Recent studies have showed that asymptomatic cerebral infarction (ACI) developed in a reasonable number of patients after cardiac catheterization. However, no study has investigated the long-term prognostic impact of ACI after cardiac catheterization. We investigated whether ACI after cardiac catheterization affects long-term mortality and subsequent cardiovascular events.We retrospectively enrolled patients who underwent cardiac catheterization before cardiac surgery and cerebral diffusion-weighted magnetic resonance imaging (DWI). The incidence and clinical features of ACI were investigated. The long-term prognosis, including all-cause mortality and subsequent major cardiovascular events (MACE; all-cause mortality, stroke, acute myocardial infarction, fatal arrhythmia, and hospitalized heart failure), was also assessed.A total of 203 patients were enrolled. Of these, 10.3% had ACI diagnosed by DWI. There were no differences in baseline characteristics between patients with and without ACI, except more frequent history of symptomatic stroke in patients with ACI. In the Kaplan-Meier analysis during a median follow-up of 1009 days, the patients with ACI showed worse mortality and a slightly higher occurrence of MACE compared with those without ACI (P = 0.01 and P = 0.08, respectively). In addition, ACI was a prognostic marker independent of age, surgery type, and history of stroke.ACI after cardiac catheterization frequently developed and was also associated with long-term prognosis. It may be an independent prognostic marker in high-risk patients who underwent subsequent cardiac surgery.

We retrospectively evaluated long-term outcomes of high dose chemotherapy followed by autologous stem cell transplant (HDC/ASCT) in patients with diffuse large B-cell lymphoma (DLBCL). Between 2004 and 2020, 46 DLBCL patients received HDC/ASCT in our institution, including 12 patients (26.1%), who received as an upfront setting (UFS). At a median follow-up time of 69 months (range, 2-169 months), the 5-year progression-free survival (PFS) rates were 82.5% (95%CI, 46.1-95.3%) in the UFS, and 57.8% (95%CI, 38.1-73.2%) in the relapsed or refractory (R/R) patients (n=34), respectively. The 5-year PFS rates were 62.3% (95%CI, 34.0-81.3%) in primary resistant (n=13) or early relapsing (within 1 year from the initial diagnosis) patients (n=4), and 53.3% (95%CI, 25.9-74.6%) in those relapsing >1 year after the initial diagnosis (n=17), with no statistically significant difference (p=0.498). In R/R patients, multivariate analysis showed that the remission status before HDC/ASCT was an independent poor prognostic factor for progression-free survival (hazard ratio [HR], 17.0; 95%CI, 3.35-86.6; p=0.000630) and high-risk category in the international prognostic index for OS (HR, 9.39; 95%CI, 1.71-51.6; p=0.0100). The incidence of non-relapse mortality by 5 years, and 10 years were 12.2%, and 15.2%, respectively. Eleven patients (23.9%) developed second malignancies, which was the most frequent late complication after HDC/ASCT, with 5-year, and 10-year cumulative incidence of 16.9%, 22.5%, respectively. In conclusion, HDC/ASCT is effective for chemo-sensitive R/R DLBCL regardless of the timing and lines of therapy. However, careful observation is required, considering the long-term complications such as secondary malignancies.

BACKGROUND: The plaques with higher grade of yellow color by angioscopy are reported to be associated with vulnerability leading to adverse outcomes in coronary artery diseases. However, no studies have been performed for peripheral artery disease (PAD). We aimed to evaluate the relationship of angioscopic findings of peripheral arteries with the long-term prognosis. METHODS: Angioscopy of iliac or femoropopliteal artery was performed before endovascular therapy in patients with PAD. The local plaque color and presence of thrombus were evaluated. Multivariable Cox regression models were used to estimate hazard ratio (HR) for all-cause mortality or major adverse cardiovascular event (MACE) related to the plaque colors as well as presence of thrombus. RESULTS: Among 67 patients, 49.3% had intensive yellow plaques (group H) and the rest had light yellow to yellow ones (group L). Thrombus was detected in 74.6% of the patients and the presence was not different between the two groups. In Kaplan-Meier analysis during a median follow-up of 976 days and 757 days, group H showed increased mortality and MACE compared with group L (p <0.01 for both). Multivariable analysis demonstrated that the intensive yellow color of plaque was independently associated with mortality and MACE [HR: 11.48, 95% confidence interval (CI): 2.19-211.1 and HR: 3.81, 95% CI: 1.36-13.48, respectively] after adjusting for the presence of thrombus. CONCLUSIONS: The yellow color intensity in local plaques by angioscopy may be a novel predictor of long-term prognosis in patients with PAD, regardless of the presence of thrombus.

We previously reported that a novel haemoglobin-platelet index (HPI) based on anaemia and thrombocytopenia was useful to predict the prognosis of patients with diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS). Here, we analyse the utility of HPI in a new validation cohort with DLBCL NOS (n  = 94). As a result, we confirm that HPI was effective for differentiating progression-free survival (PFS) and overall survival in this validation cohort. So, we further compare the utility of HPI with previously reported prognostic markers such as the National Comprehensive Center Network-International Prognostic Index (NCCN-IPI), Glasgow prognostic score (GPS), and platelet-albumin (PA) score, using a larger number of 160 patients consisting of the derivation cohort (n  = 66) and a validation cohort (n  = 94). As a result, the patients with a higher HPI score had significantly worse outcomes, and HPI predicted the prognosis of DLBCL NOS independently of NCCN-IPI. HPI was more sensitive than GPS and almost the same as PA score in predicting PFS. Moreover, the patients whose lymphoma cells were positive for interleukin-6 (IL-6) (75/111 cases) judged by immunohistochemical staining had significantly lower haemoglobin levels and platelet counts than IL-6-negative cases (36/111 cases), suggesting the involvement of IL-6 produced by lymphoma cells in anaemia and thrombocytopenia in DLBCL NOS patients.

Side population (SP) is known to include therapy-resistant cells in various cancers. Here, we analyzed SP using multiple myeloma (MM) samples. The SP accounted for 2.96% in MM cells from newly diagnosed MM (NDMM). CD34 was expressed in 47.8% of SP cells, but only in 2.11% of bulk MM cells. CD34+ MM cells expressed more immature cell surface markers and a gene signature than CD34- MM cells. CD34+ but not CD34- MM cells possessed clonogenic activities and showed long-term self-renewal activities in xenotransplantation assays. Similarly, whereas 2.20% of MM cells were CD34+ in NDMM (n = 38), this proportion increased to 42.6% in minimal residual disease (MRD) samples (n = 16) (p < 0.001) and to 17.7% in refractory/relapsed MM (RRMM) (n = 30) (p < 0.01). Cell cycle analysis showed that 24.7% of CD34+ MM cells from NDMM were in G0 phase while this proportion was 54.9% in MRD (p < 0.05) and 14.5% in RRMM, reflecting the expansion of MM. Together, CD34+ MM cells with long-term self-renewal activities persist as MRD in cell cycle quiescence or remain as therapy-resistant cells in RRMM, substantiating the necessity of targeting this population to improve clinical outcomes of MM.

We previously examined the utility of rituximab-bendamustine (RB) in patients with follicular lymphoma (FL) exhibiting less than optimal responses to 2 cycles of the R-CHOP chemotherapy regimen. The aim of this study was to identify molecular biomarkers that can predict prognosis in RB-treated patients in the context of the prospective cohort. We first analyzed the mutational status of 410 genes in diagnostic tumor specimens by target capture and Sanger sequencing. CREBBP, KMT2D, MEF2B, BCL2, EZH2, and CARD11 were recurrently mutated as reported before, however none was predictive for progression-free survival (PFS) in the RB-treated patients (n = 34). A gene expression analysis by nCounter including 800 genes associated with carcinogenesis and/or the immune response showed that expression levels of CD8+ T-cell markers and half of the genes regulating Th1 and Th2 responses were significantly lower in progression of disease within the 24-mo (POD24) group (n = 8) than in the no POD24 group (n = 31). Collectively, we selected 10 genes (TBX21, CXCR3, CCR4, CD8A, CD8B, GZMM, FLT3LG, CD3E, EOMES, GZMK), and generated an immune infiltration score (IIS) for predicting PFS using principal component analysis, which dichotomized the RB-treated patients into immune IIShigh (n = 19) and IISlow (n = 20) groups. The 3-y PFS rate was significantly lower in the IISlow group than in the IIShigh group (50.0% [95% CI: 27.1-69.2%] vs. 84.2% [95% CI: 58.7-94.6%], P = .0237). Furthermore, the IIS was correlates with absolute lymphocyte counts at diagnosis (r = 0.460, P = .00355). These results suggest that the T-cell-associated immune markers could be useful to predict prognosis in RB-treated FL patients. (UMIN:000 013 795, jRCT:051 180 181).

The aim of this trial is to evaluate the utility of rituximab-bendamustine (R-B) for untreated advanced follicular lymphoma (FL) showing non-optimal response (nOR) to R-CHOP, and to identify clinical prognostic factors for FL patients receiving R-B. Patients who failed to achieve complete response/complete response unconfirmed (CR/CRu) [nOR-group] after 2 cycles of R-CHOP subsequently received 6 cycles of R-B. The primary endpoint was the 3-year progression-free survival (PFS) rate. Secondary endpoints included determination of prognostic factors. Fifty-six patients initially received R-CHOP, 43/56 patients (76.8%) were judged as nOR, and 33/43 patients (76.7%) completed 6 cycles of R-B. At a median follow-up of 50.6 months in the nOR-group, the 3-year PFS rate was 69.0%, and the 3-year overall survival (OS) rate was 92.7%. The most common toxicities associated with R-B were grade 3-4 lymphopenia (93.0%) and neutropenia (74.4%), both of which were manageable. A multivariate analysis including dose intensity, serum soluble interleukin-2 receptor, and FL international prognostic index-2 revealed low absolute lymphocyte count (<  869/μL) at diagnosis was an independent poor prognostic factor for both PFS and OS in the R-B-treated nOR-group. This result was further confirmed in validation cohorts including R-B-treated de novo (n = 40) and relapsed (n = 49) FL patients.

We recently treated a chronic myeloid leukemia (CML) patient with liver and renal dysfunction, who was undergoing hemodialysis (HD). He was treated with 50 mg dasatinib (DAS) once daily just before HD. The maximum plasma concentration of DAS was 227 ng/mL on a non-HD day and 46.9 ng/mL on a HD day. He was subsequently treated with 200 mg bosutinib (BOS) once daily. The plasma concentration of BOS changed from 74.5 ng/mL before HD to 58.8 ng/mL after HD. Our results indicate that close monitoring of the plasma tyrosine kinase inhibitor concentrations should be considered in CML patients with organ impairment.

We evaluated the efficacy and safety of switching to nilotinib in CML-CP patients who had achieved MMR with continuous detectable BCR-ABL1 transcript levels after long-term imatinib treatment. Patients who had achieved MMR, but not deep molecular response (DMR), after > 18 months from the initiation of imatinib received nilotinib 400 mg twice daily for up to 24 months. BCR-ABL1 transcript levels were assessed every 3 months. Thirty-eight patients with a median age of 57.5 years (range 22-76 years) were evaluated. Twenty-seven patients completed 24 months of nilotinib treatment; 11 discontinued nilotinib due to retraction of consent (three patients), loss of MMR (1), intolerance (3) or AEs (5). Twenty patients [52.6%, (90% CI 38.2-66.7%)] achieved DMR. The cumulative incidence of achieving DMR by the time of 3, 6, 9, 12, 15, 18, and 21 months was 22.9, 37.7, 47.0, 53.7, 53.7, 53.7, and 53.7%, respectively. Adverse events were consistent with those reported in other nilotinib studies. Patients experienced each of the following cardiovascular complications: atrial fibrillation (G2), chest tightness and dyspnea (G1), myocardial infarction (G2) and heart failure (G3) (n = 1 for each complication). This study indicates nilotinib achieves strong, rapid induction of DMR for patients who achieved MMR after long-term imatinib therapy.

Abstract Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1–17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

Abstract Background: DNA hypomethylating agents, such as 5-azacitidine (5-aza) and decitabine, comprise the current standard in therapy for patients with high-risk myelodysplastic syndromes (MDS), with dramatic responses in some patients. However, the responses are poorly predictable and their impact on clonal dynamics has not been fully elucidated. Patients and Methods: We enrolled a total of 119 patients with high-risk MDS who were treated with 5-aza . Bone marrow samples were collected before (n = 71) and both before and after (n = 48) treatment and analyzed by targeted-capture sequencing using RNA baits designed for 67 known or putative driver genes in myeloid neoplasms and 1,674 single nucleotide polymorphisms, which enabled detection of both mutations and copy number alterations on the same platform. In 9 of the 48 patients, pre- and post-therapy samples were further analyzed by whole exome sequencing (WES). Results: Average number of driver mutations before 5-aza was 2.8 per patient and 107 (90%) patients had multiple mutations. Most frequently mutated were TP53 (27%), followed by RUNX1, TET2, DNMT3A, and ASXL1. Reflecting high-risk disease subtypes of the subjects, splicing factor mutations were relatively rare (29 %) in the current cohort. Chromosomal abnormalities were identified in 65 (55%) patients, where 7q- and /or 5q- were the most frequent. Among 48 patients with serially collected samples, 46 had one or more mutations, enabling an evaluation of clone dynamics. In total 163 and 146 mutations were detected before and after treatment, respectively. About two thirds (110/163) of the mutations before 5-aza remained detectable after treatment. By contrast, the remaining one third showed a dynamic clonal behavior; 36 mutations in 22 cases were newly acquired, whereas 53 in 28 cases disappeared. Among those newly acquired, most frequently observed were mutations in STAG2 and EP300 (n = 3), of which STAG2 (7 cases) also represented the most frequent targets of disappeared mutations after treatment. In WES in 9 patients, a total of 112 mutations were identified either before or after 5-aza treatment with a mean of 10.4 or 8.9 mutations per sample, respectively. Among these, 63 were found at both pre- and post-therapy samples, whereas 17 and 32 mutations were newly acquired or disappeared during treatment, Given that only 4 newly acquired and 8 lost mutations had been detected by targeted-capture sequencing, respectively, WES enabled more sensitive detection of alternation of clones during 5-aza treatment, which were demonstrated in 8 (89%) subjects, rather than 5 (56%) in targeted-capture sequencing. Clinical outcomes have been reported for 22 patients as of the time of abstract submission; 5 achieved complete remission (CR), 9 stable disease (SD), and 5 progressive disease (PD). Alteration in clone size was frequently associated with clinical response. The size of dominant clones significantly decreased in 4 of 5 cases with CR, whereas stable or increased in 12 of 14 patients with SD or PD. In patients with SD or PD, acquisition of new mutations was common (10/14) during 5-aza treatment and potentially implicated in the resistance to 5-aza-treatment. Of interest, newly acquired mutations were also found in 2 CR samples, albeit at low allele frequency, even though the clone size of dominant clones was substantially reduced, suggesting the evolution of alternative MDS subclones or expansion of preexisting non-leukemic hematopoietic clone. Although CR was achieved in 3 of 6 patients with TP53 mutations, the TP53-mutationsdid not totally disappeared but were still detectable in CR samples in 2 cases, suggesting that TP53 mutated clones have not been completely eradicated by 5-aza treatment. Conclusion: Our study successfully depicted the structure of clones and their dynamics in high-risk MDS on 5-aza treatment. Alteration in the size of the dominant clones was frequently associated with a clinical response. Clonal evolution was common even in patients who achieved CR. Tracking the mutations in MDS patients during 5-aza treatment provides the opportunity to detect clones resistant to 5-aza and might be used to guide 5-aza therapy. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria. Kiyoi:Celgene Corporation: Consultancy; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding. Naoe:Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Astellas Pharma Inc.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Amgen Astellas BioPharma K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene K.K.: Honoraria, Research Funding; CMIC Co., Ltd.: Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Pfizer Inc.: Research Funding. Makishima:The Yasuda Medical Foundation: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Abstract Background: Studies on germline variants responsible for cancer predisposition provide an important clue to the understanding of genetic basis of cancer and also help better prediction and management of relevant cancers. As for myeloid neoplasms, only a handful of genes, including RUNX1, CEBPA, GATA2, ETV6, and ANKRD26, have been implicated in early onset familial acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), although they are rarely seen in sporadic cases. Recently, using whole exome sequencing of familial AML/MDS, we have reported novel AML/MDS predisposing gene, DDX41, an encoding dead-box helicase gene. Conspicuously, the onset of AML/MDS was over 60 in most of the affected cases, raising a possibility that the genetic predisposition might be obscured and many cases could be diagnosed with sporadic AML/MDS. In this study, we investigated germline DDX41 mutations in sporadic cases with AML/MDS and the incidence and mutation types were compared between Asian and Western patients. Patients and Methods: We performed targeted sequencing of DDX41 in patients from Asian (N = 239) cohort of AML/MDS, where the origin of the detected variations was determined by using matched germline DNA. The result was compared to those obtained from the Western cohort (N = 1,034) in terms of frequency and type of mutation. The effect size of the mutations was estimated by calculating odds ratios of each variant for AML/MDS development using the data for DDX41 variants in Asian and Western population from the ExAC (Exome Aggregation Consortium) database (http://exac.broadinstitute.org) as controls. Results: Germline and somatic DDX41 mutations were found in 12 (5.0%) and 10 (4.7%) of sporadic cases with AML/MDS from the Asian cohort, as compared to 8 (0.8%) and 10 (1.0%) from the Western cohort. All the patients with germline variants were aged over 40 year-old with a median of 68.5, confirming the late onset of the disease also in the sporadic cases with germline variants. Eight of the 12 germline variants (67%) in the Asian cohort were accompanied by an additional somatic mutation, as compared to 2 of the 8 (25%) in the Western cohort. Biallelic involvement was demonstrated in selected cases (N = 2). In total, 8 and 3 germline variants were observed in the Asian and the Western cohorts, respectively, without no common variants between both cohorts, of which the predominant variants included p.A500fs (n=5; 42%) and p.E7X (n=2; 17%) in the Asian cohort and p.F140fs (n=6; 75%) in Western cohort. In contrast, a prominent hotspot mutation involving a highly conserved amino-acid within the helicase domain (p.R525H) was commonly observed in both cohorts, accounting for 55% of all the somatic mutations. These germline variants as a whole showed significant enrichment in AML/MDS cases compared to the respective control population (OR>171, 95% confidence interval (CI): 51-730 for the Asian variants and more than 21.7, 95%CI: 8.4-50 for the Western variants), although the enrichment of individual variants showed substantial variations, suggesting different effect size among these variants: the odds ratio was 19.5 (p<0.001) for p.F140fs, and 92.4 (p<0.001) for p.A500fs. p.E7X was detected in 2 out of 239 cases with MDS/AML, whereas not in the control Asian population. Conclusion: We demonstrated frequent germline variants of DDX41 among sporadic cases with AML/MDS from different ethnic populations. Having common ancestral origins in different ethnic populations, these alleles are found in the general population at very low frequencies (<1 in 4000), accounting for the largest congenital risk for the development of sporadic AML/MDS therein (3-5% of all sporadic AML/MDS). The onset was typically over 40 years of age and frequently accompanied by an additional somatic mutation most likely in the unaffected allele, showing a prominent hotspot at p.R525. The germline variants seem to be dominant and caused premature truncation of the protein, leading to loss-of-function in most cases, whereas somatic mutations were typically missense variants not totally abrogating protein function, suggesting the importance of less than haploinsufficiency but more than null function for leukemogenesis. At the meeting, an extended result from more than 1000 Asian cases will be presented. Disclosures Kiyoi: Kyowa-Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Pfizer Inc.: Research Funding; Novartis Pharma K.k.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Company, Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., LTD.: Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Yakult Honsha Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Teijin Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; MSD K.K.: Research Funding. Miyazaki:Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Chugai: Honoraria, Research Funding; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Naoe:Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Usuki:Boehringer Ingelheim: Other: personal fees, Research Funding; Shionogi: Other: personal fees; Fujimoto Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; SymBio Pharmaceutical: Other: personal fees, Research Funding; Eisai: Research Funding; Otsuka Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Other: personal fees, Research Funding; Shire: Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; Novartis: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; MSD: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Taiho Pharmaceutical: Other: personal fees, Research Funding; Fuji Film RI Pharma: Other: personal fees; Chugai Pharmaceutical: Other: personal fees; GlaxoSmithKline: Other: personal fees, Research Funding; Bristol-Myers Squibb: Other; Astellas: Research Funding. Miyawaki:Astellas Pharma Inc.: Consultancy, Other: personal fees; Ohtsuka Pharma Co, LTD.: Other: Safety Data Committee.

Abstract DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the >480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with >10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing >20% difference in mean methylation levels between responders and non-responders (q < 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

A 68-year-old man was referred to our hospital due to a high fever and pancytopenia. Neither tumors nor infectious lesions were detected. Hemophagocytosis was observed on the bone marrow (BM) smear, although without abnormal cells. Prednisolone therapy was ineffective for the patient's high fever. Later on, we obtained the results of a BM biopsy indicating the presence of infiltration of atypical Reed-Sternberg cells, leading to a diagnosis of HIV-negative primary bone marrow Hodgkin lymphoma (PBMHL). However, the patient died of multiple organ failure before receiving chemotherapy. As the clinical course of PBMHL is rapid, physicians must keep in mind its possibility in similar cases.

Patients with acquired haemophilia A usually show widespread subcutaneous bleeding. We describe an 86-year-old man with acquired haemophilia A associated with prostate carcinoma, showing initial localised giant haematoma and subsequent widespread subcutaneous bleeding. A localised giant haematoma may present as a first and important sign of acquired haemophilia A.

The pathogenesis of acquired immunodeficiency syndrome-associated primary central nervous system lymphoma (AIDS-associated PCNSL) remains unclear. However, cell adhesion molecules have been reported to be strongly associated with PCNSL. In this study, we established Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) from HIV-positive patients (LCL(HIV)) and normal individuals (LCL(N)). The expression of CD18 antigen by LCL(HIV) was stronger than that by LCL(N). We performed a cell adhesion assay using ISO-HAS, which is the human hemangiosarcoma cell line and expresses intercellular adhesion molecule 1 (CD54). The binding rates of LCL(HIV) and ISO-HAS without stimulation were higher than those of LCL(N). Further, we demonstrated that azidothymidine or simvastatin inhibited the binding rates of LCL(HIV) and ISO-HAS more significantly than those of LCL(N). Further, the levels of interleukin (IL)-8, a CD18 inducer, were higher in LCL(HIV) than in LCL(N). We conclude that interaction between IL-8 and CD18 may be critical to AIDS-related PCNSL.

Abstract We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-I- positive T-cell lines and fresh cells from patients with adult T-cell leukemia. We here confirmed the clinical effects of ATRA in 20 patients with ATL. Twenty patients (n=20) with median age of 56 years (range 35–68 years) diagnosed with ATL received ATRA orally. ATRA was administered for a median of 25.7 days (range 14–56 days). Efficacy was described below; no CR case, PR case was 55%, NR case was 45%. In 7 acute cases, PR case was 4 (20%) and NR case was 3 (15%). In 3 lymphoma cases, no NR case and 3 PR cases (15%) was found. In 4 chronic cases, PR case was 1 (4%) and NR case was 3 (15%). In 6 skin type. PR case was 3 (15 %) and NR case was 3 (15%). Major side effects were headache (n=5), transient liver dysfunction (n=2), hyperlipidemia (n=2) and anorexia (n=1). No major toxicity was observed. These results indicated that ATRA might be a useful agent for skin involvement of ATL with safety.

Abstract It has been known that non-Hodgkin’s lymphomas (NHL) are a major complication in human immunodeficiency virus (HIV) infection, and a major cause of death in HIV infected patients. Recently, it has been reported that highly active anti-retroviral therapy (HAART) declines the incidence of HIV-related lymphoma. In this study, we tried to make in vitro model of HIV-related lymphoma by Epstein-Barr virus transformation, and to examine the cellular and molecular characteristics of the HIV-related lymphoma cells derived from patients with HIV infection. PBMCs from 10 patients HIV infection and 10 HIV-negative normal individuals were obtained by Ficoll-Paque (Amersham Biosciences Corp. NJ) density gradient centrifugation. Lymphoblastoid cell lines (LCL) were obtained from each patient through transformation with Epstein-Barr virus (B95-8). Cells were cultured with RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (JRH Bioscences, Inc. Lenexa, Kansas), antibiotics, and L-glutamine and maintained at 37°C in an atmosphere containing 5% CO2. FACS analysis revealed the cells to be CD3−, CD4−, CD8−, CD5−, CD19+, CD20+, HLA-class I+, -class II+. After the establishment of LCLs, to obtain a highly purified population, CD19+LCLs were sorted on a FACS Vantage SE (Becton Dickinson, San Jose, CA). The purity of each CD19+LCLs population evaluated by FACS analysis was always higher than 98%. After growth advantage of LCLs, growth ability of the LCLs from HIV patients was examined by MTT assay. In results, there is no significant difference of growth ability between LCLs derived from HIV patients and those derived from normal individuals. Furthermore, the expression of cell adhesion molecules (CD11a, CD11b, CD18, CD50, and CD54) of LCLs was observed by FACS. The results showed that the expression of CD18 on LCLs derived from HIV patients was enhanced significantly in compared to those of normal LCLs. This phenomenon seems to be consistent with development of primary central nervous system (CNS) lymphoma. Furthermore, effects of anti-retroviral drugs to the expression of the adhesion molecules are examined. Somatic hypermutation of immunoglobulin heavy chain variable gene (IGHV) has been detected over 90% of HIV-related lymphoma. The effects of HAART to hypermuation of IGHV of LCL derived from HIV patients will be addressed.

Abstract We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax or pol mRNA) using the real time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT was inhibitory of proviral DNA but not NF-kB transcriptional activity and sIL-2R on HTLV-1, however ATRA was inhibitory of NF-kB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease of sIL-2R induced by ATRA may be caused by the actions of a NF-kB inhibitor acting on the NF-kB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kB inhibitor and as a RT inhibitor.

Abstract The complete remission (CR) rate in acute leukemia (AL) has been improved due to progressive chemotherapy. However, relapse frequently occurs as minimal residual disease (MRD). To eradicate MRD, immunotherapy using autologous cytotoxic T-lymphocytes has been available. Leukemia cells (LC) of the patients were stored frozen before chemotherapy. Furthermore, after achieving CR, an EBV-transformed lymphoblastoid B-cell line (LCL) was established in each case. To induce Cytotoxic T-lymphocytes (CTL), co-culture system was carried out in two types. One is co-culture of peripheral blood mononuclear cells (PBMCs) and original leukemia cells (LC), the other is co-culture of PBMCs and the patient’s own LCL. After co-culture for 5 days, cytotoxic activity was examined by FACS with PKH-26 staining. Interestingly, cytotoxicity against not only autologous LC but the patient’s own LCL was observed in almost cases. This phenomenon is called cross-killing. Moreover, regulatory T-cells (Treg) were detected in the effecter phase determined with FACS (CD4+ CD25+) and FOXP3 mRNA by RT-PCR. Enhanced cytotoxicity was observed in inverse relation to a low proportion of Treg. These results suggested that more cytotoxicity may be induced by deletion of the Treg population. In conclusion, cell therapy as an immunotherapy should be useful tool to improve the prognosis, and if stocked LC is not available, CTL activity must be induced by co-culture with the patient’s own LCL.

＜現病歴＞ 平成20年1月に右鼡径部の皮下腫瘤に気付き増大傾向となったため、大阪労災病院皮膚科受診し生検の結果、び慢性大細胞リンパ腫(DLBCL)と診断され紹介となる。PET-CTでは両腋下・右鼡径部にFDGの集積を認めた。骨髄穿刺ではDLBCL細胞の浸潤を認めなかったことよりStage Ⅲと診断した（IPIはH-I）。4月2日からR-THP-COP療法開始し副作用なく終了し2回目より外来治療となる。6月20日に４回目の同療法後、7月4日に肝障害(GOT 131IU/l, GPT 153IU/l, T-bil 0.8mg/dl, ALP 227IU/l,)を認め、7月20日にはGOT 4625IU/l, GPT 1647IU/l, T-bil 9.3mg/dl, D-bil 7.0mg/dl, ALP 283IU/lと著増し黄疸も認められた。腹部CT・超音波所見でも肝腫大・胆嚢壁肥厚あり急性肝炎で入院となる。 ＜入院後経過＞ HBs抗原 98.97IU/ml, HBs抗体陰性、Hbe抗原陰性、HBe抗体 99％、HBc抗体 10.4S/CO、HBV-DNA定量で8.7LGE/mlと上昇しており、平成15年のHBs抗原は陰性で輸血歴もないことより急性B型肝炎の再活性化(reactivation)と診断した。なおDLBCLは寛解状態を持

悪性リンパ腫治療におけるRelative Dose Intensity（RDI）の意義と、リツキシマブ導入による変化について報告した。

移植後約3年にわたり、T細胞混合キメラが持続している状態すなわちstable mixed chimerismの症例を報告した。本症例の混合キメラは、安定した免疫寛容状態を意味しており、GVHDの標的分子と考えられるHLAおよびマイナー組織適合性抗原にも免疫寛容が成立している可能性があり、移植後の免疫再構築を考える上で、どのようなサブセットのT細胞が残存しているかも興味ある症例であった。

26歳の女性に多関節炎・耳介の炎症で発症した、比較的稀な再発性多発軟骨炎について報告した。

We here show that pharmacologic inhibition of clathrin-dependent trafficking of mutated receptor tyrosine kinases (mtRTK such as FLT3-ITS and KIT D814V mutation) with chlorpromazine (CPZ) disrupts their cellular localization and inhibits their activities. CPZ suppressed the growth of primary AML cells with mtRTK, including CD34+38- AML stem cells in vitro. In mice transplanted with primary AML cells, administration of CPZ at a clinically relevant concentration inhibited the growth of AML cells with mtRTK while it showed a marginal effect on the growth of AML cells with wild-type RTK. Also, CPZ treatment eliminated AML stem cells at the periosteal region in the bone marrow of the recipient mice. These results demonstrate that the intracellular trafficking of mtRTKs would be a good therapeutic target and CPZ would be new therapeutic agent against AML with mtRTK.

TKIs have dramatically improved clinical outcomes of the patients with CML-CP. However, even if these patients maintained deep molecular responses, discontinuation of TKI results in early relapse in most cases. Therefore, new therapeutic strategies to eradicate CML stem cells (LSCs) are required to cure CML. We previously identified CD120a, CD225, and CD284 as novel surface molecules on LSCs. Our examination of clinical samples revealed that the detection of these molecules on CD34+CD38- cells might be helpful to evaluate therapeutic effects and to monitor condition changes in CML patients. Also, because TNFα-CD120a-NF-κB signaling promoted LSC proliferation, targeting these molecules may represent an attractive therapeutic approach to eradicate CML stem cells. On the other hands, expression analysis of miRNA which control the level of Abl indicated that epigenetic state of mir-203 promoter was diverse, thereby inducing various expression levels of Bcr/Abl in individual LSCs.

FLT3-ITD and KIT D816V mutation are frequently found in AML and associated with poor prognosis. In this study, we evaluated the anti-leukemic effects of an inhibitor of membrane trafficking, chlorpromazine (CPZ). Recent studies demonstrated that these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant signals to downstream. CPZ disrupted the intracellular trafficking of RTK mutants, and significantly suppressed activities of RTK mutants and their downstream molecules. Consequently, CPZ inhibited the growth and survival of AML cells with mutant RTK in a dose-dependent manner. In xenotransplantation models, administration of CPZ significantly reduced engraftment of AML cells, and also showed the cytotoxic effect to AML stem cells, while displaying minimal toxicity to normal hematopoietic cells. These results suggest that CPZ would be a promising therapeutic drug to eradicate AML cells with an established safety profile.