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One hundred and fifty-five consecutive patients with acute myeloid leukemia (AML) seen between 1980 and 1989 were studied. Patients aged 60 years or more was retrospectively compared with those aged 59 years or less, regarding clinical features and treatment results. Clinical features at presentation did not differ significantly between the elderly and the rest of patients. Attenuated chemotherapy with dose reduction was given to the elderly patients. Remission (CR) rate, first CR duration for the elderly were 58%, eight months and three months respectively, whereas in the younger patients these were 81%, fifteen months and fourteen months respectively. The difference was significant for the CR rate and survival duration, but not for CR duration.

We retrospectively analyzed the potential of Wilms' tumor gene 1 (WT1) mRNA levels in peripheral blood for predicting the prognosis of 50 patients with AML. After achieving complete remission (CR), 34 patients (69.4%) were determined to be positive and 15 (30.6%) were negative for WT1. The relapse rate of the positive and negative patients was 73.5% and 40.0% (p#x2009;=#x2009;0.02), respectively. After consolidation therapy, only 15 patients (32.6%) were positive and 31 (67.4%) were negative for WT1. Although the relapse rate of the positive and negative patients was 80.0% and 54.8% (p#x2009;=#x2009;0.10), respectively, the rate of relapse within 1 year was 73.3% in positive patients and only 33.3% in negative patients (p#x2009;=#x2009;0.01), respectively. The disease-free survival (DFS) rate at 3 years was 20.0% for positive patients and 50.0% for negative patients (p#x2009;=#x2009;0.01). The overall survival (OS) rate at 3 years was 42.8% in positive patients and 69.8% in negative patients (p#x2009;=#x2009;0.04), respectively. WT1 mRNA levels in the peripheral blood can predict relapse after CR, and its levels after consolidation therapy are closely correlated with DFS, OS, and early relapse.

We conducted a multi-institutional randomized study to determine whether high-dose daunorubicin would be as effective as standard-dose idarubicin in remission-induction therapy for newly diagnosed adult patients younger than 65 years of age with acute myeloid leukemia. Of 1064 patients registered, 1057 were evaluable. They were randomly assigned to receive either daunorubicin (50 mg/m2 daily for 5 days) or idarubicin (12 mg/m2 daily for 3 days) in combination with 100 mg/m2 of cytarabine by continuous infusion daily for 7 days as induction therapy. Complete remission was achieved in 407 (77.5%) of 525 patients in the daunorubicin group and 416 (78.2%) of 532 in the idarubicin group (P = .79). Patients achieving complete remission received intensive postremission therapy that consisted of either 3 courses of high-dose cytarabine or 4 courses of standard-dose therapy. Overall survival rates at 5 years were 48% for the daunorubicin group and 48% for the idarubicin group (P = .54), and relapse-free survival rates at 5 years were 41% and 41% (P = .97), respectively. Thus, high-dose daunorubicin and standard-dose idarubicin were equally effective for the treatment of adult acute myeloid leukemia, achieving a high rate of complete remission and good long-term efficacy. This study is registered at http://www.umin.ac.jp/ctrj/ as C000000157.

Background Patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse; the prognostic factors and optimal treatments after relapse have not been fully established. We, therefore, retrospectively analyzed data from patients with acute myeloid leukemia who had achieved first complete remission to assess their prognosis after first relapse. Design and Methods Clinical data were collected from 70 institutions across the country on adult patients who were diagnosed with acute myeloid leukemia and who had achieved a first complete remission after one or two courses of induction chemotherapy. Results Among the 1,535 patients who were treated with chemotherapy alone, 1,015 relapsed. Half of them subsequently achieved a second complete remission. The overall survival was 30% at 3 years after relapse. Multivariate analysis showed that achievement of second complete remission, salvage allogeneic hematopoietic cell transplantation, and a relapse-free interval of 1 year or longer were independent prognostic factors. The outcome after allogeneic transplantation in second complete remission was comparable to that after transplantation in first complete remission. Patients with acute myeloid leukemia and cytogenetic risk factors other than inv(16) or t(8;21) had a significantly worse outcome when they did not undergo salvage transplantation even when they achieved second complete remission. Conclusions We found that both the achievement of second complete remission and the application of salvage transplantation were crucial for improving the prognosis of patients with acute myeloid leukemia in first relapse. Our results indicate that the optimal treatment strategy after first relapse may differ according to the cytogenetic risk.

We retrospectively analyzed the clinical outcome of dasatinib in 7 patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) resistant or intolerant to imatinib. Three patients with chronic phase CML and two patients with Ph+ALL achieved major molecular response, however, two CML patients in accelerated phase (AP)/blast crisis (BC), did not. Gradeor =3 pancytopenia was seen in four patients. Among these, two AP/BC-CML patients required interruption/or dose reduction of dasatinib. As for nonhematologic adverse events, pleural effusion was seen in one patient and cytomegalovirus (CMV) colitis was observed in two patients. No patients who had been intolerant to imatinib experienced the same nonhematologic toxicity following treatment with dasatinib. We identified three patients who developed peripheral lymphocytosis, identified as natural killer cells or cytotoxic T-cells based on their large granular lymphocyte (LGL) morphologies and immunophenotypic profiles, out of six patients receiving dasatinib therapy. All three cases that developed LGL lymphocytosis achieved optimal molecular response, two of the patients, however, had pleural effusion and CMV colitis, respectively. Dasatinib inhibits off-target kinases, which may result in unexpected drug responses.

Miconazole (400-1200 mg/day) was administered to patients with deep mycosis and suspected deep mycosis, and the efficacy evaluated. beta-Glucan was determined as the early diagnostic parameter of deep mycosis, and the relationships between the clinical efficacy of miconazole and the titers of beta-glucan were also evaluated. Forty-nine cases were evaluated, including 2 cases of deep mycosis and 47 cases of suspected deep mycosis. Most of the patients had hematological malignancies. The rate of efficacy was 100% (2/2) in deep mycosis, 66.0% (31/47) in suspected deep mycosis, and 67.3% (33/49) in total. beta-Glucan was determined in 39 cases before the administration of miconazole. The rate of beta-glucan positivity was 100% (1/1) in deep mycosis and 44.7% (17/38) in suspected deep mycosis. beta-Glucan was also determined before and after the administration of miconazole in 11 cases. The titers of beta-glucan became negative in 6 cases, decreased in 2 cases and increased in 3 cases. Thus, the beta-glucan titers became negative or decreased in 72.7% (8/11) of the cases. Efficacy of miconazole was 83.3% (5/6) in the cases in which beta-glucan became negative, 50.0% (1/2) in the cases in which the titers of beta-glucan decreased, and 33.3% (1/3) in the cases in which the titers of beta-glucan increased. Miconazole was effective in the treatment of deep mycosis, and the titers of beta-glucan correlated well with the clinical efficacy of miconazole. The determination of beta-glucan appears to be useful for the diagnosis of deep mycosis.

One hundred and twenty-two patients with multiple myeloma were classified as mature, intermediate, immature, or plasmablastic subtype according to Greipp's criteria. Contrary to Greipp's report, the survival time of plasmablastic myeloma was not significantly shorter than other subtypes, nor was the plasmablastic subtype identified as a poor prognostic factor. The survival time of mature plus intermediate myeloma was significantly longer than that of immature plus plasmablastic myeloma. Between the former and latter, significant differences were found for sex, clinical stage, thrombocytopenia, bone marrow plasmacytosis, renal insufficiency, bone destruction, and response rate to treatment. Therefore, it was suspected that the immature and plasmablastic subtypes were unfavorable prognostic factors in patients with multiple myeloma.

Immunological phenotyping of acute leukemia have provided enormous and important information for the classification and lineage determination of leukemia. Forty-nine patients with CD7(+) CD56(+) acute myeloid leukemia (AML) were analyzed. There were 17 patients of M0, which corresponded to myeloid/NK cell precursor acute leukemia, and 32 patients of AML other than M0 (9 each for M1 and M2, one for M3, 3 for M4, 4 for M5 and 6 for M7). Age distribution was similar between these two groups, but CD7(+) CD56(+) M0 showed significant male predominance than CD7(+) CD56(+) M1-M7 (M:F = 15:2 vs. 15:17, P = 0.006). The disease localization and the hematological manifestations were different, showing fewer white blood cell counts and circulating leukemic blasts, less anemia, less thrombocytopenia and more frequent extramedullary involvement in M0 group. The prognosis was poor in both groups, and there was no statistical difference. These findings suggest that extramedullary involvement of myeloid/NK cell precursor acute leukemia is not directly derived from the presence of CD7 and CD56 antigens on leukemic cells. The poor prognosis of CD7(+) CD56(+) M1-M7 suggests that this phenotype may act as a prognostic factor for AML, but this should be confirmed in further studies.

A multicenter, prospective, randomized study was conducted to compare a response-oriented individualized remission induction therapy with a standard fixed-schedule induction therapy, using idarubicin (IDR) and cytarabine (Ara-C), in adult patients with acute myeloid leukemia (AML). Newly diagnosed patients with AML of age less than 65 were randomly assigned to receive either of the two schedules. Both groups received IDR (12 mg/m2) for 3 days and Ara-C (100 mg/m2) for 7 days. In the individualized group, if the bone marrow on day 8 did not become hypocellular with less than 15% blasts, patients received additional IDR for one more day and Ara-C for 2 or 3 more days. Patients achieving complete remission (CR) received the same post-remission therapy. The CR rate was 79.4% for the individualized group (n = 209) and 81.9% for the fixed group (n = 221) (p = 0.598). At a median follow-up of 81 months, 7-year predicted overall survival was 37% for the individualized group and 39% for the fixed group (p = 0.496), and 7-year predicted event-free survival was 22% for the individualized group and 23% for the fixed group (p = 0.546). Thus, the present study could not demonstrate any advantage of a response-oriented individualized induction therapy over a fixed-schedule induction therapy in this protocol setting.

Approximately one-half of the cases of Budd-Chiari syndrome (BCS) are caused by bcr/abl negative chronic myeloproliferative disorders (CMPDs). Furthermore, a mutation in the Janus kinase protein (JAK2-V617F) is detected in half of the patients with BCS. However, whether the JAK2 mutation is the primary event leading to CMPDs and BCS is controversial. We present a report concerning a young woman who suffered from BCS prior to the onset of CMPDs. Analysis of X-chromosome inactivation patterns in this patient, using the human androgen receptor gene demonstrated monoclonal haematopoiesis in her granulocytes. In contrast, she had a low burden of a JAK2-V617F mutation positive clone among granulocyte populations. These results suggest that the JAK2-V617F mutation occurs after the onset of monoclonal haematopoiesis; thus the V617F mutation of JAK2 may not be the primary event in the induction of BCS.

The clinical significance of minimal residual disease (MRD) is uncertain in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia (Ph+ ALL) treated with imatinib-combined chemotherapy. Here we report the results of prospective MRD monitoring in 100 adult patients. Three hundred and sixty-seven follow-up bone marrow samples, collected at predefined time points during a uniform treatment protocol, were analysed for BCR-ABL1 transcripts by quantitative reverse transcription polymerase chain reaction. Ninety-seven patients (97%) achieved complete remission (CR), and the relapse-free survival (RFS) rate was 46% at 3 years. Negative MRD at the end of induction therapy was not associated with longer RFS or a lower relapse rate (P = 0.800 and P = 0.964 respectively). Twenty-nine patients showed MRD elevation during haematological CR. Of these, 10 of the 16 who had undergone allogeneic haematopoietic stem cell transplantation (HSCT) in first CR were alive without relapse at a median of 2.9 years after transplantation, whereas 12 of the 13 who had not undergone allogeneic HSCT experienced a relapse. These results demonstrate that, in Ph+ ALL patients treated with imatinib-combined chemotherapy, rapid molecular response is not associated with a favourable prognosis, and that a single observation of elevated MRD is predictive of subsequent relapse, but allogeneic HSCT can override its adverse effect.

A 54-year-old woman had an episode of sudden oral bleeding and generalized petechiae 1 week after a sore throat and diarrhea. On admission, the platelet count was 0.1 x 10(4)/microl, and the platelet-associated IgG level was elevated. Hyperplasia of megakaryocytes in a bone marrow specimen and aberrant Epstein-Barr virus (EBV) antibody patterns led to a diagnosis of EBV-associated idiopathic thrombocytopenic purpura (ITP). Prednisolone (PSL) promptly restored her platelet count; however, she developed disorientation and affective lability soon after PSL was tapered. Subsequently, she ran a high fever and developed convulsive seizures. T2-weighted MRI demonstrated a high signal area in the subcortical white matter, and no abnormal findings were found on examination of the cerebrospinal fluid. The diagnosis of acute disseminated encephalomyelitis (ADEM) was made and steroid pulse therapy was started, which resulted in remission of the symptoms without recurrence in the following months. This is the first reported case of ADEM following EBV infection during treatment for ITP. Administration of PSL for ITP might mask the presenting clinical picture of ADEM. The possibility of ADEM should be investigated in patients of ITP following viral infection who develop acute encephalopathy.

The primary objective of this study was to investigate the tolerability, efficacy and pharmacokinetic profile of gemtuzumab ozogamicin (GO) in patients with relapsed and/or refractory CD33-positive acute myeloid leukemia (AML). Patients received 2-h infusions of GO twice with an interval of approximately 14 days. Tolerability was assessed using the National Cancer Institute Common Toxicity Criteria Version 2.0. Samples for pharmacokinetics were taken on day 1 and day 8 of the first treatment cycle. The dose was increased stepwise and, in each cohort, patients were treated at the same dose. Forty patients, median age 58 years (range 28–68) were treated; 20 and 20 patients were enrolled to the phase I and II parts, respectively. In the phase I part, dose-limiting toxicities (DLTs) were hepatotoxicities, and the recommended dose was established as 9 mg/m2 given as two intravenous infusions separated by approximately 14 days. The pharmacokinetic study revealed that Cmax and AUC were equivalent to those of non-Japanese patients. In the phase II part, complete remission was observed in 5 patients, and one patient had complete remission without platelet recovery. Four of these 6 in remission and one in the phase I are long-term survivors (alive for at least 44 months). GO is safe and effective as a single agent among Japanese CD33-positive AML patients. Remission lasted longer in a subset of patients than in non-Japanese patients in earlier studies. Further studies of this agent are warranted to establish standard therapy.

Regional lymph nodes of Küttner's tumor from 3 patients showed reactive follicular hyperplasia and prominent interfollicular plasmacytosis. The patients were 71-, 57-, and 73-year-old Japanese men. The polytypic nature of plasma cells was demonstrated by immunohistochemistry. There were numerous IgG-positive plasma cells with scattered IgA-positive or IgM-positive plasma cells. IgG4-positive cells comprised 25% to 40% of IgG-positive plasma cells. Prominent polyclonal hyperimmunoglobulinemia was demonstrated on laboratory test in 2 cases examined. An elevated serum IgG4 level (16%) was also demonstrated in 1 patient. The present 3 cases indicated that regional lymph node of Küttner's tumor may show reactive follicular hyperplasia and prominent interfollicular plasmacytosis and should be differentiated from various benign and malignant lymphoproliferative disorders including systemic rheumatic disease, plasma cell type of Castleman disease, and lymph node involvement of marginal B-cell lymphoma of the mucosa-associated lymphoid tissue type showing prominent plasma cell differentiation.

To identify factors associated with relapse-free survival (RFS), 80 patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia, enrolled in a phase II study of imatinib-combined chemotherapy, were analyzed. The median follow-up of surviving patients was 26.7 months (maximum, 52.5 months). Twenty-eight out of 77 patients who had achieved CR relapsed. The probability of RFS was 50.5% at 2 years. Multivariate analysis revealed that the presence of secondary chromosome aberrations in addition to t(9;22) at diagnosis constitute an independent predictive value for RFS (p=0.027), and increase the risk of treatment failure by 2.8-fold.

Deep mycotic infection (DMI) is now being recognized in adult acute leukemia (AAL) patients with increased frequency. Fever, which is resistant to antibiotics and continue despite subsequent attainment of hematological remission, in one of the characteristic feature of DMI in AAL patients. The aspergillosis of lung and candidiasis of liver are most common. fungal infection in AAL patients. The trans-bronchial lung biopsy (TBLB) is the most useful technique for the diagnosis of lung aspergillosis. The AAL patients with lung aspergillosis must be received high dose amphotericin B (Amp-B) intravenously. Additionally, trans-bronchial administration of Amp-B is one of effective therapy in patients with lung cavity. The liver abscess in successfully diagnosed by ultrasonography or CT scan. Even if percutaneous liver aspiration or surgical excision are performed, the decision of pathogenic agents is very difficult. The administration of Amp-B must be tried through a catheter inserted into the portal vein at laparotomy in these patients.

This study was designed to determine the optimal high dose for cytosine arabinoside (ara-C) in combination with fludarabine, granulocyte colony-stimulating factor, and mitoxantrone (FLAGM) in adult patients with relapsed or refractory acute myeloid leukemia. Nine patients were enrolled at increasing dosage levels of ara-C (8, 12, and 16 g/m2 per dose level). Ara-C and fludarabine were administered once a day at level 1, once or twice a day at level 2, and twice a day at level 3. All patients had grade 4 hematologic toxicity. The most common adverse events were of grade 2 or less, with nausea and vomiting being the most common (6 events), followed by diarrhea (5 events), and rash (5 events). Of the 13 grade 3 nonhematologic toxicities reported, the 2 most common were febrile neutropenia (6 events) and disseminated intravascular coagulation (3 events). No early deaths were observed. FLAGM with high-dose ara-C was considered safe for patients, and the recommended dosage of ara-C in this study was 2 g/m2 every 12 hours for a total dose of 16 g/m2.

Disseminated intravascular coagulation (DIC) is one of the important complications to develop in patients with acute myeloid leukemia (AML). While acute promyelocytic leukemia (APL) is a strong risk factor for DIC, other clinical features have not been fully defined. We retrospectively analyzed 161 consecutive adult patients with de novo non-APL AML. DIC was diagnosed in 52 patients (32%); 28 patients at diagnosis and 24 soon after the initiation of induction chemotherapy. Leukocyte counts, C-reactive protein, and lactate dehydrogenase were significantly higher in the DIC+ group. Negative expressions of CD13, CD19, CD34, and HLA-DR were more prevalent in the DIC+ group. On multivariate logistic-regression analysis, variables that were independently associated with the development of DIC were high C-reactive protein, high leukocyte count, negative expressions of CD13 and HLA-DR, and cytogenetics with a normal karyotype or 11q23 abnormality. Although DIC is considered to be associated with serious morbidity and occasional mortality, we did not find any significant differences in the complete remission rate, overall or disease-free survival between DIC+ and DIC- groups. This study is the first to define the clinical characteristics associated with DIC in patients with non-APL AML, but exactly how and when DIC should be treated remains to be determined.

A 59-year-old man was diagnosed to have idiopathic myelofibrosis (IMF) in November 2001. In April 2004, massive ascites and esophageal varices were found. IMF was considered to be the cause of portal hypertension (ascites and esophageal varices). Since ascites tend to be intractable with diuretic drugs, a transjugular intrahepatic portosystemic shunt (TIPS) was inserted in May 2004. Before TIPS, his waist measured 98 cm. On day 74 after TIPS, his waist measured 68 cm as a result of the administration of diuretic drugs alone. He eventually died due to hepatic failure on day 168 after TIPS. The autopsy findings suggest that sinusoidal fibrosis caused portal hypertension.

MDS and related disorders, including MDS/MPN and sAML that evolved from these conditions constitute disease continuum characterized by a wide spectrum of molecular lesions which often overlap. Here, we defined general mutational spectrum and clonal architecture in a large cohort (n=718) of MDS studied by whole exome sequencing (WES) and target deep sequencing. Within this cohort 97 cases were studied at multiple time points to clarify the clinical impact of clonal dynamics on phenotype commitment or outcomes. All samples were obtained after informed consent, according to protocols approved by the respective ethics boards of the participating institutions. When mean and maximum variant allele frequency (VAF) for whole mutations were at one time-point evaluated in disease phenotypes, significantly higher averaged values suggested their larger clones in sAML and CMML compared to MDS. Clustering analysis of multiple mutational events by Pyclone software discriminated the cases with multiple mutational clones (positive heterogeneity) and those with a single expansion of MDS clone (no heterogeneity detected). Over 80% of low-risk MDS and all the sAML harbored multiple clusters of mutations. These results suggest that intra-tumor heterogeneity of MDS is most likely due to various sizes of clonal and subclonal mutations, likely impacting clinical behavior. To delineate clonal dynamics in MDS, we assessed mutational burden and their temporal changes in serially collected samples (n=97). Among these, Pyclone analysis was applied to exome sequencing at two time points (n=11 pairs). All cases showed various mutational clusters with individual expansions and declines, including initially present, newly acquired or disappearing during clinical course. Initial subclones were identified at disease presentation in 55% of cases, of which in 86% the subclones expanded to occupy whole MDS population with clonal sweep. New subclones acquired during clinical course were identified in 91%, in which 60% cases harbored clonal sweep. Disappearing clones were observed in 55% of cases. Next, we applied clustering analysis on clonal size of driver mutations evaluated at multiple time points (n=97 cases) to categorize the most frequently mutated genes into 3 subtypes. Mutational burden of PTPN11 most frequently increased and were associated with leukemic evolution (an example of type I gene). Similarly, CBL, NRAS, STAG2, RUNX1, and IDH1 were categorized into the type I genes, demonstrating increased clonal size resulting in the evolutions into high-risk phenotypes. Although JAK2 mutations were related to the stable clinical course when the mutational burden decreased, cases with highly expanded JAK2 mutations resulted in leukemic evolution (occasional evolution or expansions; type II gene). DNMT3A, SRSF2, TP53, U2AF1, and ASXL1 mutations were also categorized into such type II consequences with occasional progression. The last category (type III) included clonal/founder genes EZH2, TET2, SF3B1 and PRPF8, demonstrating random shifts of clonal size and lack of association with leukemic evolution. The proposed hierarchical categorization correlates with clinical parameters. Cases with the increasing burden of type I gene mutations showed most significant increases in myeloblasts. Overall survival measured from second sampling time points in the cases with increasing type I mutations was significantly shorter in the whole cohort (HR=2.05, 95%CI; 1.14-3.79, P=0.016) and in the cases solely with IPSS INT-1 (HR=2.37, 95%CI; 1.01-5.97, P=0.048). Subcohorts classified according to the presence or absence of increasing type I mutations did not differ with regard to the IPSS categories. In contrast, increased mutational burden of type II and III genes did not correlated with any of the clinical parameters examined, even though some gene mutations including TP53, EZH2, and U2AF1 represented poor prognostic factors at disease presentation. In conclusion, this work demonstrates that detailed understanding of clonal dynamics allows for new insights into clinical significance of somatic mutations, made possible only by serial sample sequencing at multiple time points. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in more uniform and larger cohort of MDS. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Shih:Novartis: Research Funding.

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 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 ( 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.

Adult T-cell leukemia/lymphoma (ATL) is a distinct subtype of peripheral T-cell neoplasms associated with human T-cell leukemia virus type-1 retrovirus. ATL includes a heterogeneous group of patients in terms of pathological and clinical features as well as prognosis, suggesting the presence of underlying molecular pathogenesis that could explain such heterogeneity among patients. Recently, we performed an integrated molecular analysis of a large number of ATL cases and delineated a comprehensive registry of gene mutations and other genetic/epigenetic lesions in ATL. In this study, we investigated possible correlations between these genetic/epigenetic lesions and clinical/pathological phenotypes in a large set of ATL patients, with a special focus on the impact of mutations and copy number alterations (CNAs) on clinical outcome. We analyzed a total of 361 ATL samples, including acute (n = 192), lymphoma (n = 66), chronic (n = 89), and smoldering (n = 14) subtypes, for recurrent mutations and CNAs. Each subtype had characteristic genetic/epigenetic features, suggesting a distinct molecular pathogenesis therein. Aggressive (acute and lymphoma) subtypes were characterized by a higher number of mutations and CNAs including focal amplifications/deletions, hyperploid status, and CIMP phenotype, compared with indolent (chronic and smoldering) tumors. Two mutations (TP53 and IRF4) and eight focal deletions involving 1p13 (CD58), 6p21 (HLA-B), 9p21 (CDKN2A), 10p11 (CCDC7), 13q32 (GPR183), 16q23 (WWOX), 17p13 (TP53), and 19q13 (CEBPA), were more common in aggressive ATL than in indolent ATL. In contrast, showing a similar mutational distribution to those found in large granular lymphocytic leukemia, STAT3 mutations were characteristic of the indolent diseases. Gene set enrichment analysis of RNA-seq data showed a significant enrichment of MYC pathway and genes regulating cell cycle and DNA repair in upregulated genes in aggressive ATL. Next, we assessed the impact of mutations and CNVs on prognosis among 215 ATL cases, for which survival data were available. In the entire cohort, mutation in CCR4 and IRF4, focal amplification in 9p24 (CD274) and 14q32 (BCL11B), and focal deletion in 9p21 (CDKN2A) were found to be significant predictors of poor overall survival, after adjustment for disease subtype and age. Multivariate analysis revealed that disease subtype (aggressive vs. indolent) was the most significant predictor of clinical outcome in ATL. Subsequent multivariate analysis according to disease subtype showed that within the patients with aggressive ATL, older age (≥ 70 years), CCR4 mutations, and 9p24 amplification were independently associated with an adverse outcome. Based on the number of the risk factors they owned, patients with aggressive ATL were classified into three categories showing marked difference in 3-year overall survival (OS) (P < 0.001): those with no risk factors (OS, 32%), with one risk factor (18%), and with two or more (0%). Among the patients with indolent ATL, we found IRF4 and TP53 mutations, 9p24 amplification, and deletions in 9p21 and 10p11 were independently associated with reduced survival. Interestingly, these alterations, except for 9p24 amplification, were also identified as genes more frequent in aggressive ATL. More importantly, based on these risk factors, the patients with indolent ATL can be classified into two categories showing very different prognostic profiles: patients with no risk factors (OS, 89%) and those with one or more risk factors (21%) (P < 0.001, HR = 16.8, 95% CI:5.4-52.5), suggesting that patients with indolent ATL having a genetic feature of the aggressive subtypes might genetically and biologically represent a distinct subset, which should be better managed as having an aggressive disease. Among these poor prognostic factors, 9p24 amplification and CCR4 mutation are especially interesting, because these lesions might be plausible targets of available agents, including anti-PD1/PD-L1 and anti-CCR4 antibodies. In conclusion, based on the comprehensive genetic profiling, we demonstrated that the known subtypes of ATL can be further classified into genetically and biologically distinct subsets of tumors characterized by discrete sets of genetic lesions and substantially different prognosis. Our results suggest that molecular profiling can improve the prediction of prognosis in ATL patients and better guide therapy. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Shin-bio: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Background: FMS-like tyrosine kinase 3 (FLT3) mutations in patients with acute myeloid leukemia (AML) are strongly associated with poor prognosis and leukocytosis. Approximately 15-35% of AML patients have internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence and 5-10% have a missense mutation of D835 within the tyrosine kinase domain. ASP2215 potently inhibits FLT3 with established preclinical activity against both ITD and D835 mutations. Preliminary results from a Phase 1/2, first-in-human study suggest ASP2215 is generally well tolerated and associated with antileukemic activity in FLT3 mutation-positive (FLT3+) subjects with relapsed or refractory AML (R/R AML). The safety and antileukemic activity of ASP2215 is currently being evaluated in an ongoing, Phase 1 study in Japanese subjects with relapsed or refractory (R/R) AML. Methods: This open-label, dose-escalation study, conducted in adult Japanese subjects (≥18 years) with R/R AML, consisted of a single- and a repeated-dose periods. ASP2215 (20-300 mg) was orally administered to subjects as a single dose (Cycle 0) and then once daily (Cycle 1 and subsequent cycles, each cycle was 28 days) until one of the discontinuation criteria were met. Dose escalation proceeded in a step-wise manner, based on the onset of dose-limiting toxicity (DLT) during Cycles 0 and 1, with the next dose level determined sequentially using the Bayesian Continual Reassessment Method (CRM). The study design allowed that at least one subject would receive the 20 mg dose and at least three subjects would receive higher doses (40-300 mg) of ASP2215. Primary endpoints included ASP2215 tolerability and determination of maximum tolerated dose (MTD, defined as the dose where posterior mean DLT incidence is ~33% when calculated using Bayesian CRM). Secondary endpoints included clinical response (based on Cheson's criteria) and pharmacokinetic profile. Results: As of June 24, 2015, 18 subjects with a median age of 71.5 years (range: 60-81 years) and 94% with ECOG 0-1 have received ASP2215 at dosages of 20 mg (n=1), 40 mg (n=4), 80 mg (n=4), 120 mg (n=4), and 200 mg (n=5). Three subjects were FLT3-ITD mutation positive, eight were FLT3-ITD negative, and the FLT3-ITD status for the other seven was unknown; FLT3 point mutation status for the majority (94%) of these subjects is not yet available. Only one DLT (tumor lysis syndrome), experienced at the 120 mg dose, has been reported suggesting MTD has not yet been reached. Abnormal hepatic function (n=5) and stomatitis (n=3) were the only treatment-related AEs occurring in ≥3 subjects. Grade ≥3 AEs were thrombocytopenia (n=2), febrile neutropenia (n=1), device-related infection (n=1), tumor lysis syndrome (n=1) and pneumonia (n=1). Five serious AEs deemed to be possibly or probably treatment related by the investigators (febrile neutropenia, edema, pneumonia, device-related infection, subdural hematoma) occurred in three subjects. ASP2215 plasma concentrations increased with doses from 20 mg to 200 mg with median time to maximal concentration of 2-6 hr. A total of 13 subjects had evaluable bone marrow to assess antileukemic activity; six of whom achieved at least partial response (Table). Conclusions: These preliminary findings in Japanese patients with R/R AML suggest ASP2215 is a well-tolerated FLT3 inhibitor with dose dependent increase in PK and has shown antileukemic activity in Japanese patients. ASP2215 tolerability, PK profile, and antileukemic activity are consistent with the ongoing dose-escalation/dose-expansion study being conducted in the US and Europe. Based on the positive findings, randomized Phase 3 trials have been planned. Table 1. Best Clinical Response by ASP2215 Dose and FLT3 Mutation Status Dose (mg) FLT3-ITD mutation status Best response Assessment day with first report of best response 20 Unknown CRi Day 28 40 Negative NR Day 29 Negative PR Day 29 Negative NR Day 29 80 Negative CR Day 28 Negative NR Day 11 Positive PR Day 28 Unknown NR Day 30 120 Negative NR Day 29 Positive CRi Day 57 200 Negative CRp Day 39 Positive NR Day 21 Unknown NR Day 27 CR, Complete remission; CRi, complete remission with incomplete hematologic recovery; CRp, complete remission with incomplete platelet recovery; NR, no response; PR, partial response. Disclosures Naoe: Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding. Off Label Use: ASP2215 is an investigational product for the treatment of AML. Sakura:Astellas: Other: Principal Investigator of the clinical research; study fee. Kobayashi:Gilead Sciences: Research Funding. Usuki:Kyowa Hakko Kirin: Other: personal fees, Research Funding; Shire: Research Funding; Takeda Pharmaceutical: Research Funding; Fujimoto Pharmaceutical: Research Funding; Shionogi: Other: personal fees; SymBio Pharmaceutical: Other: personal fees, Research Funding; Eisai: Research Funding; Otsuka Pharmaceutical: Research Funding; MSD: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; Novartis: Other: personal fees, Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; GlaxoSmithKline: Other: personal fees, Research Funding; Taiho Pharmaceutical: Other: personal fees, Research Funding; Fuji Film RI Pharma: Other: personal fees; Chugai Pharmaceutical: Other: personal fees; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Boehringer Ingelheim: Other: personal fees, Research Funding; Bristol-Myers Squibb: Other; Astellas: Research Funding. Iida:Astellas: Other: Principal Investigator of the clinical research; study fee. Morita:Astellas: Honoraria, Other: Personal fees. Bahceci:Astellas Pharma Global Development: Employment. Kaneko:Astellas Pharma, Inc.: Employment. Yamada:Astellas Pharma, Inc.: Employment. Takeshita:Astellas Pharma, Inc.: Employment. Miyawaki:Astellas Pharma Inc.: Consultancy, Other: personal fees; Ohtsuka Pharma Co, LTD.: Other: Safety Data Committee.

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples. A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H). The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers. Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed. RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator. Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

We evaluated the clinical course of acute myeloid leukemia (AML) and the levels of WT1 mRNA in 191 AML patients. Of 114 previously untreated patients with AML, 107 cases were positive for WT1 mRNA (93.9% : 107/114). WT1 mRNA expression-levels declined to below 50 copies/microg RNA ("negative") after remission was achieved in all 66 patients who achieved remission and 84.8% (47/54) cases were "negative" at the end of the follow-up periods. On the other hand, WT1 mRNA was expressed in 87.0% of non-remission cases (47/54), maintaining 50 copies/microg of RNA or higher ("positive"). In all 29 cases who relapsed during the follow-up observation period after achieving remission, WT1 mRNA levels declined transiently approximately around the time of achieving remission and then rose again when the disease relapsed. Moreover, we determined the time of elevation of WT1 mRNA in 29 relapsed cases. In 79.3% of relapsed cases (23/29), WT1 mRNA levels rose above 200 copies/microg RNA, 43 days (median) before the diagnosis of "relapse". Given the percent of the correct diagnosis, WT1 mRNA at 200 copies/microg RNA appeared to be a reasonable cut-off level for early detection of AML-relapse. The WT1 mRNA level reflected the clinical condition. Taken together, these findings indicate that WT1 mRNA levels allow us to detect the presence of so-called "minimal residual disease" (leukemic cells) that cannot be detected by morphological examination. Besides these promising data, this kit is suitable for routine monitoring of AML because this kit utilizes peripheral blood as a test specimen, reducing the patient's burden at the time of collection of clinical samples as compared with bone marrow aspirate.

A 38-year-old man was diagnosed as having acute myelogenous leukemia in December 2001. He achieved a complete remission after undergoing three courses of induction chemotherapy. As the patient's prognosis was considered to be poor, he was then treated with a bone marrow transplant from his HLA 1 antigen mismatched sister in May 2002. Grade 2 skin graft-versus-host-disease (GVHD) developed on day 14 but reduced with methylprednisolone (mPSL). Prednisolone (PSL) was discontinued on day 104, and the patient was discharged on day 112. There was no evidence of clinical chronic GVHD, the serum creatinine level remained high, and cyclosporine (CsA) was gradually tapered off and discontinued on day 165. However chronic GVHD of the skin appeared on day 179, and CsA (100 mg/day) was restarted. On day 186 he was admitted to our hospital complaining of fever. A CT scan of the chest demonstrated bilateral interstitial infiltrates, which were considered as lung GVHD lesions and PSL was started, following which chronic GVHD of the skin and liver improved. The lung GVHD worsened, however, subcutaneous and mediastinal emphysema developed and the patient died on day 206. Interstitial pneumonia had progressively worsened as the manifestation of the chronic lung GVHD. We concluded that this clinical course was rare.

A 47-year-old male had symptoms of coughing and with fever and was admitted to our hospital where tests revealed he had anemia and thrombocytopenia. Following the results obtained from a bone marrow aspiration, he was diagnosed as having acute myeloid leukemia (AML). A chest radiograph and a CT scan demonstrated nodular shadows and pleural exudate in both lungs. We suspected atypical pneumonia, or fungal pneumonia, and he was subsequently given antibiotics and antifungal agents, which were, however, ineffective. On the third day after admission, he was put on mechanical ventilation, and a bronchoalveolar lavage examination revealed no germs. His respiration, however, progressively worsened and he died on the twelfth day after admission. Although the autopsy findings revealed no infectious lesions in his lungs, a PAS-positive intra-alveolar eosinophilic material, which was positive for surfactant apoprotein A staining, was present. As a result of the autopsy findings, a diagnosis of secondary pulmonary alveolar proteinosis (PAP) associated with AML was made. Among the reported cases of PAP in hematological malignancy, there has not been one case detected at the time AML was diagnosed. If there is evidence of an unknown cause of lung infiltration when AML is diagnosed, attention should be paid to the possibility of the presence of PAP.

Purpose A novel therapeutic approach is urgently needed for BCR-ABL–positive acute lymphoblastic leukemia (ALL). In this study, we assessed the efficacy and feasibility of chemotherapy combined with imatinib. Patients and Methods A phase II study of imatinib-combined chemotherapy was conducted for newly diagnosed BCR-ABL–positive ALL in adults. Eighty patients were entered into the trial between September 2002 and January 2005. Results Remission induction therapy resulted in complete remission (CR) in 77 patients (96.2%), resistant disease in one patient, and early death in two patients, as well as polymerase chain reaction negativity of bone marrow in 71.3%. The profile and incidence of severe toxicity were not different from those associated with our historic chemotherapy-alone regimen. Relapse occurred in 20 patients after median CR duration of 5.2 months. Allogeneic hematopoietic stem-cell transplantation (HSCT) was performed for 49 patients, 39 of whom underwent transplantation during their first CR. The 1-year event-free and overall survival (OS) rates were estimated to be 60.0%, and 76.1%, respectively, which were significantly better than those for our historic controls treated with chemotherapy alone (P < .0001 for both). Among the current trial patients, the probability for OS at 1 year was 73.3% for those who underwent allogeneic HSCT, and 84.8% for those who did not. Conclusion Our results demonstrated that imatinib-combined regimen is effective and feasible for newly diagnosed BCR-ABL–positive ALL. Despite a relatively short period of observation, a major potential of this treatment is recognized. Longer follow-up is required to determine its overall effect on survival.

Several lymphoproliferative disorders may be interpreted as multicentric Castleman's disease (MCD) clinicopathologically. These include HIV infection, autoimmune-disease-associated lymphadenopathy, idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia, "idiopathic MCD", POEMS syndrome (polyneuropathy, anasarca, organomegaly, endocrinopathy, M-proteins, and skin lesions), and non-Hodgkin's lymphomas. Among these, idiopathic MCD appears to be relatively rare. We report on the clinicopathologic and immunohistologic findings of five cases of idiopathic MCD and discuss the problems regarding their differential diagnosis. Some of the characteristic clinical findings of POEMS syndrome, including hepatosplenomegaly, skin change, endocrine abnormalities and anasarca, were present in all five cases. However, during the course of disease, minimal diagnostic criteria for POEMS syndrome, i.e., monoclonal plasma cell proliferation and sensory motor neuropathys, were absent in all five cases. The serum interleukin-6 level and the vascular endothelial growth factor level were found to be elevated in two of the cases examined. Various autoantibodies were detected in three cases. However, none of them fulfilled the diagnostic criteria for any of the definite autoimmune-disease. Histologically, three lesions exhibited a mixed type of Castleman's disease, and two exhibited the hyaline-vascular type. The majority of the germinal centers were of the hyaline-vascular or epithelioid germinal type, with a few hyperplastic germinal centers. The interfollicular area was characterized by prominent vascularity. Moderate to large sheets of plasma cells were observed in three mixed type cases. The polytypic nature of B-lymphocytes was demonstrated by immunohistochemistry and polymerase chain reaction. Immunohistochemical study demonstrated that the majority of germinal centers exhibited a tight/concentric pattern of FDC network. Few CD57-positive T-cells were observed in the hyaline-vascular and epithelioid follicles. The lack of CD57-positive T-cells appears to be related to the formation of abnormal germinal centers in the MCD.

Little information is available regarding the effect of the conditioning regimen on the outcome of bone marrow transplantation (BMT) from an unrelated donor. Therefore, we retrospectively compared the outcome after a cyclophosphamide/total body irradiation (Cy-TBI) regimen, an intensified Cy-TBI regimen (Cy-TBI+), a busulfan and cyclophosphamide (Bu-Cy) regimen, and a Bu-Cy regimen with total lymphoid irradiation (Bu-Cy-TLI). Clinical data of 1875 adult patients who underwent unmanipulated unrelated BMT for leukemia or myelodysplastic syndrome by using 1 of the 4 regimens between 1993 and 2002 were extracted from the database of the Japan Marrow Donor Program. The effect of the conditioning regimen was adjusted for other independent significant factors by multivariate analyses. The Cy-TBI regimen was significantly better than the Bu-Cy regimen with regard to the incidence of engraftment failure (odds ratio, 2.49; P = .046) and overall survival (relative risk [RR], 1.31; P = .050). The Bu-Cy-TLI regimen decreased relapse (RR, 0.13; P = .039) but increased nonrelapse mortality (RR, 1.89; P = .0061). The Cy-TBI+ regimen resulted in increased nonrelapse mortality (RR, 1.48; P = .0003) and inferior survival (RR, 1.45; P < .0001). The results of this retrospective study suggested that the Cy-TBI regimen was superior to other regimens in unrelated BMT.

We conducted a retrospective survey of multiple myeloma (MM) patients who underwent reduced-intensity conditioning allogeneic stem cell transplantation (RIST) at 11 hospitals participating in the Japan Myeloma Study Group. Forty-five patients (median age, 53 years) were included in this study. The conditioning regimen consisted of a fludarabine-based regimen in 24 patients and a regimen based on total body irradiation (1–2 Gy) in 18 patients. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and tacrolimus in 28 and 17 patients, respectively. All patients showed myeloid engraftment. Complete chimerism was obtained in 42 patients. Grade II to IV acute GVHD developed in 28 (65%) of 43 patients evaluated, and chronic GVHD developed in 31 (76%) of 41 patients. Early death before day 100 was observed in 4 patients (8.8%). A complete response (CR) was obtained in 12 patients. The factors affecting overall survival were severe acute GVHD and the response after RIST. To date, 18 patients are alive, with 9 patients remaining in CR at a median follow-up of 25 months. The overall and progression-free survival rates at 3 years were 38.5% and 18.8%, respectively. These observations suggest that RIST is feasible with reliable donor engraftment and relatively low transplantation-related mortality in Japanese MM patients.

The purpose of this study was to evaluate patients who contracted the varicella zoster virus infection (VZV) following their allogeneic stem cell transplants. We retrospectively reviewed the incidence and the timing of varicella zoster virus (VZV) infections, including the clinical course, complications, and associated clinical risk factors. Between January 1998 and April 2003, a total of 71 patients received allogeneic stem cell transplants in our hospital. For prophylaxis of the herpes virus infection, all patients were given a daily oral 1000 mg dose of acyclovir from day -7 to day +35. Among the 71 patients, 28 of them (39.4%) developed VZV infection between day 77 and day 980 (median 182 days) following their allogeneic stem cell transplants. In 21 of these infected patients (75%) the occurrence was within the first 300 days after the transplant. Twenty-two patients (78.5%) were under treatment with immunosuppressive agents. Twenty-six patients developed only one episode of the VZV infection after their transplants, but two other patients developed two episodes. Twenty one patients (75%) stricken with the VZV infections had cutaneous reactivation infections of a single dermatome, and in one patient two dermatomes were affected. Five patients (17.8%) developed disseminated cutaneous zoster, and one patient (3.6%) developed a visceral infection. Treatment with acyclovir (oral or drip infusion) was successful in 25 patients. Two patients improved with vidarabine treatment, however the patient with the visceral infection died despite the use of acyclovir. The incidence of visceral infection was low, but the one case was fatal.

A 51-year-old female with acute myeloid leukemia was admitted to our hospital in December 2001. Though she had undergone two courses of induction chemotherapy (idarubicin hydrochloride + cytarabine), she failed to achieve a complete remission. In April 2002, while in non-complete remission, she subsequently underwent total body irradiation (TBI) and treatment with cyclophosphamide (CY) and etoposide (VP-16) before receiving an allogeneic peripheral blood stem cell transplant from her HLA-identical brother. For graft-versus-host disease (GVHD) prophylaxis, she was given tacrolimus and methotrexate. The infused CD34 positive cells provided 8.1 x 10(6) cells per kg. Engraftment was obtained on post-transplant day 14, and there was no evidence of clinical acute GVHD. The use of tacrolimus was discontinued on post-transplant day 60. As there was no occurrence of clinical acute GVHD, the patient received a donor lymphocyte infusion (CD3 cells 0.57 X 10(7) cells per kg) on post-transplant day 105. On day 132, however, she complained of coughing and fever, and on day 135, she was admitted to our hospital again for dyspnea. A CT scan demonstrated ground-glass opacity in the right pulmonary lobe. After considering her clinical course, symptoms, blood gas, CT scans, etc., we suspected interstitial pneumonia. The dyspnea progressively worsened, however, and despite the use of mechanical ventilation from day 143, the patient died on day 149. From the day she was admitted till the day she was intubated, she was unable to produce sputum. Autopsy findings revealed yellow-white tracheal pseudomembranes, as well as Aspergillus hyphae in the trachea, bronchus, and bilateral lungs. These findings are characteristic of Aspergillus tracheobronchitis. The clinical course of Aspergillus tracheobronchitis in allogeneic stem cell transplant recipients is, however, different from that of the usual invasive Aspergillus infection, and although Aspergillus tracheobronchitis is a very rare disease, attention should be paid to the possibility of its occurrence.

The association of ethnicity with the incidence of graft-versus-host disease (GVHD) and other clinical outcomes after transplantation is controversial. We compared the results of HLA-identical sibling bone marrow transplantations for leukemia, performed between 1990 and 1999, among different ethnic populations, including 562 Japanese, 829 white Americans, 71 African Americans, 195 Scandinavians, and 95 Irish. Results for adults and children were analyzed separately. Multivariate analyses of adult patients showed that white Americans, African Americans, and Irish cohorts were at significantly higher risk for acute GVHD than Japanese or Scandinavian cohorts (relative risk [RR] = 1.77, P < .001; RR = 1.84, P < .006; RR = 2.22, P < .001, respectively). White Americans, African Americans, and Irish, but not Scandinavians, were at significantly higher risk for early (within 3 months of transplantation) transplant-related mortality (TRM) compared with Japanese (RR = 2.99, P < .001; RR = 5.88, P < .001; RR = 2.66, P < .009, respectively). No differences in the risk for chronic GVHD, relapse, and overall survival were noted. In the pediatric cohort (limited to Japanese and white Americans), white Americans were at significantly higher risk for acute (RR = 1.93; P = .04) and chronic (RR = 3.16; P = .002) GVHD. No differences in other clinical outcomes were noted. Our findings suggest that ethnicity may influence the risk for GVHD, though overall survival rates after transplantation remain similar.

A-36-year-old male diagnosed with acute myelogenous leukemia (AML, M2) failed to achieve a complete remission after having undergone two courses of induction chemotherapy (idarubicin hydrochloride + cytarabine, high-dose cytarabine), and moreover, his cerebrospinal fluid (CSF) then revealed abnormal blasts. Dosages of 30 mg of methotrexate, 80 mg of cytarabine, and 40 mg of prednisolone acetate were given via lumbar puncture, and following this treatment, the leukemic cells were no longer detectable in his CSF. As a prophylaxis for meningeal leukemia relapse, he continued to receive intrathecal chemotherapy (methotrexate, prednisolone acetate) twice a week, and later he underwent reinduction therapy (second high-dose cytarabine). He displayed mental disorientation on the thirty-seventh day after he had been given a high-dose of cytarabine (araC). Up to the thirty-seventh day, he had also been given a total of 135 mg of intrathecal methotrexate. On the thirty-ninth day he fell into a coma, and his brain computed tomography (CT) demonstrated low density areas in the white matter, in addition to edema. These findings were compatible with leukoencephalopathy. With the administration of glycerin and methylprednisolone, his condition, however, began to reverse, and by the fifty-third day his brain edema had subsided and his mental condition had improved. On the ninetieth day he began to walk without assistance, and after the third course of re-induction chemotherapy, he achieved complete remission. He is currently doing well.

Although FLT3 mutations are essentially found in myeloid-lineage leukemia cells, a high level of FLT3 expression was recently observed in MLL gene-rearranged acute lymphoblastic leukemia without FLT3 mutations. Here, we analyzed the biologic and clinical significance of the FLT3 transcript level in comparison with several gene alterations in 181 de novo acute myeloid leukemia (AML) cases. The mean expression level in AML was higher than that in normal mononuclear cells, whereas the range varied widely. A high level of FLT3 is related to internal tandem duplication of the FLT3 gene (FLT3/ITD), the mutations within the activation loop of FLT3 (FLT3/D835Mt), and tandem duplication of the MLL gene (MLL-TD) but not to p53 or N-RAS gene mutations. Furthermore, a high expression level in AML cases with FLT3 mutations was not related to MLL-TD. Overexpressed FLT3 revealed autophosphorylation and had the same sensitivity to the FLT3 inhibitor as FLT3/ITD. Overexpression of FLT3 (more than 200,000 copies/microgRNA) was an unfavorable prognostic factor for overall survival in 91 AML cases without FLT3/ITD. These results indicated that FLT3 overexpression may distinguish a novel disease entity in AML without FLT3 mutations and serve as a therapeutic target for FLT3 inhibitors.

By retrospectively analyzing 288 patients with de novo myelodysplastic syndrome (MDS), we sought to determine the prevalence and clinical characteristics of bone marrow eosinophilia and basophilia that were detected at presentation. Bone marrow eosinophilia and basophilia were defined as a differential count of each cell type exceeding 5.0% and 1.0%, respectively. Of 288 patients with MDS, 36 (12.5%) fulfilled this criterion for bone marrow eosinophilia (MDS-Eos); 34 patients (11.8%) showed basophilia (MDS-Bas), and 11 (3.8%) satisfied both criteria (MDS-EosBas). The remaining 229 patients had neither eosinophilia nor basophilia in their bone marrow (MDS−/−) at presentation. Cytogenetic analysis was carried out on unstimulated bone marrow cells obtained from 264 patients. When the cytogenetic categorization of the IPSS (International Prognostic Scoring System) for MDS was applied, significantly higher numbers of MDS-Eos and MDS-Bas patients had chromosomal abnormalities carrying intermediate or poor prognosis, compared with the MDS−/− patients. Specific chromosomal abnormalities and complex karyotypes were associated with MDS-Eos and/or MDS-Bas. In accordance with these results, the overall survival rate was significantly lower, and the evolution to acute myelogenous leukemia (AML) occurred more frequently in the MDS-Eos and MDS-Bas than in the MDS−/− patients. Multivariate analysis demonstrated that bone marrow basophilia was an independent risk factor for evolution to AML. Our study indicates that bone marrow eosinophilia and basophilia in patients with MDS predict a poorer prognosis.

Acute myeloid leukaemia (AML) with trilineage dysplasia (AML/TLD) is de novo AML recognized by the morphological dysplasia of three mature cell lines in the presence of leukaemic blasts. We studied the karyotypes of AML/TLD of patients with de novo AML, except for those with the French-American-British classification M3, who were enrolled onto the Japan Adult Leukaemia Study Group (JALSG)-AML 92 trial. Morphological and cytogenetic analyses were performed in 559 patients and TLD phenotype was found in 155 patients (27.7%). The 511 patients with informative morphological and cytogenetic data were classified into three groups according to karyotype: favourable, intermediate and adverse risk groups (92, 375 and 44 patients respectively). Normal karyotype was the most frequent as a total, and among both the non-TLD and TLD patients (164 patients 45.3% and 78 patients 52.7% respectively). All but one patient with AML/TLD was classified into the intermediate or adverse cytogenetic risk group. TLD phenotype was associated with lower remission rate and shorter overall survival but it did not influence disease-free survival. Although we did not find any specific cytogenetic abnormalities for AML/TLD, the rarity of favourable karyotypes among TLD patients indicates the biological difference between AML/TLD and AML/non-TLD.

Because of progress in supportive therapies, the upper limit of age for conventional allogenic stem cell transplantation (allo-SCT) is rising. We retrospectively evaluated the impact of age on transplant outcomes in patients older than 50 years of age who underwent conventional allo-SCT in 8 institutions in Japan. The median age was 52-years old (range 50 to 65). The underlying diseases included severe aplastic anemia (n = 3), acute myelogenous leukemia (n = 20), acute lymphoblastic leukemia (n = 10), chronic myelogenous leukemia (n = 11), myelodysplastic syndrome (n = 18), and non-Hodgkin lymphoma (n = 3). Forty two patients (67%) with hematological malignancies received allo-SCT in an advanced disease stage at the time of transplant. The two-year overall survival and disease-free survival rate were 50.1% and 43.6%, respectively. In patients with hematological malignancies, the two-year probability rates of survival were 54.3% with standard risk patients, and 45.9% with poor risk patients. The severity of acute GVHD, the kind of grafts, and age (or = 55) were related to poor prognosis. Our data suggest that prophylaxis of acute GVHD and selection of the graft is more important for older patients, and that patients less than 55-years old can be candidates for conventional allo-SCT.

In an attempt to determine the pathological significance of a long arm deletion of chromosome 13 (13q-) in bone marrow failure syndrome, we reviewed the clinical records of nine patients who were initially diagnosed with aplastic anaemia due to bone marrow hypoplasia without dysplasia. Six patients responded to immunosuppressive therapy and the other three improved with steroids. None of the patients developed acute leukaemia (follow up: 54-129 months) and the estimated 5-year survival was 78%. These findings indicate that pancytopenia with 13q- represents bone marrow failure of a benign nature, similar to aplastic anaemia without karyotypic abnormalities, rather than preleukaemia.

Clinical features were investigated in 114 patients with de novo myelodysplastic syndrome (MDS) diagnosed over the past 10 years, and eight cases (7%) were complicated with eosinophilia in the bone marrow. Two patients had refractory anaemia (RA), five had RA with excess of blasts (RAEB), and one had RAEB in transformation. Their bone marrow cells exhibited tri-lineage dysplasia and morphological abnormalities in eosinophils. Cytogenetics revealed major karyotype abnormalities (MAKA) in five patients. Survival durations were significantly shorter than those of other MDS patients. Our study suggests that marrow eosinophilia in MDS is strongly related to the coexistence of MAKA.

Background: A combination of all-trans retinoic acid (ATRA) and chemotherapy (CT) has dramatically improved the prognosis of acute promyelocytic leukemia (APL). Nevertheless, considerable number of patients are either refractory to the treatment or relapse after an initial complete remission (CR). Although prognostic factors for APL have been studied, the influence of chromosomal variations in addition to t(15;17) remains controversial. One of the reasons is due to the numbers of cases studied were relatively small (47 to 513 cases). Here, we analyzed clinical features and outcomes of 775 APL caseswith or without additional chromosome abnormalities (ACAs) who were treated with ATRA and CT in the JALSG-APL studies including a large number of cases analyzed for karyotype. Methods: 1,024 cases aged between 15 and 70 yrs with newly diagnosed APL were enrolled in the JALSG APL92, 95, 97 and 204 studies and 775 patients were assessable for karyopypes. All protocols included induction therapy with ATRA and CT, following several courses of post-remission chemotherapy including anthracyclines. Arsenic trioxide (ATO) was not included. Clinical and biological characteristics such as age, gender, initial leukocyte count, platelet count, number of APL cells, DIC score, lack of Auer-rod and incidence of variant type were analyzed in relation to chromosomal abnormalities in 766 cases. CR rate, relapse rate (RR), overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were assessed and compared between patients with or without ACAs. Each variation of ACAs was also analyzed with clinical and biological features. This work was supported in part by the National Cancer Center Research and Development Fund (26-A-24), Grants-in-Aid from the Cancer Research from the Japanese Ministry of Health, Labor and Welfare (#23-004 and #25100501). These studies were approved by our IRB. Results: ACAswere noted in 235 patients (30%). Sanz score and the initial leukocyte count were significantly lower in patients with ACAs (p=0.027 and p=0.027, respectively). No other clinical or biological differences were found between patients with and without ACAs. The subgroups of ACAs were shown in Figure 1. Trisomy 8 was found in 76 cases (32%). Other ACAs were found involving chromosome 15 in 37 cases (16%), both chromosomes 15 and 17 in 31 cases (13%), chromosome 7 in 19 cases (8%), chromosome 9 in 12 cases (5%), chromosome 6 in 8 cases (3%), chromosome 21 in 7 cases (3%) and alternative ACAs in 43 cases (18%). A low initial leukocyte count ( Conclusions: The present study is the largest to date to focus on the influence of ACAs on clinical outcomes of patients with APL treated with ATRA and CT. The analysis revealed exact variation and frequency of ACAs. We found that patients with ACAs were associated with the lower initial leukocyte count and the lower survival outcomes in elderly patents, suggesting a possible link to age and post-remission chemotherapy. Some promising agents, such as ATO, tamibarotene and gemtuzumab ozogamicin might change the prognostic factors, including ACAs. Careful chromosomal analyses, especially ACAs related to chromosome 15 and/or 17, need to be analyzed by molecular methods and performed in future prospective studies with alarge number of cases. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Kiyoi: Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Kobayashi:Ohtsuka: Research Funding; Behringer: Research Funding; Simic: Research Funding. Asou:Chugai Pharmaceutical Co., Ltd.: Research Funding. Miyazaki:Nippon-Shinyaku: Honoraria.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner. In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL. RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants. In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. Disclosures No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in > 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.