: Honoraria; Otsuka Pharmaceutical.: Honoraria; AbbVie GK: Honoraria; Janssen Pharmaceutical KK: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Uoshima: Janssen: Honoraria; Eisai: Honoraria. Usuki: MSD K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Amgen-Astellas Biopharma K.K.: Research Funding; Mundipharma K.K.: Research Funding; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Celgene K.K.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; Incyte Biosciences Japan G.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding. Kuroda: Otsuka Pharmaceutical: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Abbvie: Consultancy, Honoraria; Ono Pharmaceutical: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sysmex: Research Funding; Pfizer: Honoraria, Research Funding; Fujimoto Pharmaceutical: Current Employment, Honoraria, Research Funding; Taiho Pharmaceutical: Research Funding; Asahi Kasei: Research Funding; Shionogi: Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Janssen Pharmaceutical K.K: Consultancy; Bristol-MyersSquibb: Consultancy, Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Dainippon Sumitomo Pharma: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding. Ono: Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; Mundipharma K.K.: Honoraria; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Merck Sharp & Dohme: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb Company: Honoraria; Celgene: Honoraria, Research Funding. Fujimaki: CSL Behring K.K.: Honoraria; Mundipharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Meiji Seika Pharma Co., Ltd.: Honoraria; Janssen Pharmaceutical KK: Honoraria; AbbVie GK: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis KK: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Pfizer Japan Inc.: Honoraria; Otsuka Pharmaceutical.: Honoraria. Shibayama: Chugai: Research Funding; Eizai: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Kyowa Kirin: Speakers Bureau; AbbVie: Research Funding; Celgene: Research Funding; Novartis: Research Funding, Speakers Bureau; Ono: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Mundi Pharma: Speakers Bureau; Otsuka: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; AstraZeneca: Research Funding, Speakers Bureau; Takeda: Speakers Bureau. Kondo: Abbvie: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Otsuka Pharmaceutical: Consultancy, Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy; Pfizer: Honoraria. Minami: Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria; Astellas: Honoraria; Ono: Research Funding; CMIC: Research Funding. Kimura: Eisai: Speakers Bureau; Kyowa-Kirin: Research Funding, Speakers Bureau; PharmaEssentia: Speakers Bureau; Astellas: Speakers Bureau; Sanifi: Speakers Bureau; Gilead: Research Funding; Nippon-Boehringer-Ingelheim: Research Funding; Celgene: Research Funding, Speakers Bureau; Sumitomo-Dainippon: Research Funding; Chugai: Research Funding, Speakers Bureau; Mundi: Research Funding; Yakult: Research Funding, Speakers Bureau; MSD: Research Funding, Speakers Bureau; Ono: Research Funding, Speakers Bureau; SymBio: Research Funding, Speakers Bureau; Daiichi-Sankyo: Research Funding, Speakers Bureau; Janssen: Research Funding; Astellas-Amgen-Biopharma: Research Funding; Takeda: Research Funding, Speakers Bureau; Nippon-Shinyaku: Research Funding, Speakers Bureau; Amgen: Research Funding; Alexion: Research Funding, Speakers Bureau; Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Ohara Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; AbbVie: Research Funding, Speakers Bureau; Apellis: Research Funding.

Cytogenetic/molecular heterogeneity is the hallmark of multiple myeloma (MM). However, we recently showed that the serine/threonine kinase PDPK1 and its substrate RPS6KA3 (also termed RSK2) are universally active in MM, and play pivotal roles in myeloma pathophysiology. In this study, we assessed involvement of aberrant miR-375 repression in PDPK1 overexpression in MM. An analysis of plasma cells from 30 pre-malignant monoclonal gammopathies of undetermined significance and 73 MM patients showed a significant decrease in miR-375 expression in patient-derived plasma cells regardless of the clinical stage, compared to normal plasma cells. Introduction of miR-375 reduced PDPK1 expression in human myeloma cell lines (HMCLs), indicating that miR-375 is the dominant regulator of PDPK1 expression. In addition, miR-375 introduction also downregulated IGF1R and JAK2 in HMCLs. CpG islands in the MIR375 promoter were pathologically hypermethylated in all 8 HMCLs examined and in most of 58 patient-derived myeloma cells. Treatment with SGI-110, a hypomethylating agent, and/or trichostatin A, a histone deacetylase inhibitor, increased miR-375 expression, but repressed PDPK1, IGF1R and JAK2 in HMCLs. Collectively, these results show the universal involvement of overlapping epigenetic dysregulation for abnormal miR-375 repression in MM, which is likely to contribute to myelomagenesis and to subsequent myeloma progression by activating oncogenic signalling pathways.

Imatinib mesylate (IM) is a first generation ATP-competitive selective inhibitor for tyrosine kinases (TK) such as ABL, ARG, PDGFRα and b, and c-KIT.[1][1] While use of IM has markedly improved treatment outcomes of Philadelphia chromosome (Ph)-positive leukemias, including chronic myeloid leukemia

Expression of intragenic exon rearrangements (IERs) has reportedly been detected in both normal and cancer cells. However, there have been few reports of occurrence of these rearrangements specific to neoplasms including malignant lymphoma. In this study, we detected IERs of ten genes (NBPF8, SOBP, AUTS2, RAB21, SPATA13, ABCC4, WDR7, PHLPP1, NFATC1 and MAGED1) in non-Hodgkin B cell lymphoma (B-NHL) cell line KPUM-UH1 using a high-resolution single nucleotide polymorphism array and reverse transcription polymerase chain reaction using reversely directed divergent primers within exons involved in genomic intragenic gains followed by sequencing analysis. Among them, the IERs involved in SOBP (6q21) exon 2 and 3 and AUTS2 (7q11.22) exon 2–4 were the molecular lesions specific to tumors and were frequently detected in B-NHL samples. These IERs constitute novel genetic alterations of B-NHL, which might be associated with tumorigenesis and be useful as genetic biological markers.

Disease-specific cytogenetic abnormalities involving BCL2 gene rearrangement frequently co-exist with other cytogenetic abnormalities, contributing to disease progression in follicular lymphoma (FL). In the present study, we retrospectively investigated the prognostic impact of BCL2-unrelated cytogenetic abnormalities in FL. Of 139 consecutively diagnosed patients with FL at two independent institutes, metaphase spreads of tumor cells were obtained for use in G-banding analysis in 77 patients. The recurrent additional cytogenetic abnormalities included chromosome gains +5 (n = 8), +7 (n = 16), +12 (n = 10), and +X (n = 12), and losses -8 (n = 7), -13 (n = 12) -15 (n = 7), and 6q- (n = 7). While -15 was associated with shorter progression-free survival (PFS) in all 77 analyzed patients with evaluable G-banding results (p = 0.04), this negative impact was not evident in 42 patients treated using an R-CHOP-like regimen as first-line treatment. By contrast, 6q- was predictive for shorter PFS in patients who were initially treated with R-CHOP-like regimens without maintenance therapy (p

The combinations of melphalan, bortezomib, and prednisolone (VMP) and of lenalidomide and dexamethasone (Rd) are standard treatment strategies for transplant-ineligible newly diagnosed multiple myeloma (NDMM). To make the most of these two strategies, we investigated the efficacy and feasibility of first-line treatment with 4 cycles of VMP followed by continuous Rd therapy in a multi-institutional phase 2 study in Japanese patients with transplant-ineligible NDMM. Thirty-six patients of median age 74 years old with NDMM initially received 35-day cycles of VMP: oral melphalan (6 mg/m2) and prednisolone (60 mg/m2) on days 1 to 4 and bortezomib (1.3 mg/m2) on days 1, 8, 15, and 22. After 4 cycles of VMP, treatment was switched to 28-day cycles of Rd, which was continued until disease progression or emergence of an unacceptable adverse event (AE) in 33 patients, while one patient who achieved CR after VMP continued VMP at the physician's discretion. The overall response rates after VMP and after Rd were 66.7% and 86.1%, including CR rates of 5.6% and 36.1%, respectively. In a median follow-up period of 34.3 months, the progression-free survival and overall survival rates at 3 years were 43.2% and 81.3%, respectively. Grade 3-4 hematological AEs included neutropenia (39% with VMP and 24% with Rd) and thrombocytopenia (11% with VMP and 3% with Rd). There was no death due to an AE. In conclusion, sequential therapy with VMP followed by Rd is effective and mostly feasible for transplant-ineligible NDMM. The study is registered as UMIN000034815.

Cyclin D1 (CCND1) overexpression is an early and unifying oncogenic event in mantle cell lymphoma (MCL) and multiple myeloma (MM) with chromosome 11q13 abnormalities. Herein, we report newly discovered transcript variants of the CCND1 gene in MCL and MM cells with chromosome 11q13 abnormalities. These transcript variants, designated CCND1.tv., covered the full-length coding region of CCND1 with longer 5'-untranslated regions (5'-UTRs) of CCND1 and occasionally contained a novel exon. CCND1.tv. was specifically detectable in patient-derived primary MCL or MM cells with chromosomal translocation t(11;14)(q13;q32), but not in t(11;14)-negative cells. The lengths of the 5'-UTR sequences of CCND1.tv. differed among patients and cell lines. Introduction of CCND1.tv. led to increased expression of normal-sized CCND1 protein in HEK293 cells. Furthermore, mTOR inhibition by rapamycin or serum starvation reduced ectopic expression of CCND1.tv.-derived CCND1 protein, but not 5'-UTR less CCND1-derived CCND1 protein in HEK293 cells, suggesting that the protein expression of CCND1.tv. is regulated by the mTOR pathway. Our results suggest that the aberrant expression of CCND1.tv. may contribute to the understanding of the pathogenesis of MCL and MM with 11q13 abnormalities.

Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.

We retrospectively analyzed efficacy and safety of therapy with rabbit antithymocyte globulin (rATG) in combination with cyclosporine A (CsA) in 30 Japanese adult patients with acquired aplastic anemia (AA) in the Kyoto Clinical Hematology Study Group. The median observation period was 31 months and the median age of the patients was 54 years. The objective response rates (ORRs) to rATG plus CsA increased over time until 18 months after the start of treatment; the rate of achievement of better than partial response at 18 months was 66.7%. The 2-year overall survival (OS) rate was 79% in all patients. In eight patients aged ≥ 75 years old, the ORR was 62.5% and the 2-year OS rate of 50% was not significantly inferior to that in patients aged ≤ 74 years old. The overall mortality rate was 16.7% in our cohort, while the mortality rate in patients aged ≥ 75 years old was 37.5%, which was higher than that in patients aged ≤ 74 years old (9.1%), although the difference was not statistically significant. Collectively, rATG combined with CsA is an effective and feasible treatment for AA, while patients should be appropriately selected.

Mantle cell lymphoma (MCL) is a relatively rare subtype of B-cell non-Hodgkin lymphoma (NHL) that has a poor prognosis despite recent advances in immunochemotherapy and molecular targeted therapeutics against NHL. Therefore, the development of a new therapeutic strategy for MCL is urgently needed. In this study, we show for the first time that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), an oncogenic serine–threonine protein kinase, is commonly expressed in its phosphorylated active form in patient-derived tumor cells of various types of B-cell NHL cells, including diffuse large B-cell lymphoma, follicular lymphoma, and MCL. Blockade of PDPK1 activity by small-molecule inhibitors specific for PDPK1 (BX-912 and GSK2334470) or by RNA interference exerted antiproliferative effects in all four MCL-derived cell lines examined and these growth-inhibitory effects were mediated by both induction of apoptosis and G 2 /M cell cycle blockade. In addition, blockade of PDPK1 led to inactivation of its downstream effector kinase RSK2, but not AKT, suggesting the importance of the PDPK1/RSK2 signaling pathway in the proliferation and survival of MCL cells. Finally, when combined with anticancer agents, including genotoxic agents, a proteasome inhibitor, and a BH3 mimetic in vitro, the PDPK1 inhibitor BX-912 showed additive growth-inhibitory effects against MCL-derived cell lines in most settings. In particular, the combination of BX-912 and ABT-263, a BH3 mimetic, resulted in the enhancement of the induction of apoptosis. In conclusion, our results suggest that PDPK1 is a potential novel therapeutic target in MCL and indicate that clinical development of PDPK1-targeted therapy for MCL is desirable.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of non-Hodgkin lymphoma characterized by aggressive symptoms and various abnormal laboratory test results. One of the rare immunologic abnormalities in AITL is exuberant polyclonal plasmacytosis, but its clinical significance has not been evaluated. This report concerns three AITL cases with exuberant polyclonal plasmacytosis and investigates its clinical impact by comparison with 12 patients without plasmacytosis. Our study found that the performance status (PS) of the former was significantly worse and their serum immunoglobulin levels were significantly higher. All other parameters, including B symptoms, various prognostic scores, blood cell counts other than plasmacyte, and serum levels of lactate dehydrogenase, C-reactive protein and soluble interleukin-2 receptor, showed no significant differences. More importantly, although the diagnosis of AITL with plasmacytosis was not straightforward in our series, outcomes of treatment with conventional chemotherapy or immunosuppressive therapy with cyclosporine A were favorable. To conclude, AITL should be considered a candidate underlying disease of exuberant polyclonal plasmacytosis. Provided a correct diagnosis is made early and is followed by adequate treatment, the prognosis for AITL with plasmacytosis may not be worse than that for those without plasmacytosis despite the severe exhaustion at first presentation.

Among various cytogenetic abnormalities involved in lymphomagenesis and disease progression of highly aggressive and treatment-resistant B-cell lymphomas (HABCLs), classified as the so-called aggre...

Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by the coemergence of microangiopathic hemolytic anemia, thrombocytopenia, and thrombosis-mediated ischemic injuries of various organs, such as the central nervous system and kidneys. Acute myocardial infarction (AMI) has also occasionally been reported as a complication with TTP as the secondary thrombotic event; however, its emergence as the initial thrombotic event in TTP is extremely rare. This report describes an 80-year-old male patient with acquired TTP, who was affected by AMI without any clinically apparent damage to other organs or abnormal laboratory findings that would be suggestive of TTP at the first presentation. Although AMI was successfully treated by percutaneous coronary intervention (PCI), the patient developed marked thrombocytopenia with acute kidney injury and hemolytic anemia 5 days after PCI. The patient was diagnosed as having acquired TTP based on decreased ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13) below the level of detection and the presence of the inhibitor against ADAMTS13, and eventually died of multiorgan failure due to TTP despite undergoing repeated plasma exchanges and immunosuppressive therapies, including corticosteroid and rituximab. Although caution is often paid to therapy-related thrombocytopenia or renal damage after PCI, that is, those caused by antiplatelet drugs, heparin, or contrast agents, our report alerts us to the presence of TTP as an extremely rare, but underlying cause for AMI that could be subclinical at the initial presentation.

Intravascular B-cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma, which is characterized by the growth of lymphoma cells within blood vessel lumina without nodular lesions, and which predominantly affects elderly patients. IVLBCL is characterized by B-symptoms and a variety of systemic symptoms due to focal obstruction of blood flow, but may be difficult to diagnose due to its peculiar intravascular localization and the lack of nodular lesions. While hypercalcemia is one of the complications of various types of cancerous diseases, it has rarely been reported as the first presentation of IVLBCL. In this report, we present the case of a 71-year-old male with IVLBCL who showed hypercalcemia accompanied by elevation of serum parathyroid hormone-related protein (PTH-rP) as the initial presentation. Interestingly, immunohistochemical staining revealed that the intravascular lymphoma cells expressed high levels of PTHrP. Six courses of immunochemotherapy, consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), and two courses of high-dose methotrexate induced complete remission (CR) and retained CR for 4 months. We also reviewed other IVBCL cases in which hypercalcemia was the initial presentation. We suggest that IVLBCL, although rare, should be considered as a possible causative in hypercalcemia of unknown underlying disease.

Transient thrombocytopenia is common after autologous peripheral blood stem cell transplantation (auto-PBSCT). Although patients underwent auto-PBSCT usually show the recovery of peripheral leukocytes and platelets counts within a couple of weeks after auto-PBSCT, delayed platelet engraftment (DPE) is occasionally observed despite prompt neutrophil engraftment. However, there are few studies evaluating the risk factors for DPE. In this study, to investigate the association between clinical characteristics and duration required for post-auto-PBSCT platelet engraftment, we retrospectively analyzed 144 patients with B-cell non-Hodgkin lymphomas who underwent auto-PBSCT between January 2000 and December 2017 at 5 independent institutes involved in the Kyoto Clinical Hematology Study Group. Platelet recovery was defined as a recovery of platelet count greater than 50.0 × 109/L without transfusion support for 3 consecutive days, and the first day of 3 consecutive days was regarded as the day of platelet engraftment. The median observation period was 930 days (range, 25 - 5272 days). Median age at auto-PBSCT was 61, and 144 patients included 109 diffuse large B cell lymphoma, 11 follicular lymphoma, 22 mantle cell lymphoma. Ninety one patients were in complete remission at the time of auto-PBSCT. The mobilization regimens for stem cell collection were chemotherapy such as high-dose VP16 (46.5%), CHASE (cyclophosphamide, AraC, VP16, and dexamethasone) (40.3%), and high-dose AraC (8.3%) followed by G-CSF. All 144 patients except one achieved neutrophil engraftment with the median engraftment day of 10 days (range, 4-41 days), and 139 patients achieved platelet engraftment with the median engraftment day of 19 days (range, 10-1125 days). Multivariate analysis showed that infused CD34-positive cell number (≥ 2.0 × 106/kg vs. < 2.0 ×106/kg: HR 3.06: P < 0.001), pre-apheresis absolute lymphocyte count (PA-ALC), which was the mean number of lymphocytes during aphaeresis procedure (≥ 1.0 × 109/L vs. < 1.0 × 109/L: HR 2.62: P < 0.001), and the days required for apheresis procedure (≥ 3 days vs. 1 or 2 days: HR 0.49: P = 0.02) were independently associated with the duration for platelet engraftment after auto-PBSCT. Age, gender, diagnosis, Ann Arbor stages, previous history of bone marrow infiltration, performance status, international prognostic index (IPI)-defined disease risk, disease status at the time of transplant, the number of chemotherapy regimens, and mobilization regimen were not associated with platelet engraftment period. On the basis of these results, we next tried to create a new prediction system for post-transplant platelet recovery by incorporating the three factors, i.e., infused CD34-positive cell number, PA-ALC, and days required for apheresis procedure. Risk groups were determined by the sum of predictive factors for platelet recovery: favorable (3 factors), intermediate (2 factors), unfavorable (1 factor), and very unfavorable (no factor). As the results, the median duration for platelet engraftment with favorable, intermediate, unfavorable, and very unfavorable groups were 15, 19, 31, and 148 days, respectively (P < 0.001) (Figure). Furthermore, 14 of 144 patients did not achieve platelet engraftment by day 80 after auto-PBSCT. We defined those as patients with DPE. Median duration for platelet engraftment in patients with DPE was 137 days (range, 82-1125 days), while that for neutrophil engraftment was 11 days. Intriguingly, PA-ALC was less than 1.0 × 109/L in all 14 patients with DPE, while 6 required apheresis for ≥3 days, and 5 received Figure Figure. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding.

Abstract 4418 Chromosomal translocations play a crucial role in tumorigenesis, often resulting in the formation of chimeric genes or in gene deregulation through position effects. Hematologic malignancies are associated with a large number of such rearrangements. In this study, we have identified 28S ribosomal DNA (RN28S1) gene as a novel fusion partner of the B-cell leukemia/lymphoma 11B (BCL11B), immunoglobulin kappa variable 3–20(IGKV3–20), and component of oligomeric golgi complex 1 (COG1) locus in T-cell acute lymphoblastic leukemia (T-ALL), Burkitt lymphoma (BL) and multiple myeloma (MM), respectively. RN28S1-BCL11B fusion gene was identified in T-ALL with t(6;14)(q25;q32) by cDNA bubble PCR using BCL11B primers, whereas the breakpoint of 14q32 was near, but out of, the BCL11B. BLAST search revealed that the RN28S1 was fused to BCL11B within exon3 (coding region). During the expression analysis of RN28S1-BCL11B, We identified the same type of RN28S1-BCL11B fusion transcripts in a BL and 2 MM cell lines, and unexpectedly identified other RN28S1-fusion transcripts in hematologic malignancy cell lines. Fusion of RN28S1 and IGKV3–20 exon2 (coding region) was detected in 2 BL and a MM cell lines, and that of RN28S1 and COG1 within exon 13 (coding region) in a MM cell line. Furthermore, RN28S1 breakpoint was concentrated in between 1.4∼1.8kb. RN28S1 is located in the p12 region of chromosomes 13, 14, 15, 21 and 22. The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large clusters of tandemly repeated copies in the chromosome, and as the product, ribosomal RNA accounts for ∼70% of total RNA in the cell. rDNA exists in high copy number in most eukaryotic cells. Repeatitive genes tend to lose copies through homologous recombination among the repeats. The rDNA has a gene amplification system, thought to be present in most eukaryotes, that function to keep high copy numbers. In the tumor cells, rDNA is transcribed higher than normal cells for cell proliferation. Thus, that may be associated with chromosome instability. RN28S1 degradation was reported to be associated with apoptosis of hematologic malignancies, and previously, another group was detected RN28S1-BCL6 fusion genes in gastric lymphoma. This case was no expression of BCL6 protein. In conclusion, these results suggest that RN28S1 fusions are concerned with the play something role of tumorigenesis. Because of RN28S1 fused to BCL11B, IGKV3–20 and COG1 whose breakpoints were involved in the coding region, we speculate that these fusion genes are not produce normal structure. Disclosures: No relevant conflicts of interest to declare.

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

Galectin-3 is induced in chronic myelogenous leukemia (CML) cells by co-culture with bone marrow stromal cells, making paracrine growth promotion of CML cells in conditioned medium (CM) from galectin-3 overexpressing CML cells more potent. We used gel filtration chromatography to demonstrate that the bovine SERPINA1-fetal bovine serum albumin (BSA) complex was specifically suppressed in CM from galectin-3 overexpressing cells. The SERPINA1-BSA complex as well as human plasma SERPINA1 inhibited the growth of CML cells, while exogenous galectin-3 partly offset this effect. These findings suggest that galectin-3 overexpression promotes paracrine growth of CML cells by interfering with the action of the growth inhibitory SERPINA1-albumin complex.

Disseminated intravascular coagulation (DIC) is a life-threatening complication, and its control is essential for therapeutic success. Recombinant human soluble thrombomodulin alfa (rTM) is a novel therapeutic agent for DIC. The efficacy of rTM in the treatment of DIC is reportedly superior to that of conventional anti-DIC treatments, such as unfractionated heparin or low molecular weight heparin, but hemorrhagic events occasionally interfere with the therapeutic benefits of rTM. We assessed the clinical features of 20 consecutive patients who were given rTM for DIC associated with various hematologic disorders. Eight patients achieved remission of both primary disease and DIC, eight died due to progression of the primary disease, and four died of various hemorrhagic complications. Assessment of 16 biomarkers for coagulation showed that the four patients who died of hemorrhagic complications despite remission of their primary disease showed lower ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin Type 1 motif, member 13) plasma activity than other patients (P = 0.016). The optimal cut-off level of ADAMTS-13 for predicting risk of hemorrhagic complications was 42 % (P = 0.007). Plasma ADAMTS-13 activity determined at diagnosis of DIC may help predict the risk of hemorrhagic events during and/or following DIC treatment with hematologic disorders.

[Introduction] Multiple myeloma (MM) remains mostly incurable despite major advances in treatment strategies. The complex interplay among various cell-intrinsic and -extrinsic molecular mechanisms confers inter-patients diversity and intraclonal heterogeneity in MM. However, as the universal and relevant therapeutic target molecule against MM, we have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and its major downstream substrate RSK2 were universally active in MM (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014), while we also identified that the repression of miR-375 was found to be the universal underlying mechanism for the overexpression/activation of PDPK1 in MM (Tatekawa S, ASH meeting 2015). In this study, we further extended our study to assess the clinical relevance of miR-375 repression and the molecular mechanisms for the miR-375 repression in MM for the future clinical translation of miR-375/PDPK1/RSK2 signaling axis in the diagnosis and treatment development for MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 113 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells; N=10, MGUS; N=30, newly diagnosed MM (NDMM); N=34, relapsed/refractory (RRMM); N=39). The level of miR-375 expression was calculated with 2-ΔΔCt methods. Human U6 snRNA was examined as the reference and cDNA of RPMI8226 was used as a calibrator sample. The methylation status of miR-375 upstream regions including promoter site were analyzed by methylation-specific PCR (MSP) and bisulfite sequence. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine the histone modification status in miR-375 upstream regions. The circulating plasma RNA samples were converted to cDNA libraries followed by sequencing using the multiplex small RNA library primer set. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent. [Result] When compared to normal plasma cells, the miR-375 expression was significantly decreased in CD138 positive plasma cells from MGUS (p [Conclusion and Discussion] This study revealed that the expression level of miR-375 decreased specifically in clonal plasma cells from MGUS to RRMM patients, and the repression of miR-375 was caused by the overlapping epigenetic dysregulations. The abnormal repression of miR-375 which is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM is likely to be involved in the myelomagenesis in MGUS as well as the disease progression in MM, and the epigenetic deregulations may be a novel therapeutic target for MM. Disclosures Iida: Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding. Kuroda:Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Astra Zeneca: Research Funding; Janssen: Honoraria.

Follicular lymphoma (FL) is the most prevalent among indolent subtypes of non-Hodgkin lymphomas. While the histologic transformation associates with disease aggressiveness in variant FL, a minor population of non-transformed FL patients also shows a shorter period for disease control despite the use of rituximab (Rit) immunochemotherapy. In this study, we investigated the clinical and genetic features of high-risk non-transformed FL. With the median follow-up period of 61 months for 103 consecutively treated patients with histologically non-transformed FL including 85 patients treated with the first-line Rit-containing therapies and 18 with watch and wait, the 5-year overall survival (OS) and progression-free survival (PFS) of the entire cohort were 93.3% and 54.4%, respectively. Chemotherapy was initiated according to the GELF criteria. Although neither FLIPI nor FLIPI2 sufficiently predicted the risks for either OS or PFS, the presence of two extranodal involvements, including peripheral blood (PB) (N=7, p=0.006) and/or bone (N=6, p=0.007), at diagnosis were found to be significantly associated with poor PFS, but not OS, in our cohort. Indeed, 13 high-risk patients with PB and/or bone involvements at diagnosis showed a significantly shorter median PFS of 27.2 months compared with that of the 90 standard-risk patients, who have not yet reached the median (pTG substitution to be the most common mutational signature, while there was no difference with regard to the composition of the signature between the high- and standard-risk FLs. "Kataegis", a pattern of clustered hypermutations, was observed mostly in the target motif of activation-induced cytidine deaminase (AID), recurrently on chromosome 2p12 (involving the IGK locus), 14q32 (IGH), 18q21 (BCL2), 21p11, and 22q11 (IGLL). The mean number of C>T mutations in the AID-motif was significantly increased in the high-risk FLs over that in the standard-risk FLs (17 vs. 11, p=0.02), whereas there was no difference in the mean number of mutations in the target motif of APOBEC, which is considered to be the major driving agent responsible for kataegis in various solid cancers, between the two risk groups (8 vs. 7, p=0.49). For functional assessment of mutated genes, CREBBP and TNFRSF14 mutations were identified as the driver mutations of FL regardless of the disease risk using background mutation rate analysis. Hypergeometric test on all of the non-synonymous mutated genes from 14 FLs resulted in 71 significantly enriched gene ontologies (GOs), including the terms "cell development and differentiation", "regulation of cell death", "immunity mediated by lymphocytes, including B cells", "hematopoiesis", and "differentiation of leukocytes, including lymphocytes". In addition, pathway analysis demonstrated that mutated genes were enriched in the Notch and B cell receptor signaling pathways. However, we were not able to identify GOs or signaling pathways specific to the high-risk FL patients that could explain the different outcomes of the disease. Non-synonymous mutations in the high-risk FLs were found in genes, such as MEF2B and FUBP1, and were suggested to be associated with cancer aggressiveness, but not with the AID pathway. Overall, our results suggested that genomic instability, which allows for the emergence of distinct mutations not only by kataegis due to AID activity but also by additional mechanisms that increase mutations, underlies development of the high-risk phenotype in FLs. Disclosures Kuroda: Bristol Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Astra Zeneca: Research Funding; Janssen: Honoraria.

[Introduction] Multiple myeloma (MM) is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. We have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, and its major downstream substrate RSK2, a member of the 90 kDa ribosomal S6 kinase family of serine threonine kinases, were universally active in eleven human MM-derived cell lines (HMCLs) examined regardless of the type of cytogenetic abnormality, the mutation state of RAS, RAF and FGFR3 genes and myeloma cells of approximately 90% of symptomatic patients at diagnosis. Our study also disclosed that PDPK1/RSK2 signaling axis played pivotal roles in myeloma pathophysiology by regulating series of downstream molecules, such as c-MYC, IRF4, D-type cyclins, or PLK1, while the inactivation of either PDPK1 or N-terminal domain of RSK2 resulted in the induction of apoptosis in myeloma cells which was accompanied by the activation of BH3-only proteins BIM and BAD (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014). Here we assessed the underlying mechanism for PDPK1 overexpression in MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 92 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells (N=10), MGUS (N=21), newly diagnosed MM (NDMM) (N=27), relapsed/refractory (RRMM) (N=34). The pre-miR-375 precursor molecule (miR-375 mimics), the siRNA targeted against PDPK1, or a negative control RNA-oligonucleotides was transfected into 8 cell lines by utilizing Hemagglutinating Virus of Japan (HVJ)-envelope vector. The copy number abnormality of PDPK1 gene was assessed by double-color FISH for PDPK1 gene and the centromere of chromosome 16. The methylation status of miR-375 promoter site was analyzed by methylation-specific PCR (MSP). This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent, and normal bone marrow plasma cells were obtained from volunteers who were not affected by hematologic disease. [Result] The level of miR-375 expression was calculated with 2-ΔCt methods. Human U6 snRNA was examined as the reference. The median log102-ΔCt ± SD of normal plasma cells, MGUS, NDMM, RRMM and HMCLs were -2.46 ± 0.67, -3.64 ± 0.68, -4.23 ± 0.95, -3.92 ± 1.24 and -3.69 ± 0.29 respectively. When compared to normal plasma cells, the miR-375 expression was significantly decreased in NDMM and RRMM (p [Conclusion and Discussion] PDPK1 is activated by autophosphorylation and, therefore, its expression level is the crucial determinant for its activity, Because our study revealed the miR-375 expression as the major regulator of PDPK1 expression, it is suggested that the abnormally repressed miR-375 is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM. Moreover, since the decreased miR-375 expression was observed in plasma cells of MGUS and was more pronounced in MM, miR-375 repression by epigenetic deregulation may be involved in both disease development and progression of MM. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a highly molecularly heterogeneous hematologic malignancy and remains mostly incurable despite recent improvement of treatment outcome by novel agents. Therefore, the identification of novel and universal targetable therapeutic molecules is a core component for the therapeutic development. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a member of serine threonine kinase, is a rationalistic candidate of a novel therapeutic target against MM. PDPK1 is universally phosphorylated in all eleven MM-derived cell lines examined regardless of type of cytogenetic/genetic abnormalities or the activation/mutation state of FGFR3, RAS, ERK and AKT. PDPK1 promotes the cell proliferation of myeloma cells by activating RSK2Ser227 at N-terminal kinase domain which is a pivotal regulator of molecules that are essential for myelomagenesis, such as c-MYC, IRF4, or Cyclin Ds (Shimura Y, Mol Cancer Ther 2011), and AKTThr308. In contrast, PDPK1 inhibition by a selective inhibitor, BX-912, caused G2/M arrest, which was accompanied by the inactivation of PLK1, and resulted in cell death via induction of apoptosis which was accompanied by the activation of pro-apoptotic BH3-only proteins, BIM and BAD, in myeloma-derived cell lines. These molecular and cytological effects of PDPK1 inhibition in myeloma cells were also validated by gene knockdown by means of RNA interference using two different siRNAs specific for PDPK1. In addition, the cytotoxic effect of BX-912 was not hampered in two cell lines acquiring resistance to bortezomib (BTZ) (Ri M, Leukemia 2010), and PDPK1 inhibition by BX-912 showed additive to synergistic in vitro cytotoxic effects on myeloma cells with melphalan, etoposide, bortezomib or BAY11-7085, an inhibitor for NF-κB. BX-912 also induced cell death in eleven patient-derived primary myeloma cells those were positively isolated by CD138-labelling from bone marrow aspirates, while normal peripheral lymphocytes were significantly less sensitive to PDPK1 inhibitor compared with MM cells. In the clinical setting, PDPK1 was active in myeloma cells of 57 of 65 (87.7%) symptomatic MM patients at diagnosis. While patients backgrounds, such as age, gender, and the type of M-protein, were not significantly different between PDPK1-negative (PDPK1(-)) and PDPK1-positive (PDPK1(+)) patients, patients with the disease stage III according to International Staging System were significantly more frequent in the PDPK1(+) cohort compared with PDPK1(-) cohort. The PDPK1(-) patients tended to exhibit longer overall survival (OS) than the patients with PDPK1(+) (median OS: 2925 days vs. 2155 days, p=0.069) with a median follow-up period of 1310 days (range: 228–3317 days). Of particular, the 8-year OS of PDPK1(-) patients was statistically significantly more favorable than those of PDPK1(+) patients (83% vs. 17%, p=0.041). In addition, when we analyzed the impact of PDPK1 activity on the treatment outcome of patients treated by BTZ and dexamethasone therapy (BD), our results revealed that progression free survival (PFS) of patients with PDPK1(-) was a significantly longer than those of patients with PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), not reached vs. 167 days, p=0.049). The PFS of PDPK1(-) patients treated by high-dose therapy/autologous stem cell transplantation (HDT/ASCT) tended to be longer than those of PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), 972 days vs. 567 days, p=0.125). In conclusion, our study provides the rationale for PDPK1 as a possible universal molecular target for MM with various molecular/cytogenetic features. Especially, PDPK1 is a potential therapeutic target for not only newly diagnosed patients but also patients who are resistant or refractory to currently available anti-myeloma therapies. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. Patient-derived samples were obtained with informed consent. Disclosures No relevant conflicts of interest to declare.

Background Cell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. Methods Bone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. Results We first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. Conclusion Our results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. Disclosures: Kodama: Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

Little is known about the molecular pathogenesis of acute myeloid leukemia (AML) with high-level DNA amplifications commonly defined as the presence of double minutes (dmins) and/or homogeneously staining regions (hsr), although dmins and hsr are found in nearly 1% of karyotypically abnormal AML and myelodysplastic syndromes (MDS). It is sometimes difficult to precisely assign dmins, hsr, and marker chromosomes (mars) to specific chromosomal bands, while most of them consist of an amplified segment from chromosome band 8q24 or 11q23. In AML, the 8q24 amplicon has been previously identified in a commonly involved segment of about 4 Mb, which contains several genes such as TRIB1 (C8FW), NSMCE2 (non-SMC element 2), PVT1, and MYC. To further define the pathogenetic lesions of 8q24 amplicons in AML, we performed comprehensive molecular studies on leukemic cells from a patient with AML (patient 1) with two mars and leukemic cell lines HL60 and K562, identifying two novel chimeric transcripts, PVT1-NSMCE2 and BF104016-NSMCE2. Regarding PVT1-NSMCE2 fusion transcripts, PVT1 exon 1a fused to NSMCE2 exon 3 in patient 1, PVT1 exon 3a to NSMCE2 exon 4 in patient 2, and PVT1 exon 4b to NSMCE2 exon 4 in K562. Patient 2 was identified from the screening of an additional 50 patients with AML or MDS by RT-PCR. As for BF104016-NSMCE2 fusion transcript, BF104016 exon 1 fused to NSMCE2 exon 6 in HL60 harboring hsr and dmins. BF104016 is located inside the CCDC26 gene, sharing the same exon of CCDC26. In patient 1, fluorescence in situ hybridization identified the amplification of 5’PVT1 on the mars and dmins, and the amplification of NSMCE2 only on the mars. In HL60, PVT1 and NSMCE2 were amplified on dmins. Although real-time quantitative PCR showed the amplification of the aberrant NSMCE2 chimeric transcripts, western blot analysis demonstrated the depletion of NSMCE2 protein. High-resolution oligonucleotide array analysis demonstrated two amplicons at 8q24 commonly found in patient 1 and HL60. Furthermore, the coding directions of these three fusion genes were different at the 8q24; NSMCE2 and PVT1 are transcribed from centromere toward telomere, opposite to the direction of transcription of the CCDC26 gene. These results suggest that the chimeric formation of these genes were caused by chromothripsis. PVT1 and CCDC26 were known as large intervening non-coding RNAs, both of which were considered to be associated with the oncogenesis. NSMCE2 is known as a small ubiquitin-like modifier (SUMO) E3 ligase and is required for DNA repair. Functional analysis of NSMCE2 chimeric transcripts with PVT1 or CCDC26 will contribute to understanding of the leukemogenesis in AML harboring 8q24 amplicons. Disclosures: Taniwaki: Novartis: Honoraria.

Abstract 2945 Background: Despite recent advances of therapeutic strategies, multiple myeloma (MM) still remains mostly incurable due to its drug resistance by both various cell intrinsic molecular abnormalities and the supports of myeloma microenvironment factors. The search for new therapeutic target molecule upon which various MM-promoting signalings converge is essential for the development of more effective therapy for MM. RSK2 is a member of the 90 kDa ribosomal S6 kinases (RSKs) family of serine (Ser) /threonine (Thr) kinases and lies downstream of RAS/ERK-1/2 signaling cascade. In MM, it has been shown that RSK2 mediates signaling initiated by FGFR3 activation for cell survival, but the functional role and the value as the therapeutic target of RSK2 have not been clearly defined. In this study, we precisely studied the association with RSK2 activation and chromosomal status, especially focusing on RSK2Ser227, which is responsible for downstream signaling phosphorylation, and the value of RSK2 as the therapeutic target in MM. Methods: Twelve human myeloma-derived cell lines and primary MM cells were utilized in this study. The use of human samples was approved by the ethical review board of our institute. Cell killing assay was performed using flow cytometry. Growth inhibitory assay was performed with modified MTT assay. Protein levels were examined by Western blotting. BI-D1870, a RSK2 inhibitor, was purchased from Symansis Limited (Auckland, New Zealand). RNA interference for RSK2 was performed by transfection of small interfering RNA (siRNA) into NCI-H929 cells by means of CLB-Transfection Kit (Lonza, Swiss) with protocol 9. Drug combination assays were evaluated with CalcuSyn software (Biosoft, Cambridge, UK). Results: RSK2Ser227 was shown to be phosphorylated in all 12 MM cell lines and 6 of 9 primary MM cells. t(4;14) involving IgH/FGFR3 was detected only in 7 cell lines and high FGFR3 expression was identified only in 6 cell lines. RSK2Ser227 was phosphorylated even in the absence of ERK1/2 or RSK2Tyr529 phosphorylation in several MM cell lines, indicating that their activation was not mandatory to RSK2Ser227 phosphorylation. Our study also did not show any positive relationship between RSK2Ser227 phosphorylation and other so-called “high-risk” cytogenetic abnormalities, such as 13q-, t(11;14), t(8;14), t(14;16), 1q21+, or 17p-. Treatment with BI-D1870 (RSK2 inhibitor) resulted in complete dephosphorylation of RSK2Ser227, but not RSK2Tyr529. The phosphorylation status of ERK1/2, or related signaling kinases, such as AKT, p38 MAPK, or JNK was also not affected. BI-D1870 showed dose-dependent growth inhibition effect in 6 MM-derived cell lines by inducing apoptosis. At molecular level, Mcl-1, p21WAF1/CIP1, cyclin D2 and c-Myc was significantly down-regulated and Bim was up-regulated by BI-D1870 intervention in NCI-H929 cell. BI-D1870 didn't modulate expression level of IRF-4. These change were also observed in other MM cell lines (KMS-34, AMO-1). To exclude the possibility that above BI-D1870 induced changes in protein expression and cell death are due to its off-target effects, we performed gene knockdown experiments of RSK2. Transient gene knockdown of RSK2 by means of RNAi caused downregulation of cyclin D2, p21WAF1/CIP1, c-Myc, while it induced cell death in NCI-H929 cells. In contrast, Bim expression was not altered by RSK2 knockdown. Lenalidomide (LEN) is the most powerful currently available IMiD for MM treatment. While cell death induction by LEN (100μM) was partly prevented by IL-6 (50ng/ml) in NCI-H929 cells, co-administration of BI-D1870 (0.2 μM, at the degree of low concentration not to affect cell viability when used solely) overcame the resistance to LEN induced by IL-6. KMS-11/BTZ were highly resistant to bortezomib (BTZ)-induced cell death, by contrast, it showed the similar sensitivities to BI-D1870-induced cell death when compared with their parental cells. And more, BI-D1870 showed synergistic or additive effects with upcoming new anti-MM agents, such as RAD001, MS-275 and ABT-263 in MM cell lines. Conclusions: RSK2, especially RSK2Ser227, may be a universal therapeutic target for MMs with diverse molecular signatures. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria.

Abstract 3744 [Introduction] Tyrosine kinase inhibitors (TKIs) for BCR-ABL have dramatically improved the treatment outcome of chronic myelogenous leukemia (CML) during this decade, however, the resistance to TKIs due to various molecular mechanisms, such as BCR-ABL mutations, dysfunction of cell killing mechanisms like BIM deregulation, or the supports by leukemia microenvironment factors, has remained to be overcome. We have identified that BIM, a member of BH3-only proteins, is the essential pro-apoptotic mediator under the blockade of BCR-ABL signaling (Kuroda J, PNAS, 2006). One of the attractive therapeutic strategies for complete eradication of CML cells by simultaneously targeting both TKI-sensitive and TKI-resistant CML cells may be the use of agents that can activate BIM as well as other pro-apoptotic BH3-only protein which is not utilized in apoptosis by TKIs. In this study, we assessed the therapeutic potential of the protein phosphatase 2A (PP2A) activator Fingolimod (FTY720), an immunosuppressive agent currently used for multiple sclerosis, in CML. [Materials and methods] Human CML-derived cell lines, K562, MYL, KBM5, KCL22, and their various subclones, were utilized in this study. Murine Ba/F3 lymphoid cells were transfected with either wild type or mutated bcr-abl gene. FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Cell death analyses were performed using flow cytometry. Protein levels were determined by conventional Western blotting. [Result and discussion] Treatment with FTY720 impairs phosphorylation of BCR-ABL and its downstream targets, AKT, ERK and STAT5, and inhibited the growth of CML cell lines through the induction of apoptosis. The apoptosis induced by FTY720 was accompanied by the activation of both caspase-8 and caspase-9, while enforced expression of either BCL2 or dominant-negative protein of FADD (FADD.DN) by gene introduction partly protected K562 cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways in FTY720-induced apoptosis. FTY720 treatment resulted in the activation of Bim as well as the processing of Bid into tBid, a BH3-only protein which plays crucial role in accelerating death-receptor-mediated extrinsic apoptosis pathway and is not utilized in apoptosis by TKIs. Importantly, gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the degrees of cell protection by those were not as much as the degree of cell protection by overexpression of either BCL2 or FADD.DN. Moreover, double knockdown of BIM and BID did not further protect K562 cells from apoptosis by FTY720, when compared with single knockdown of BIM. These results indicate that BIM and BID compensate each other for promoting apoptosis by FTY720 in CML cells, especially when either of them is functionally disturbed. Through these mechanisms, FTY720 overcomes TKI resistance by various mechanisms, such as ABL kinase domain (KD) mutations in BCR-ABL-transfected Ba/F3 cells (T315I, G250E, E255K or H396P) and in KBM5 cells with T315I. FTY720 also overcomes TKI resistance due to dysfunction of BIM by gene deletion polymorphism in KCL22 cells. Also, although galectin-3 overexpression has been shown to mediate bone marrow environment-mediated drug resistance in CML cells (Yamamoto-Sugitani M, PNAS, 2011), the addition of FTY720 to imatinib overcomes resistance to imatinib in galectin-3 overexpressing K562 cells and MYL cells. In conclusion, FTY720 induces apoptotic cell death in CML via dual activation of both BIM and BID, and is potent in overcoming various types of cell intrinsic and tumor microenvironment-mediated resistance to TKI. Disclosures: No relevant conflicts of interest to declare.

Abstract 4450 In B-cell malignancies, the genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In the present study, we have identified BTB and CNC homology 2 (BACH2) as a novel translocation partner gene of the immunoglobulin heavy chain (IGH) locus, resulting in chimeric Cδ-BACH2 gene in a patient with MYC-IGH-positive highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). A 71-year-old male was diagnosed as having highly aggressive B-cell lymphoma/leukemia. SKY analysis revealed the representative karyotype of tumor cells as 45,X,-Y,der(3)t(3;X)(p21.2;q24),del(6)(q15),der(14)t(8;14)(q24;q32),del(16)(q22),der(20) t(3;20)(q21;p13). FISH and long-distance PCR analyses identified a fusion of MYC with Cγ. FISH analysis also detected a small IGH signal on del(6), and a VH on del(6)(q15). Genome copy number analysis using SNP-array detected an approximately 60Mb deletion in 6q15–25 region, and its centromeric breakpoint within BACH2 gene. The cDNA bubble PCR using BACH2 primers detected multiple PCR products, and sequence analysis has revealed that one of the products contained a sequence of the first exon of IGHCδ fused to 5’ untranslated region of BACH2 exon 2. Genomic fusion point of Cδ-BACH2, was within intron 1 of Cδ and intron 1 of BACH2. Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, indicating that whole coding region of BACH2 was fused to Cδ. This suggested that Cδ-BACH2 fusion cause constitutive activation of BACH2. We next analyzed the expression levels of BACH2, MYC, PRDM1, and IRF4 genes in the patient using real-time PCR and compared them with those of several hematologic malignancy cell lines, including 14 non-Hodgkin's lymphoma (NHL), 10 multiple myeloma (MM), and 3 B-lineage acute lymphoblastic leukemia, and 3 EB-virus transformed B-cell lines from normal healthy volunteers. IGH-MYC-positive MM cell lines showed increased levels of MYC expression compared with the other cell lines. The MYC expression level in our patient was lower than those in IGH-MYC-positive MM cell lines; however, it was similar to 5 IGH-MYC-positive NHL cell lines [3 Burkitt lymphoma (BL) and 2 diffuse large B-cell lymphoma (DLBCL)]. On the other hand, BACH2 expression levels were higher in NHL cell lines, especially in BL cell lines, but not correlated with the IGH-MYC translocation status. Our IGH-BACH2-positive case also showed a higher level of BACH2 expression. The expression levels of IRF4 and PRDM1 were higher in MM cell lines than in other cell lines. PRDM1 is activated by MYC through IRF4, and the MYC is negatively regulated by activated PRDM1. In MM cell lines with IGH-MYC translocation, MYC was highly expressed regardless of high expression of PRDM1, indicating that MYC activated by IGH translocation could not be inhibited by PRDM1. Unexpectedly, the expression levels of PRDM1 and IRF4 were very low in NHL including our patient, suggesting that the regulation of MYC in NHL is different from that in MM. BACH2 is a B cell-specific transcription repressor, and is specifically required for class switch recombination, somatic hypermutation, and germinal center formation. One of the target genes of BACH2 is PRDM1 at 6q21-q22.1 that is required for plasma cell differentiation. In this patient, deletion of 6q15–25 was found, indicating loss of one PRDM1 allele. It was reported that PRDM1 is inactivated by chromosomal alterations in 24% of activated B cell–like DLBCL, suggesting that PRDM1 acts as a tumor suppressor gene, and its inactivation may contribute to lymphomagenesis by blocking post–GC differentiation. The combination of BACH2 and MYC in double IGH translocations is unique and consistent with previous reports demonstrating that each partner gene found in double or multiple IGH translocations is exclusively specific to certain types of B-cell lymphoma. These results suggest that the promoter of IGHCδ and/or Eμ enhancer of IGH activate the expression of BACH2, and that BACH2 may act as oncogene in some cases with B-cell lymphoma. Although the IGH-BACH2 translocation is rare in NHL, our data suggest that the BACH2 plays a critical role in B-cell lymphomagenesis through not only IGH translocation but also activation by some other mechanisms. Disclosures: No relevant conflicts of interest to declare.

The AML1/RUNX1 gene is frequently rearranged by chromosomal translocations in acute leukemia. To date, more than 10 fusion genes involving AML1 have been cloned, such as AML1-MTG8 in acute myeloid leukemia (AML) with t(8;21), AML1-EVI1/MDS1 in therapy-related AML/myelodysplastic syndrome with t(3;21), and TEL/ETV6-AML1 in B precursor ALL with t(12;21). We analyzed a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22), and identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on 21q22 using the bubble PCR method for cDNA. The genomic breakpoints were within intron 7 of AML1 and intron 7 of LAF4 resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. LAF4 gene is a member of the AF4/FMR2 family, and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. These findings provide new insights into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. In this study, we first applied the panhandle PCR method that is usually used for cloning the fusion partners of MLL or NUP98; however, no fusion transcripts could be obtained. Therefore, we searched for another method for cloning the fusion transcripts and successfully adapted the bubble PCR method for cloning the novel AML1-LAF4 fusion transcript. To date, bubble PCR has been performed for cloning unknown genomic fusion points but not fusion cDNAs. Using double-strand cDNA, we could apply the bubble PCR method for cloning fusion cDNA with rare non-specific products. Bubble PCR for cDNA could amplify in both 5′ to 3′ and 3′ to 5′ directions of the gene or transcript and handle any exons fused to unknown partners for amplification easily. This method will contribute to identifying numerous novel translocation partners more easily, and these findings may help to clarify the role of the fusion proteins in leukemogenesis.

The pathogenetic roles of 8q24 amplified segments in leukemic cells with double minute chromosomes remain to be verified. Through comprehensive molecular analyses of 8q24 amplicons in leukemic cells from an acute myelogenous leukemia (AML) patient and AML-derived cell line HL60 cells, we identified two novel fusion genes between NSMCE2 and long noncoding RNAs (lncRNAs), namely, PVT1-NSMCE2 and BF104016-NSMCE2. Our study suggests that 8q24 amplicons are associated with the emergence of aberrant chimeric genes between NSMCE2 and oncogenic lncRNAs, and also implicate that the chimeric genes involving lncRNAs potentially possess as-yet-unknown oncogenic functional roles. Electronic supplementary material The online version of this article (doi:10.1186/s13045-014-0068-2) contains supplementary material, which is available to authorized users.