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The chromosomal abnormalities associated with follicular lymphoma (FL) prognosis are not fully elucidated. Here, we evaluated the pattern of chromosomal abnormalities in FL, and clarified the correlations between the cytogenetic features and clinical outcome. Cytogenetic analysis was performed using standard methods of Giemsa-banding at diagnosis for 201 FL patients admitted to our hospitals between 2001 and 2013. The identified chromosomal abnormalities were: t(14;18)(q32;q21) (59·2%), +X (17·9%), del(6)(q)/-6 (16·9%), +7 (14·4%), abnormality of 1q12-21/1q (12·9%), del(13)(q)/-13 (11·9%), abnormality of 3q27 (10·4%), abnormality of 10q22-24 (10·0%), +12/dup(12)(q) (10·0%), abnormality of 1p21-22/1p (9·0%), +18 (9·0%), del(17)(p)/-17 (5·0%), and a complex karyotype (54·7%). Patients with trisomy 21 had a significantly shorter progression-free survival (P = 0·00171) and overall survival (OS) (P < 0·001) than those without trisomy 21; additionally, patients with trisomy 21 in the rituximab-treated cohort also had a significantly shorter OS (P = 0·000428). Multivariate analysis identified trisomy 21 as an independent risk factor in our cohorts with or without t(14;18) (P = 0·015). In conclusion, the presence of trisomy 21 was an independent risk factor for in FL. Chromosomal analysis of FL patients at diagnosis can provide useful information about their expected survival.

Congenital combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in lectin mannose-binding type 1 (LMAN1) or multiple coagulation factor deficiency 2 (MCFD2) encoding chaperone molecules involved in the intracellular transport of FV and FVIII. Here, we report a case of F5F8D in an elderly patient diagnosed with hematoma after a right thigh injury. A 71-year-old male had a history of abnormal bleeding after tooth extraction and cholecystectomy. The patient injured his right thigh with a kitchen knife; he was urgently hospitalized to a referral hospital 8 days later due to the occurrence of hematoma at the same site. Owing to prolongation of the coagulation time (PT 16.1 s, 1.72; APTT, 66.1 s), he received hemostatic treatment with fresh-frozen plasma. He was then referred to our hospital for examination of PT and APTT prolongation. FV and FVIII activities were moderately decreased to about 15%, and no inhibitor was detected. Whole-exome sequencing identified a previously reported homozygous nonsense mutation in LMAN1, revealing F5F8D in the proband. In this case, FFP infusion alone was not sufficient for increasing coagulation factor activities. Definitive diagnosis of F5F8D provides him with the treatment option with FVIII concentrates.

Recent studies have revealed the clinical and biological features of stage I follicular lymphoma (FL), but information about patients with stage I FL who underwent total resection after tissue biopsy is limited. Among 305 FL patients diagnosed between 2001 and 2013, clinical stage I disease was observed in 36 patients. Of these, 18 patients underwent total resection after diagnostic tissue biopsy. We used (18)F-fluorodeoxyglucose positron emission CT for staging assessment in 13 of 18 patients (72.2%). The median age was 56.5 years. Six patients (33.3%) were male. The soluble interleukin-2 receptor alpha concentration was significantly lower than in patients with residual disease. Among these 18 patients, 7 patients (38.9%) were treated with a “watch-and-wait” (WW) policy, 7 (38.9%) were treated with involved-field irradiation, and 4 (22.2%) received systemic chemotherapy. Patients with resected disease were treated with significantly different strategies from those with residual disease (p = 0.0026). Five patients experienced relapse during follow-up (median follow-up: 48.2 months). All relapses were distant from the primary site, irrespective of treatment strategy. Among all stage I patients, disease resection was not a significant factor for survival (p = 0.9294). Collectively, the choice of treatment strategy was significantly influenced by patient status. Resection status was not significantly associated with survival after several treatment strategies.

90Y-ibritumomab tiuxetan (90Y-IT) is widely used, but the factors responsible for its optimal treatment effects are unknown. We enrolled 34 patients with relapsed indolent lymphoma treated with 90Y-IT monotherapy at Gunma University Hospital between 2003 and 2014 in the present study. Clinical data including computed tomography and 18-Fluoro-deoxyglucose positron emission tomography were retrospectively analyzed. The overall response rate and complete response rate were 91% and 82%, respectively. The median progression-free survival (PFS) and overall survival were 32 months and not reached, respectively. In univariate analysis, tumor long-axis diameter ≤ 2.5 cm, maximum standardized uptake value (SUVmax) ≤ 6.5, localized disease, normal levels of serum soluble interleukin-2 receptor, and the number of involved nodal sites ≤ 3 immediately prior to 90Y-IT were associated with median PFS greater than 6 years. However, in multivariate analysis, only tumor long-axis diameter ≤ 2.5 cm and SUVmax ≤ 6.5 affected PFS [hazard ratio (HR) 0.130, P = 0.0021 and HR 0.283, P = 0.0311, respectively]. Patients with only one prior regimen needed less granulocyte colony-stimulating factor and platelet transfusion. Thus, 90Y-IT treatment should be considered for patients with indolent lymphoma in first relapse who have tumor long-axis diameter ≤ 2.5 cm and SUVmax ≤ 6.5.

Background:Although the addition of rituximab to CHOP regimen improved prognosis in DLBCL patients with more than 80% of a long-term survival rate, CNS relapse did not decrease and 5 to 10% of patients experienced CNS relapse after rituximab-containing chemotherapy. Risk factors for CNS relapse after standard chemotherapy have been aggressively investigated, and a risk model, CNS-international prognostic index (IPI), has been widely used. However, risk factors for CNS relapse after high-dose chemotherapy following ASCT, which is recognized as an important treatment option for high-risk DLBCL patients, have not been elucidated. So, we performed this retrospective analysis to address this unsolved issue. Patients and methods:This study analyzed 87 adult patients who underwent ASCT against chemo-sensitive DLBCL including intravascular large B-cell lymphoma (IVLBCL) between 1997 and 2015 at the four institutions in Gunma, Japan. There was no restriction on the type of conditioning regimens. CNS-directed regimens were defined as chemotherapy or conditioning regimens containing high dose cytarabin, high dose methotrexate, busulfan, ranimustine, or total body irradiation. Only the first relapse after ASCT was assessed in this study. Fisher's exact test was used to compare binary variables. Cumulative incidences (CIs) of CNS relapse were compared using the stratified Gray test, considering relapse without CNS lesions and death without the event as a competing risk. The Fine-Gray proportional hazard model was used for multivariate analysis of risk factors for CNS relapse. The potential risk factors evaluated in this analysis were age at transplant, gender, clinical stage, IPI (high-intermediate/high or not), and CNS-IPI (high or not) at diagnosis, CD5 positivity, CNS involvement prior to ASCT, CNS-directed chemotherapy prior to ASCT, and CNS-directed conditioning regimen. Values of p < 0.05 were considered significant. Results:Of the 87 patients assessed in this study, 48 were male and 39 were female, and the median age was 57 years (range: 23 to 66 years). CD5 was expressed in 19% of the patients, and 10% were diagnosed as IVLBCL. CNS-IPI at diagnosis was high in 53%, and rituximab and CNS-directed chemotherapy was administered prior to ASCT in 83% and 16%, respectively. CNS involvement was observed prior to ASCT and at the time of ASCT in 9% and 0%, respectively. Disease status at the time of transplant was first complete remission (CR) in 47%, advanced CR in 23%, and partial remission in 30%. CNS-directed conditioning regimens were used in 38%. With a median observation time of 21.9 months, seven patients experienced CNS relapse as the first relapse after ASCT. The 3-year CI of CNS relapse was 7.3% with 5.7 months of median duration from ASCT (range: 2.7 to 69.0 months). In univariate analysis, only CD5 positivity was identified as a significant risk factor for CNS relapse (3-year CIs in patients with and without CD5 expression: 27.0% vs. 2.2%, respectively; p < 0.01). In multivariate analysis, CD5 positivity, CNS-IPI at diagnosis, CNS-directed chemotherapy prior to ASCT, and CNS-directed conditioning regimen were evaluated, and only CD5 positivity was identified as an independent risk factor for CNS relapse (relative risk=20.1; p < 0.01). Of the seven patients with CNS relapse after ASCT, four expressed CD5 and five received CNS-directed chemotherapy prior to ASCT and/or conditioning regimens. All seven patients died from DLBCL within two years after CNS relapse. Conclusion:These results suggested that CNS relapse occurred in DLBCL patients even after ASCT with similar incidence to that after chemotherapy. Although prophylactic strategies for CNS relapse should be investigated especially in patients with CD5-positive patients, use of CNS-directed chemotherapy prior to ASCT and/or conditioning regimens did not affect the CIs of CNS relapse. A future study with a larger cohort is warranted to develop a risk model for CNS relapse after ASCT in DLBCL patients. Disclosures Handa: Ono: Research Funding.

We evaluated the prognostic significance of the serum level of the soluble form of interleukin-2 receptorα (sIL-2Rα) and investigated its association with CD25 expression on tumor cells in diffuse large B-cell lymphoma (DLBCL). Three hundred and thirty-eight adult patients with newly diagnosed DLBCL were eligible for this retrospective study. 32.2% of patients were treated with CHOP-like regimen and 67.8% with R-CHOP-like regimen. CD25 expression on the surface of tumor cells was evaluated in 143 cases and its relationship with sIL-2Rα level was also investigated. Both overall survival (OS) and progression-free survival (PFS) were poorer in patients with higher sIL-2Rα, in both R-CHOP and CHOP groups. sIL-2Rα > 1,000 U/mL and performance status (PS) ≥ 2 were independently associated with poorer OS, and sIL-2Rα > 1,000 U/mL, age > 60 years, and ≥ 2 extranodal sites were independently associated with poorer PFS in the R-CHOP group. The sIL-2Rα level was higher in the CD25-positive group than in the CD25-negative group in stage 3 or 4 disease (p = 0.010). Multiple linear regression analysis showed CD25 expression to be independently correlated with sIL-2Rα levels. High sIL-2Rα is an important risk factor for survival in DLBCL treated with not only CHOP-like, but also R-CHOP-like regimens, regardless of the tumor's expression of CD25.

A 64-year-old woman,with more than a 20 year history of polycythemia vera(PV),developed portal hypertension,myelofibrosis and extramedullary hematopoiesis accompanied by massive ascites. Portal hypertension resulted not only from infiltration of the liver sinusoids by hematopoietic cells but also from nodular regenerative hyperplasia of the liver. Wright-stained smears of ascites samples consisted of mesothelial cells and macrophages. However,cultures of mononuclear cells from the ascites showed the presence of hematopoietic progenitor cells including megakaryocyte colony formation and burst forming units. The JAK2-V617F mutation was positive in granulocytes. Contrary to other reports,radiation therapy was not effective and severe myelosuppression continued for more than one month. We present the unusual clinical course for this case of PV and discuss the pathophysiology of refractory ascites. (Kitakanto Med J 2012;62:159~162)

Objectives: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by the production of autoreactive antibodies against platelet antigens. Although dysfunction of multiple aspects of cellular immunity is considered to be important in the pathogenesis of ITP, it has not been clarified which cell types play a principal role. Methods: We enrolled 46 untreated patients with chronic ITP and 47 healthy adult volunteers, and investigated by flow cytometry the percentage and absolute number of cells in their peripheral blood that participate in the regulation of cellular immunity. These included plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), natural killer (NK) cells, natural killer T (NKT) cells, regulatory T (Treg) cells, and Th17 cells. Results: We found a significant reduction in the absolute number of pDCs, but not of mDCs, in patients with ITP when compared with healthy controls (P

There have been conflicting reports over the JAK2-V617F mutation status of platelets in chronic myeloproliferative diseases (CMPDs). The aim of this study was to analyse JAK2-V617F status, not only in granulocytes but also in platelets. The JAK2-V617F mutation was analysed in both granulocytes and platelets in 115 patients with CMPDs using direct sequencing. JAK2-V617F was detected in granulocytes from 71 of those patients, all 71 of whom also had platelet JAK2-V617F expression. The remaining 44 patients showed negative JAK2-V617F expression on granulocytes, but positive JAK2-V617F expression was detected on the platelets from nine of the 33 essential thrombocythaemia (ET) patients, one of the eight polycythaemia vera patients, and two of the three primary myelofibrosis patients. When ET patients were divided into three groups according to granulocyte and platelet JAK2-V617F status (both-positive, platelets-only positive and both-negative), the both-positive and platelets-only positive groups shared the clinical features of higher white blood cell count and frequent thrombosis. These results suggest that analysis of platelets is a more sensitive approach for detecting JAK2-V617F in CMPD patients than analysis of granulocytes. They also suggest that previous reports of the incidence of JAK2-V617F in CMPD patients, obtained using only analysis of granulocytes, could be underestimations.

A 40-year-old man was diagnosed with Langerhans cell histiocytosis (LCH) in October 2010. His LCH was refractory to conventional chemotherapy, and thus worsened to Langerhans cell sarcoma (LCS) in May 2011. Although we repeated combination chemotherapies, new infiltration of the liver and bone marrow, as well as primary lesions of the bone, lymph nodes, and skin, appeared. These intensive chemotherapies caused candida liver abscesses, invasive aspergillosis, disseminated varicella zoster virus infection and bacterial sepsis. We administered bendamustine for chemotherapy, which resulted in a partial response (PR) with no severe adverse events. Because of pancytopenia caused by secondary myelodysplastic syndrome, we stopped the bendamustine chemotherapy after two courses. PR was maintained for 4 months. We plan to perform allogeneic hematopoietic stem cell transplantation from a sibling donor after a conditioning regimen. Optimal therapy for adult LCH, which is a rare and treatment-resistant disease, has yet to be established. Bendamustine is a potential chemotherapeutic agent for standard treatment of LCS.

We evaluated the prognostic significance of the serum level of the soluble form of interleukin-2 receptorα (sIL-2Rα) and investigated its association with CD25 expression on tumor cells in diffuse large B-cell lymphoma (DLBCL). Three hundred and thirty-eight adult patients with newly diagnosed DLBCL were eligible for this retrospective study. 32.2% of patients were treated with CHOP-like regimen and 67.8% with R-CHOP-like regimen. CD25 expression on the surface of tumor cells was evaluated in 143 cases and its relationship with sIL-2Rα level was also investigated. Both overall survival (OS) and progression-free survival (PFS) were poorer in patients with higher sIL-2Rα, in both R-CHOP and CHOP groups. sIL-2Rα1,000 U/mL and performance status (PS) ≥ 2 were independently associated with poorer OS, and sIL-2Rα1,000 U/mL, age60 years, and ≥ 2 extranodal sites were independently associated with poorer PFS in the R-CHOP group. The sIL-2Rα level was higher in the CD25-positive group than in the CD25-negative group in stage 3 or 4 disease (p = 0.010). Multiple linear regression analysis showed CD25 expression to be independently correlated with sIL-2Rα levels. High sIL-2Rα is an important risk factor for survival in DLBCL treated with not only CHOP-like, but also R-CHOP-like regimens, regardless of the tumor's expression of CD25.

Background: An increased risk of secondary myelodysplastic syndrome and acute myeloid leukemia (sMDS/AML) with a cumulative incidence of 4 to 18% has been described in adult patients with malignant lymphoma who undergo high-dose chemotherapy with autologous stem cell transplantation (ASCT). Although a high-dose VP-16 regimen has been reported to have greater stem cell mobilization potential than a high-dose cyclophosphamide regimen, this regimen was identified as a potential risk factor for sMDS/AML in several studies. Thus, this multi-center retrospective analysis of ASCT recipients with malignant lymphoma was performed to assess the safety of high-dose VP-16 as a stem cell mobilization regimen. Patients and methods: This study analyzed 153 adult patients who underwent ASCT against malignant lymphoma at four institutions in Gunma, Japan between 1997 and 2012. Patients with progressive disease were excluded. There was no restriction on the type of pathological diagnosis and conditioning regimens. The high-dose VP-16 regimen consisted of 350 mg/m2 of VP-16 (days 1 to 4) and 16 mg/body of dexamethasone (days 1 to 4). Overall survival (OS) was defined as the interval from the date of transplant to the date of death. Fisher's exact test was used to compare binary variables. Cumulative incidences (CIs) of sMDS/AML were compared using the stratified Gray test, considering death without the event as a competing risk. The Fine-Gray proportional hazard model was used for multivariate analysis of risk factors for sMDS/AML. The potential risk factors evaluated in this analysis were age at transplant, sex, pathological diagnosis, prior malignancy, conditioning regimens (LEED or not), number of chemotherapy regimens before ASCT, prior radiation therapy, disease status, and cyclophosphamide equivalent dose (CED). Values of p < 0.05 were considered significant. Results: Of the 153 patients assessed in this study, 93 were male and 60 were female, and the median age was 56 years (range: 18 to 68 years). Underlying diseases were diffuse large B-cell lymphoma (including IVLBCL) in 87 patients, follicular lymphoma in 8, other B-cell lymphoma in 24, Hodgkin lymphoma in 16, and T-NK-cell lymphoma in 18, and 81 patients were conditioned with LEED regimens. Disease status at the time of transplant was first complete remission (CR) in 77, advanced CR in 34, and partial remission in 42. The high-dose VP-16 regimen was used to mobilize stem cells in 85 patients. Of the 68 patients not using the high-dose VP-16 regimen, stem cells were mobilized with (R-)CHOP in 17, CHASE(-R) in 28, and others in 23. There were no significant differences in patient characteristics, disease features, and transplant procedures between patients with and without the high-dose VP-16 regimen. With a median observation time of 55.2 months, eight patients developed sMDS/AML, and the 5-year cumulative incidence of sMDS/AML was 3.5%. Type of sMDS/AML was MDS in six, chronic myelomonocytic leukemia in one, and AML in one, and median duration from ASCT was 52.9 months (range: 31.2 to 104.4 months). On univariate analysis, only age over 50 years was identified as a significant risk factor for sMDS/AML (5-year CIs in age under and over 50 years: 0.0% vs. 5.3%, respectively; p = 0.043), but not stem cell mobilization with the high-dose VP-16 regimen (5-year CIs with and without high-dose VP-16 regimen: 1.5% vs. 6.6%, respectively; p = 0.528). On multivariate analysis, age, using high-dose VP-16, and CED were evaluated. Age over 50 years (hazard ratio [HR] = 44140; p < 0.001) was identified as an independent risk factor for sMDS/AML, but not the high-dose VP-16 regimen (HR = 0.58; p = 0.432). The 5-year OS rates were not significantly different between patients with and without the high-dose VP-16 regimen (56% vs. 54%, respectively; p = 0.845). Conclusion: These results suggest that stem cell mobilization with a high-dose VP-16 regimen was not associated with an increased risk of sMDS/AML and did not affect the long-term survival rate in adult patients who underwent ASCT against malignant lymphoma. Therefore, considering this regimen's greater potential to mobilize stem cells, a high-dose VP-16 regimen is considered a preferable stem cell mobilization regimen in these patients. Disclosures Matsumoto: Janssen Pharmaceutical: Honoraria; Celgene: Honoraria.

[Introduction] Follicular lymphoma (FL) is an indolent neoplasm that is generally characterized by nodular proliferation of B cells arising from the follicular center of the lymph node.The typical chromosomal abnormality in FL is the translocation t(14;18)(q32;q21), which is found in more than 75% of cases. Several other chromosomal abnormalities, which might contribute to tumor progression, have been found. However, the prognostic significance of cytogenetic features of FL has not been clearly established in the Rituximab Era. The purpose of this study was to evaluate the pattern of chromosomal abnormalities in FL and to clarify the correlation between cytogenetic features and clinical outcome. [Patients and Methods] Cells from lymph nodes or other sites of disease at diagnosis from 201 patients with FL admitted to our hospital and affiliated hospitals between 2001 and 2013 were cytogenetically analyzed using standard methods of G-banding. Ninety nine (49.3%) men and 102 (50.7%) women with a median age of 59 years (range, 28 - 83 years) were included in the analysis. The median follow up period was 48.3 months. Forty three patients (21.4%) were Stage I or II; 156 patients (77.6%) were Stage III or IV; and 2 patients (1%) were unknown. Eighty patients (39.8%) were follicular lymphoma international prognostic index (FLIPI) low, 55 patients (27.4%) were intermediate, and 43 patients (21.4%) were high; and 23 patients (11.4%) were unknown. The distribution of FL pathological subgroups was as follows: FL Grade 1 - 2, 142 patients (70.6%); FL Grade 3a, 30 (15.0%); and unknown, 29 (14.4%). One hundred and fifty seven patients received Rituximab-containing chemotherapy as an initial treatment. [Results] t(14;18)(q32;q21) was the most common abnormality observed in 119 patients (59.2%); however, t(14;18) showed no correlation with clinical outcome. Other numerical or structural abnormalities that were identified in more than 5% of the patients were as follows: +X (17.9%), del(6)(q) / −6 (16.9%), +7 (14.4%), abnormality of 1q12-21 / 1q (12.9%), del(13)(q) / -13 (11.9%), abnormality of 3q27 (10.4%), abnormality of 10q22-24 (10.0%), +12 / dup(12)(q) (10.0%), abnormality of 1p21-22 / 1p(9.0%), +18 (9.0%), del(17)(p) / −17 (5.0%), and the number of cytogenetic aberrations higher than 3 (54.7%). Patients with +21 (p = 0.00171) or with >3 cytogenetic aberrations (p = 0.00269) had a significantly shorter progression free survival (PFS) in univariate analysis. Patients with +21 (median OS 29.9 Mo vs. not reached, p < 0.001), with 3q27 abnormality (p < 0.001), with del(17)(p) / −17 (p = 0.00659), with +7 (p = 0.0369), and with >3 cytogenetic aberrations (p = 0.0301) were associated with a shorter overall survival (OS). We also found an association between trisomy 21 or 3q27 abnormality and poor OS with and without t(14;18) (p < 0.001). Multivariate analysis identified +21 and >3 cytogenetic aberrations as independent prognostic factors for PFS in this population. We also identified 3q27 abnormality and +21 as independent prognostic factors for OS in this population. When patients with or without +21 were compared, t(14;18) positivity was not significantly different between the two groups, but 3q27 positivity was significantly higher in the patients with +21 than in those without +21 (30.8% vs. 9.0%, p = 0.034). Patients with +21 were significantly older than patients without +21 (64.5 years vs. 58.4 years, p= 0.027). There were no differences between the groups in other characteristics such as stage, FLIPI, pathological grade, and laboratory data. [Conclusion] Both the presence of trisomy 21 and the presence of 3q27 abnormality were independent risk factors for overall survival in FL with and without t(14;18). Chromosomal analysis of FL at the time of diagnosis can provide important information about survival. Disclosures No relevant conflicts of interest to declare.

We retrospectively investigated pathological types, clinical backgrounds, treatments and prognoses in 726 adult patients with newly diagnosed malignant lymphoma in Gunma Prefecture. They consisted of 679 patients with non-Hodgkin lymphoma (B-cell type, 603; T- and NK-cell type, 76) of which 376 patients had diffuse large B-cell lymphoma (DLBCL) and 47 patients with Hodgkin lymphoma. When comparing the prognosis of DLBCL between patients receiving rituximab (R-CHOP group; n=212) and not using rituximab (CHOP group; n=126), both 3-year overall survival (73.5% vs 61.7%, p=0.010) and 3-year progression-free survival (65.1% vs 45.8%, p0.001) were statistically better in the R-CHOP group compared to the CHOP group. Our results suggest that more than half of patients were DLBCL and the rituximab-containing regimen results in an improved prognosis for DLBCL patients.

Objective While the somatic mutation of Janus Kinase 2 (JAK2) and the thrombopoietin receptor (c-MPL) gene are thought to affect the pathogenesis of bcr/abl negative chronic myeloproliferative neoplasm (MPN), the relationship between the mutation and the clinical features remain obscure. Methods The mutation status of these genes in granulocytes, platelets, T-cells, and erythroid colonies (BFU-E) was obtained from 115 MPN patients, and then the clinical features of the MPN subtypes were compared. Results The JAK2-V617F mutation was observed in three lineages of granulocytes, platelets, and BFU-E in almost all polycythemia vera (PV) and primary myelofibrosis (PMF) patients. In contrast, 68% of essential thrombocythemia (ET) patients have the JAK2-V617F mutation in at least one of the lineages, of which 70% of these patients have the JAK2-V617F mutation in three lineages; the remaining ET patients with the JAK2-V617F mutation only exhibited the mutation in one or two lineages. Further, the ET patients that exhibited the JAK2-V617F mutation in three lineages had higher WBC and granulocyte counts as compared to the ET patients that did not have the JAK2-V617F mutation or only had the mutation in one or two lineages. Concerning the MPL gene, two ET patients had the MPL-W515L gene mutation in their platelets, although the lineage of the JAK2-V617F mutation involved differed from case to case. Conclusion The progenitor cells that are involved with the JAK2-V617F mutation in MPNs are different in each subtype and this difference may also affect the clinical features of MPNs.

Ten years after being diagnosed with polycythemia vera, a 55-year-old woman required frequent blood transfusion due to secondary myelofibrosis. She underwent reduced-intensity stem cell transplantation (RIST) from an HLA-identical sibling donor. Since mixed chimerae were identified in the peripheral blood at day 35, cyclosporine was withdrawn. At day 73, she developed acute graft-versus-host disease of the liver, while simultaneous resolution of splenomegaly occurred and complete donor chimerism in the peripheral blood was achieved. Frequent red blood cell transfusion was required until day 300 after transplantation. Thus, RIST for an older patient with secondary myelofibrosis was successful without severe treatment-related morbidity. This case suggests that RIST could be an effective treatment modality for secondary myelofibrosis.

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.

Background: Analysis of prognostic factors and clinical trials of novel agents for Waldenstrӧm macroglobulinemia (WM) are ongoing in Western countries, but few studies of WM have been performed in Japan. As a step toward future investigations, we retrospectively analyzed clinical features and prognostic factors in Japanese patients with WM. Methods: We retrospectively analyzed clinical and laboratory characteristics, treatment and outcomes of 110 patients with WM, IgM-MGUS or lymphoplasmacytic lymphoma (LPL) diagnosed from January 2001 to March 2013 at 12 institutes. Overall survival (OS) was analyzed using Kaplan-Meier methods and survival was compared using log-rank testing. Several clinical characteristics at diagnosis were assessed by Cox regression for uni- and multivariate analysis for OS. Results: Median age at diagnosis was 69 (range, 41-96) years, 73.6% were male, 12.0% had an ECOG performance status 2-4 and 6.4% presented with B-symptoms. Hyperviscosity, peripheral neuropathy, amyloidosis, cryoglobulinemia and cold agglutinin disease were shown in 9.1%, 4.5%, 1.8%, 4.5% and 2.7%, respectively. In 94 patients with available CT findings at diagnosis, lymphadenopathy, hepatosplenomegaly, pleural effusion, lung involvement, bone involvement and skin involvement were shown in 41.5%, 14.9%, 8.5%, 4.3%, 4.3% and 6.4%, respectively. Median serum monoclonal protein level was 2.62 g/dl (range, 0.70-9.35 g/dl). Symptomatic WM was present in 76 patients, asymptomatic WM in 23 and IgM-MGUS in 2 according to criteria of the Second International Workshop on WM. Seven patients showed IgG- or IgA-secreting LPL and 2 showed LPL without bone marrow infiltration. In patients with symptomatic WM, international prognostic scoring system for WM (ISSWM) was low in 9.2%, intermediate in 34.2%, high in 39.5% and unknown in 17.1%. Among patients with asymptomatic and symptomatic WM, watchful waiting was performed in 91.3% and 40.0%, respectively, with 61.9% and 36.7% remaining untreated, respectively. Median time to treatment from diagnosis of asymptomatic or symptomatic WM was 240 days (range, 3-1238 days) and 31 days (range, 0-2011 days), respectively. Oral alkylating agents were administered to 34.7% of patients with WM, 19.4% were treated with CHOP or CHOP-like regimen with or without rituximab, 8.2% received fludarabine mono- or combination therapy and 6.1% received rituximab monotherapy. Rituximab-containing therapy was administered as the initial treatment in 33.8% of patients who received treatment. Overall response rate (ORR) (complete + partial response rate) was 48.6%, and patients treated with rituximab-containing therapy displayed higher ORR (64.0%) compared to those with non-rituximab therapy (40.8%). Plasmapheresis was performed in 3.7% of patients. Three patients (2.7%) showed transformation to diffuse large B-cell lymphoma, and 7 (6.4%) developed second primary malignancies. Median follow-up was 38 months, 5-year OS rate for all patients was 74.9% (95% confidence interval (CI) 62.5-83.7) and rates for those with symptomatic WM, asymptomatic WM and other LPL were 66.0% (95%CI 50.6-77.6), 100% and 88.9% (95%CI 43.3-98.4), respectively. Significant differences in survival between risk groups of ISSWM in patients with symptomatic WM were not seen (5-year OS: high, 62.4%; intermediate, 64.3%; low, 75.0%; p=0.86). Although no significant difference in OS was observed compared to initial treatment (p=0.265), patients treated with rituximab during the observation period showed significantly prolonged OS compared to those treated without rituximab (5-year OS rates: 78.9% vs. 45.6%, p=0.036). In univariate analysis, age, pleural effusion, serum albumin, C-reactive protein and serum IgM levels were poor prognostic factors for OS. In multivariate analysis, age >65 years (hazard ratio (HR)=3.294; 95%CI 1.097-9.888, p=0.0336) and pleural effusion (HR=4.55; 95%CI 1.602-12.930, p=0.0045) were identified as significant prognostic factors for OS. Conclusion: Prognostic factors for WM in Western countries may not be applicable to Japanese patients. This study suggested presence of pleural effusion at diagnosis is associated with poor clinical outcomes. Further investigations including histopathological examinations and molecular analyses are required to elucidate prognostic factors in Japan. Disclosures No relevant conflicts of interest to declare.

Abstract 1935 Poster Board I-958 Introduction: Several reports have identified soluble-form IL-2 receptor á (sIL-2Rá) as a significant prognostic factor in patients with non-Hodgkin lymphoma treated using chemotherapy, particularly in rituximab-containing regimens. However, the clinical significance of sIL-2R is not fully understood, as only small populations have been studied to date. The rationale for increasing of serum level of sIL-2Rá in non-Hodgkin lymphoma is also unclear. Patients and Methods: We analyzed 409 patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL) between January 2001 and July 2008. Treatment comprised CHOP-like regimen with (R-CHOP-like) or without rituximab. Levels of sIL-2R were evaluated with enzyme-linked immunosorbent assay at diagnosis. Overall survival (OS) and progression-free survival (PFS, death from any cause, relapse and refractory disease) were analyzed using Kaplan-Meier methods and survival was compared using log-rank tests. To estimate the survival impact of several factors, including sIL-2Rá level, PS, LDH, B symptoms, extranodal sites ≥2 and age, we performed multivariate analysis using Cox proportional hazards. In 166 of 409 patients, CD25 (IL-2Rá) expression on tumor cells was evaluated using a lymphoma sample from the lymph node, bone marrow, blood or other extranodal organ by flow cytometry. To estimate CD25 expression of tumor cell, CD45 bright cells (mature lymphocyte gate) were gated and considered positive if positivity was seen in >20% of the population excluding CD4-positive cells) using three-color flow cytometry. Results: Median age was 68 years (range, 17-91 years), males/females 1.18, and 28.9% of patients were treated with CHOP-like regimen and 60.2% with R-CHOP-like regimen. Clinical stage was I in 24.4%, II in 24.2%, III in 13.1%, and IV in 38.8%. International Prognostic Index (IPI) was Low in 33.5%, LI in 23.5%, HI in 18.7% and H in 24.3%. Median follow-up for CHOP-like and R-CHOP-like groups was 924 days (range, 16-2878 days) and 799 days (range, 29-2688 days), respectively. Median sIL-2Rá value was 1360 U/L (range, 170-59,500 U/L). For the entire population, CR rate was 71.9%, 3-year OS was 67.6% and PFS was 58.8%. OS differed significantly between sIL-2R >1000 U/L and ≤1000 U/L, between >2000 U/L and ≤2000 U/L and between >3500 U/L and ≤3500 U/L, (p1000 U/L and ≤1000 U/L, between >2000 U/L and ≤2000 U/L and, between >3500 U/L and ≤3500 U/L (p3500 U/L is extremely poor even if treated with R-CHOP (53.2%). We showed that one rationale for increasing level of serum sIL-2Rá level in DLBCL is to remove from the tumor cell surface. Disclosures: No relevant conflicts of interest to declare.

Background: Dendritic cells (DCs), natural killer (NK) cells, and invariant NKT (iNKT) cells play important roles in innate immune systems. These cells have recently been shown to be involved in immunoregulation, and some studies have suggested associations with various kinds of autoimmune disease. Conversely, regulatory T cells (Tregs) that are important for peripheral tolerance and Th17 cells that play a central role in maintenance of chronic inflammation are also associated with the pathogenesis of several autoimmune diseases. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies, but relationships to innate immunity are unclear. In addition, the pathogenesis of ITP associated with Helicobacter pylori remains obscure. In particular, the regulation of immune responses by these cells in patients infected with H. pylori has not been investigated. This study analyzed DCs, NK cells, iNKT cells, Tregs and Th17 cells in patients with ITP. Methods: Subjects comprised 31 patients with ITP and 22 healthy donors. Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all subjects. Flow cytometry was used to investigate amounts of circulating plasmacytoid DCs (pDCs) (CD123+ HLA−DR+) and myeloid DCs (mDCs) (CD11c+ HLA−DR+) from whole white blood cells, and NK cells (CD3− CD56+), iNKT cells (Vα24+ Vβ11+), Tregs (CD4+ CD25+ Foxp3+) and Th17 cells (CD4+ IL−17A+) from mononuclear cells. The intracellular interleukin (IL)-17A production in CD4+ T-cells activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin was assessed to detect Th17 cells. Results: Both the percentage and numbers of pDCs were significantly reduced in patients compared to healthy controls (p Conclusion: These results suggest that alterations in innate immunity as a reduction of pDCs could be associated with the pathogenesis of ITP. Furthermore, as in some autoimmune diseases that have been considered as Th1 diseases, Th17 cells may play an important role in ITP.

Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p < 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

The incidence of chronic lymphocytic leukemia (CLL) is low in Asian countries including Japan, while it is the most common type of leukemia in western countries. It has been evident that the immunoglobulin heavy chain variable region (IGHV) gene mutation status can predict the prognosis in CLL; unmutated IGHV genes correlate with a worse prognosis than mutated genes. Over-representation of selected IGHV genes is noted in western CLL, in particular IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-21. Although their relative frequencies vary between cohorts, the most frequent gene in western countries is IGHV1-69, which is found in about 10–20% of all CLL patients. Several studies have shown very unusual Ig characteristics in CLL: highly restricted IGHV gene usage and very similar antigen-binding sequences (stereotyped antigen receptors), suggesting a role for antigen selection during the development and maintenance of the disease. For the purpose of clarifying the characteristics of CLL in the Japanese population, we analyzed both IGHV and Ig light chain (κ-chain, IGK and λ-chain, IGL) genes in 81 CLL cases and compared the findings with cases of 52 leukemic chronic lymphoproliferative disorders (CLPD) including 6 hairy cell leukemia (HCL), 1 prolymphocytic leukemia (PLL), 31 indolent lymphoma in leukemic phase (15 mantle cell lymphoma (MCL), 7 follicular lymphoma (FL), 5 splenic marzinal zone lymphoma (SMZL), and 4 lymphoplasmacytic lymphoma (LPL)) and 14 cases that could not be classified further. Of the 81 Japanese CLL patients, 17 (21.3%) had unmutated IGHV, and 63 (78.7%) had mutated IGHV. The number of CLL with mutated IGHV was at a higher frequency compared to previous reports from western countries. It may be partly explained by the fact that the commonly unmutated IGHV1-69 type was rare (1.2%), but the commonly mutated IGHV4-34 type was frequent (27.2%) in the Japanese CLL patients. We previously reported that IGHV1-69 CLL is rare in Japan (1/44), which is confirmed by the present study of newly diagnosed cases (0/37). Moreover, only 1 of 65 CLL patients was reported to use IGHV1-69 in China. These findings suggest that IGHV1-69 is extremely rare in Asia. Similar to reports from Scandinavian countries, IGHV3-21 cases showed biased λ-chain expression (5/6), but were not associated with overuse of IGLV3-21 (V2-14) in our cohort. Recently, studies of B-cell antigen receptors (BCRs) in patients with CLL identified that subsets of cases expressed almost identical BCRs. We also found a pair of CLL patients who had the same IGHV4-39, IGHD6-13, IGHJ5 (heavy chain) and IGKV1-39 (O12), IGKJ1 or 2 segment with remarkably similar H and L CDR3 sequences. The use of IGHV, IGKV and IGLV was significant different when compared between CLL and leukemic CLPD. IGHV4-34, which was the most preferentially used in CLL patients (21/81, 26.0%), was used rarely in CLPD patients (4/52, 7.7%, p = 0.007). Of the 4 CLPD patients with IGHV4-34, 3 were MCL (CD5+) and 1 was unclassified CLPD (CD5 −). As leukemic cells of all CLL cases were CD5+, only 1 of the 25 patients using IGHV4-34 had CD5 negative cells. In normal B-cell development, naive IGHV4-34 B-cells are positively selected and mostly restricted to the follicular mantle zone but these cells are largely excluded from the germinal centers. This mechanism may be relevant to IGHV4-34 usage being underrepresented in CLPD other than MCL, which mainly consisted of GC- or post-GC-derived lymphomas/leukemias. In CLPD patients, only 1 patient with SMZL used the IGHV1-69 gene. Interestingly, IGHV1-69 was associated with IGHD5-24, IGHJ3, IGKV3-20 and IGLKJ1, which have been previously identified to comprise one of the stereotypical BCR gene subsets in patients with CLL. In the CLL patients, IGKV3-11 (L6) and IGLV3-21 (V2-14) were the most frequent IGKV (7/43) and IGLV (11/35), respectively. However, in the CLPD patients, IGKV3-11 and IGLV3-21 were used by none (0/26, p = 0.03) and only 1 MCL patient (1/22, p = 0.002), respectively. To date little data has been obtained on CLL in Japan and other Asian countries, where the susceptibility to CLL is very low. Thus, it is important to investigate genetic and environmental differences between Asian and western countries to identify risk factors that give rise to this disease. In addition, a comparison of the disease features of CLL with other lymphoproliferative disorders will further elucidate the clinical and pathogenetic characteristics of CLL.

BCR/ABL-negative chronic myeloproliferative disorders (CMPDs) are considered to arise in a pluripotent progenitor cell. High prevalence of a point mutation in Janus Kinase 2 (JAK2) exon12 gene (JAK2-V617F) in CMPDs has been observed in many reports. Since most studies, however, are restricted mainly in granulocyte lineage, information about the JAK2 gene status in other cell lineage is limited. Heparinized peripheral blood was obtained from 49 patients with polycythemia vera (PV), 109 with essential thrombocytosis (ET), and 5 with chronic idiopathic myelofibrosis (CIMF). After centrifugation, platelets were collected from the upper plasma layer. The remaining blood layer was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet and T-cells recovered from the interface were further positively selected using either anti-CD3 or anti-CD4 immunoconjugated magnetic beads (Miltenyi Biotec, Germany). After DNA and RNA extraction, PCR amplified JAK2 exon 12 gene was sequenced to check the presence of JAK2-V617F mutation. Furthermore, X-chromosome linked clonal analysis using human androgen receptor (HUMARA) gene was carried out in 58 females. Correlation between the mutation status and clinical data was analyzed using statiscal software R (The R Development Core team). In PV, the frequency of JAK2-V617F mutation in granulocytes and platelets was 72.3 % and 74.3%, respectively (p-value 0.844). In ET, this mutation rate was 51.9% in granulocytes and 73% in platelets and the difference was significant (p-value 0.005). In all CMPDs, the cases showing JAK2-V617F mutation in granulocytes always had the mutation in platelets as well, but in one case of PV and 11 cases of ET, the JAK2-V617F mutation was observed only in platelets. T cell fraction was negative for JAK2-V617F mutation in all cases examined. White blood cell count was significantly higher in cases of JAK2-V617F mutation in their platelets compared to that in cases without this mutation (12.6 × 109/l vs. 9.2 × 109/l), but hemoglobin level and platelet count did not differ between the two groups (14.9g/l vs. 14.6 g/l, 81.4 × 109/l vs. 95.2 × 109/l). In addition, cases showing this mutation in platelets were more likely to be associated with thrombosis than cases showing the wild type gene (p-value 0.018). On clonal analyses using the HUMARA gene, 6/8 patients with PV, 15/27 patients with ET, and 1/1 patient with CIMF demonstrated a monoclonal pattern in their granulocytes. Interestingly, in 23 cases showing the JAK2-V617F mutation, 7 patients showed the polyclonal HUMARA pattern; in 13 cases without mutation, 7 showed the monoclonal HUMARA pattern. The discrepancy of JAK2-V617F mutation rate between platelets and granulocytes in ET suggests that analysis of platelet fraction is advantageous for detecting the mutation. This finding also suggests that the JAK2 mutation could be involved only in megakaryocyte lineage in some ET patients. HUMARA analysis confirmed that some ET patients with monoclonal disease lack the JAK2-V617F mutation, which is consistent with previous reports.