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Extranodal NK/T-cell lymphoma, nasal type (ENKTL) is an Epstein-Barr virus-positive, aggressive lymphoma with a heterogeneous cell of origin and variable clinical course. Several clinical prognostic indices have been proposed for ENKTL; however, there are few pathological biomarkers. This multi-institutional study sought to identify histologically assessable prognostic factors. We investigated mutation profiles by targeted next-generation sequencing (NGS) and immunohistochemical assessments of expression of MYC, Tyr705-phosphorylated (p-)STAT3, and CD30 in 71 ENKTL samples. The median age of the patients was 66 years (range, 6-100). The most frequent mutations were in STAT3 (27%), JAK3 (4%), KMT2D (19%), TP53 (13%), BCOR (10%), and DDX3X (7%). Immunohistochemistry (IHC) revealed that ENKTLs with STAT3 mutations exhibited higher expression of pSTAT3 and CD30. BCOR mutations were associated with increased MYC expression. Univariate analysis in the entire cohort showed that stage (II, III, or IV), BCOR mutations, TP53 mutations, and high MYC expression (defined as ≥40% positive neoplastic cells) were associated with reduced overall survival (OS). Multivariate modeling identified stage (II, III, or IV) and high MYC expression as independent adverse prognostic factors. In a subgroup analysis of patients treated with anthracycline (AC)-free chemotherapy and/or radiotherapy (RT) with curative intent, BCOR but not high MYC expression was an independent adverse prognostic factor. In conclusion, activating STAT3 mutations are common in ENKTLs and are associated with increased CD30 expression. MYC overexpression is, at least in part, associated with deleterious BCOR mutations, and this BCOR–MYC linkage may have prognostic significance, underscoring the potential utility of IHC for MYC in risk stratification of patients with ENKTL.

A 71-year-old woman with a four-year history of chronic lymphocytic leukemia (CLL) received ibrutinib as initial treatment due to progressive anemia and thrombocytopenia. Eleven months after the start of the treatments, although her cytopenia had ameliorated, she developed classic Hodgkin lymphoma, a rare form of Richter's transformation. She was successfully treated with two courses of adriamycin, vinblastin, bleomycin and dacarbazine followed by radiotherapy. In general, several clinical, genetic and molecular factors are associated with Richter's transformation. In addition, our present case suggested that ibrutinib could be a potential risk factor for Richter's transformation in CLL patients.

We report an exceptionally rare case of mantle cell lymphoma (MCL) that transdifferentiated into sarcoma with limited neuromuscular differentiation. An 81-year-old man with t(11;14)-positive MCL was treated with rituximab and bendamustine and achieved complete remission; however, just 2 months later, the patient developed multiple systemic tumors. Pathologic studies revealed round cell sarcoma expressing synaptophysin, CD56, and myogenin without any B-cell markers. The CCND1 translocation and an identical IGL gene rearrangement were shared by both the MCL and sarcoma. Whole-exome sequencing detected 189 single nucleotide variants (SNVs) in the MCL and 205 SNVs in the sarcoma; 160 SNVs including NSD2, ATM, RB1, and TP53 mutations were shared between MCL and sarcoma cells. An additional PTPN11 mutation was specifically found in the sarcoma. These findings confirmed the shared clonal origin of MCL and sarcoma in this patient and indicated that MCL can transdifferentiate into sarcoma in rare cases.

We conducted a multicenter study on anti-programmed cell death-1 monoclonal antibodies (anti-PD-1 mAbs) before/after allogeneic hematopoietic cell transplantation (allo-HCT) for Hodgkin lymphoma. Anti-PD-1 mAbs were administered to 25 patients before allo-HCT and to 20 after allo-HCT. In pre-allo-HCT setting, the median interval from the last administration to allo-HCT was 59 days. After allo-HCT, 12 patients developed non-infectious febrile syndrome requiring high-dose corticosteroid. The cumulative incidences of grade II-IV acute graft-versus-host disease (aGvHD) were 47.1%. Eight patients who had GvHD prophylaxis with post-transplant cyclophosphamide (PTCy) had less frequent aGvHD (grade II-IV, 14.6% versus 58.8%; P = 0.086). The 1 year overall survival (OS), relapse/progression, and non-relapse mortality rates were 81.3%, 27.9%, and 8.4%. In post-allo-HCT setting, the median interval from allo-HCT to the first administration was 589 days. The overall and complete response rates were 75% and 40%. At 100 days after anti-PD-1 therapy, the cumulative incidences of grade II-IV aGvHD, moderate-to-severe chronic GvHD, and grade 3-4 immune-related toxicity were 15.0%, 30.0%, and 30.0%. While the 1 year relapse/progression rate was 47.4%, the 1 year OS probability was 89.7%. In conclusion, immune-related complications were frequent despite modifications of GvHD prophylaxis or anti-PD-1 mAb dosing. In anti-PD-1-mAb-pretreated patients, PTCy-based GvHD prophylaxis may be effective.

Boltzmann machines (BMs) are useful in various applications but are limited by their requirement to generate random numbers. In contrast, chaotic Boltzmann machines (CBMs) are neural networks that imitate the stochastic behavior of BMs with the chaotic dynamics and deterministic behavior, without random numbers. CBMs can potentially require fewer hardware resources than the original algorithms due to the unnecessity of random number generators. In this study, hardware-oriented algorithms and a differential multiply-accumulation operation are proposed to overcome the difficulties of implementing CBMs on field-programmable gate arrays (FPGAs). A hardware-oriented algorithm for CBMs, which includes fixed-point operations and shift operations, is proposed to reduce hardware resource utilization in the implemented circuits. In particular, the differential multiply-accumulate operation allows us to implement the multiply-accumulate operation with block random access memory and digital signal processors to reduce the consumption of lookup tables and flip-flops in FPGAs without losing the calculation speed. Our proposed approach was evaluated in numerical simulations, logical synthesis, and FPGA implementation. The calculation speed of FPGA-implemented CBMs was compared with software-implemented CBMs, which resulted in 1 / 6,500 of calculation time reduction in a 300-neuron CBM. Moreover, 2,048 neurons of CBM were realized by the logical synthesis. Therefore, the proposed hardware implementation of CBMs was shown to be feasible. The proposed CBMs can solve combinatorial optimization problems at a larger scale with fewer resources.

We analyzed the incidence of bone marrow fibrosis in 91 newly diagnosed Japanese multiple myeloma (MM) patients and evaluated the impact of fibrosis on clinical characteristics and therapeutic outcomes. Thirty-four (37%) patients had greater than grade 1 bone marrow fibrosis. The presence of bone marrow fibrosis did not affect laboratory data, the percentage of plasma cells in bone marrow or cytogenetic findings. It also had no significant effect on response to initial treatment, engraftment after autologous hematopoietic stem cell transplantation or overall survival. Interestingly, the incidence of extramedullary disease at diagnosis was significantly higher in patients with bone marrow fibrosis (p = 0.006). Analysis of biological characteristics of MM cells revealed that expression of CD49e, an alpha5/beta1 integrin, was downregulated in MM cells derived from patients with bone marrow fibrosis (p = 0.026). When seven of the original 34 patients were re-evaluated for fibrosis grading after treatment, five (71%) showed a reduction in fibrosis. Our present findings suggest that the presence of bone marrow fibrosis may predict development of extramedullary disease in MM.

A multicenter phase II study was conducted in 44 elderly (≥ 65 years) Japanese patients with newly diagnosed acute myeloid leukemia (AML) to evaluate whether azacitidine is also effective and feasible in Japanese AML patients. The 28 patients with AML with poor-risk cytogenetics and/or myelodysplasia-related changes (unfavorable AML) were randomly assigned to receive either azacitidine or conventional care regimens (CCR), and the other 16 patients without unfavorable AML received azacitidine alone. The primary endpoint was overall survival. At the median follow-up of 29 months, among the 26 evaluable patients with unfavorable AML, the median survival time (MST) of patients who received azacitidine (N = 14) was 9.6 months and that of patients who received CCR (N = 12) was 5.3 months (HR 0.73; 95% CI 0.31-1.69; log-rank P = 0.459). The MST of all 29 patients who received azacytidine, including the 15 evaluable patients without unfavorable AML, was 12.4 months. Adverse events of azacitidine were manageable and consistent with its established safety profile. Azacitidine tended to prolong survival in newly diagnosed elderly Japanese patients with AML, and was feasible as a front-line therapy for elderly AML patients.

The prognosis of patients with aggressive adult T cell leukemia-lymphoma (ATLL) is dismal even with intensive chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is a promising option for patients with aggressive ATLL, but the posttransplant outcome remains unsatisfactory. Hence, to further improve clinical outcomes, novel therapeutic approaches are needed. The clinical significance of immune checkpoint protein expression has not been well-established in aggressive ATLL. This study aims to identify the association between the expression profile of immune checkpoint proteins on ATLL cells and clinical outcomes. This retrospective study cohort included 65 patients with aggressive ATLL diagnosed between 2001 and 2015 at the National Cancer Center Hospital, Tokyo, Japan. Formalin-fixed paraffin-embedded tissue was used to immunohistochemically determine the expression of immune checkpoint proteins and assess the impact of expression profile on the probability of overall survival from diagnosis or HSCT. The current analysis shows that cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) expressions were adverse prognostic factors in patients with aggressive ATLL. Experiments that assess the efficacy of immune checkpoint inhibitors are warranted to alleviate the adverse impacts associated with negative immune checkpoints.

Metastasis of cancer cells to the bone marrow is relatively rare, despite being one of the most important causes of myelosuppression in patients with solid tumours. A bone marrow examination via a biopsy is the standard method of diagnosing cancer cell invasion into the bone marrow. However, it is sometimes challenging to distinguish neuroendocrine carcinoma cells from haematopoietic cells due to their small, round shape and chromosomal abnormalities resembling haematological malignancies. We herein report a case of bone marrow invasion of small cell neuroendocrine carcinoma of the endometrium mimicking therapy-related myeloid malignancy.

Post-transplant lymphoproliferative disorder (PTLD) usually develops with systemic symptoms, such as fever, generalized lymphadenopathy, and elevation in the lactate dehydrogenase level. Here, we present the case of a 65-year-old female patient with PTLD localized to the colon; the patient only had mild diarrhea without systemic symptoms. She had myelodysplastic syndrome and was treated with cord blood transplantation (CBT). She had a past medical history of sigmoid colon cancer treated with colonosectomy and adjuvant chemotherapy. After CBT, she achieved complete remission and was discharged after 60 days. Further, 79 days after CBT, she presented with abdominal pain. Computed tomography scan revealed adhesive ileus. The abdominal pain was resolved in 1 day with conservative treatment, however, mild diarrhea persisted. Therefore, we performed colonoscopy and found multiple ulcerative lesions in the upper colon. A pathological examination revealed PTLD. Furthermore, elevation of EBV-DNA in the blood was also confirmed. There was no detectable lesion on positron emission tomography-computed tomography (PET-CT) outside the colon; thus, we diagnosed PTLD localized into the colon that was successfully treated with rituximab. Our present experience suggests that it might be important to perform endoscopy and monitoring of EBV-DNA for early detection of PTLD, especially localized in the gastrointestinal tract.

Angioimmunoblastic T-cell lymphoma (AITL) is frequently associated with immunological abnormalities, such as hypergammaglobulinemia, autoimmune cytopenia, and the presence of various autoantibodies. Few reports on AITL have also described the development of myelofibrosis resulting from the invasion of lymphoma cells that produced various cytokines, including TGF-β. Interestingly, recent studies demonstrated that autoimmunity can directly cause autoimmune myelofibrosis (AIMF). Usually, bone marrow fibrosis associated with AIMF is rapidly improved by treatment. Here, we describe our experience with a case of AITL complicated with the presence of numerous autoimmune abnormalities, including positive Coombs, anti-nuclear antibody, anti-ds-DNA antibody, anti-phospholipid antibody, and cold agglutinin tests. The patient presented with severe bone marrow fibrosis (MF-3) at the initial diagnosis. After two courses of the CHASE therapy, myelofibrosis rapidly disappeared, and the autoimmune abnormalities were ameliorated. These findings suggest that the bone marrow fibrosis observed in this case was partly attributable to an AIMF-like mechanism.

Coagulation abnormalities are a rare but critical complication associated with plasma cell diseases. We herein present a case of multiple myeloma (MM) with complicated coagulopathy. Initially, the patient showed severe bleeding tendency due to concomitant acquired hemophilia A and acquired von Willebrand syndrome. Interestingly, the patient also exhibited hyperactivation of factor IX. During treatment for MM, the bleeding complications were ameliorated; however, the patient had central retinal vein occlusion. All of the coagulation abnormalities were completely resolved after the complete remission of MM. This case suggests that MM patients may have concomitant risks for both bleeding and thromboembolic complications.

Double-expressor lymphoma (DEL) is a diffuse large B cell lymphoma that exhibits co-expression of MYC and BCL2 proteins by immunohistochemistry. Patients with double-expressor lymphoma have a poor prognosis after standard chemoimmunotherapy or after high-dose chemotherapy with autologous transplantation, but the prognostic impact of DEL after allogeneic hematopoietic cell transplantation has not been well characterized. We retrospectively analyzed 60 consecutive patients with de novo diffuse large B cell lymphoma or transformed follicular lymphoma who underwent allogeneic transplantation at our center and had available immunohistochemistry data. Thirty-seven patients (62%) had DEL. The 2-year progression-free and overall survival rates were lower in patients with DEL than in those without DEL (20% versus 78%; overall P .001 and 46% versus 77%; overall P = .016, respectively). The cumulative incidence of disease progression at 2 years was higher in patients with DEL (60% versus 13%; overall P = .005). The cumulative incidence of nonrelapse mortality did not differ statistically in the 2 groups. Even in patients with DEL and chemosensitive disease at transplantation, the 2-year progression-free survival rate was only 27% due to early disease progression. Multivariate analysis showed associations between DEL and increased risks of progression-free survival events (hazard ratio [HR], 4.58; 95% confidence interval [CI], 2.07-10.2; P .001), overall mortality (HR, 2.29; 95% CI, 1.03-5.09; P = .042) and disease progression (HR, 3.60; 95% CI, 1.38-9.44; P = .009). Patients with DEL had poor outcomes after allogeneic transplantation. Innovative strategies are needed to improve outcomes in this population.

The development of tumor lysis syndrome (TLS) in association with treatment for myeloproliferative neoplasms (MPNs) is relatively rare. We herein present the case of a post-polycythemia vera (PV) myelofibrosis patient with massive splenomegaly who developed laboratory TLS after treatment with ruxolitinib, a potent JAK1/JAK2 inhibitor. She also exhibited a rapid reduction of spleen volume. Our present case suggests the potential risk of TLS development after ruxolitinib treatment, particularly in patients with massive splenomegaly.

Hexokinase II (HXKII) is a key regulator of glucose metabolism that converts glucose to glucose 6-phosphate. Furthermore, HXKII blocks mitochondria-dependent apoptosis by inhibiting the release of cytochrome c. HXKII overexpression is frequently observed in several types of cancer and confers chemoresistance to cancer cells. In the present study, we found that compared with cell lines generated from diffuse large-B-cell lymphoma (DLBCL) patients, cell lines with features of Burkitt lymphoma have higher levels of HXKII because of the activation of both c-MYC and HIF-1. Under normoxia, HXKII levels were correlated with the growth ability of each B-cell lymphoma cell line. HXKII levels were further enhanced when the B-cell lymphoma cells were cultured under hypoxia. The high levels of HXKII induced by hypoxia conferred cisplatin resistance in all tested B-cell lymphoma cell lines. The HDAC inhibitor panobinostat significantly suppressed HXKII expression under both normoxic and hypoxic conditions. Importantly, panobinostat reversed the anti-lymphoma action of cisplatin, and this effect was diminished by hypoxia. These data suggest that HXKII plays different roles, including in the regulation of glycolysis and inhibition of apoptosis, depending on its expression levels. Furthermore, inhibition of HXKII expression by panobinostat may represent a new and attractive strategy to overcome cisplatin resistance.

We report a case of anaplastic large cell lymphoma (ALCL) with involvement of bone marrow, exhibiting extreme leukocytosis leading to death due to multi-organ failure within 1 week after admission. The patient had a history of rheumatoid arthritis, and had severe pneumonia at admission. To elucidate the basis for the observed extreme neutrophilia, we analysed the levels of several cytokines in serum samples taken from the patient at diagnosis. The patient exhibited an extreme increase in interleukin-17 (IL-17), one of the major regulatory cytokines for inflammation and neutrophil migration. Interestingly, a recent study revealed that anaplastic lymphoma kinase (ALK)-positive ALCL cells produce IL-17. IL-17 also contributes to treatment resistance in multiple types of cancer. Given these previous findings, our case may suggest a possible link between overproduction of IL-17 and an aggressive ALCL phenotype. Further studies will be required to determine whether serum IL-17 levels serve as a useful prognostic marker for ALCL.

Metformin suppresses the growth of a variety of malignant hematologic cells. It is widely accepted that metformin inhibits the growth of malignant cells primarily by suppressing the mTOR pathway or regulating autophagy. In contrast, we found another possible mechanism that inhibits the growth of malignant cells, suppression of the activity of the oncogenic kinase JAK2V617F. We identified at least two distinct mechanisms involved in metformin-induced JAK2V617F inhibition. First, metformin increases reactive oxygen species levels in these cells, leading to the inhibition of SHP-2, a positive regulator of JAK2V617F. These effects of metformin require AMPK. Second, metformin activates protein tyrosine phosphatase PP2A, a negative regulator of JAK2V617F. Furthermore, we determined that among the numerous PP2A subfamily members, the PP2A complex containing the B56α subunit is responsible for the inhibition of JAK2V617F. In contrast, the B56α-containing PP2A complex functions as a positive regulator of JAK2V617F by inhibiting AMPK. Finally, we determined that metformin enhances the antileukemic action of ruxolitinib in HEL and SET-2 cells. Our present observations suggest that the combination of metformin with ruxolitinib might be a new therapeutic option for treating JAK2V617F-induced myeloproliferative neoplasms. In addition, activators specific for PP2A complexes containing the B56α subunit may be useful for the treatment of JAK2V617F-induced myeloproliferative neoplasms.

The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. Disclosures Kirito: Novartis Pharma KK: Honoraria.

がん性胸水に対して一般的には胸水穿刺, 胸腔ドレナージや胸膜癒着療法などを行うが, 成功率, 処置操作やカテーテル留置に伴う苦痛の程度や入院期間などにおいて必ずしも満足される結果が得られているとはいえない. 今回われわれは高齢者肺がん患者に生じたがん性胸水症例に対して皮下埋め込み型の胸腔―腹腔シャント造設を行い, 患者の希望する在宅移行に有効であった症例を経験した. 本法はがん性胸水に対する緩和医療, 特に在宅緩和医療の有効な手段の1つとなりうる可能性がある. Palliat Care Res 2008; 3(2): 326-330

Adult T cell lymphoma-leukemia (ATL) is a highly aggressive disease and allogeneic hematopoietic transplantation (allo-HSCT) is the only therapeutic option for achieving a cure. However, some ATL patients cannot undergo HSCT. One of the important reasons for restricting HSCT in ATL is the high incidence of pulmonary complications associated with ATL including opportunistic infections, infiltration of ATL cells, and HTLV-1 associated bronchopneumonopathy. Herein, we report an ATL case with pulmonary infiltration of ATL cells successfully treated with allo-HSCT after improvement of pulmonary function with administration of the anti-CCR4 antibody mogamulizumab. To our knowledge, this is the first ATL case showing improvement of pulmonary invasion of ATL cells after treatment with mogamulizumab. In addition, this case suggests that mogamulizumab treatment might be useful as a bridge to allo-HSCT in ATL patients.

gastric body. The patient was treated with red blood cell transfusion and a proton-pump inhibitor. Two days after the initial observation, esophagogastroduodenoscopy was performed again. As shown in Fig. 1a, irregular thickened folds were found at the gastric corpus. The biopsy specimens revealed significant invasion of round cells in the submucosal area (Fig. 1b). Immunohistochemical studies revealed that these cells were positive for CD 138 (Fig. 1c) and κ chain (Fig. 1d), and negative for CD56, CD20 and CD79a. There was no evidence of deposition of amyloids in the stomach wall. Based on these findings, we diagnosed the patient with gastrointestinal invasion of MM. Although we continued the treatment with protonpump inhibitor, we were unable to control the gastrointestinal bleeding and she developed multi-organ failure. She died 3 weeks after admission. Gastrointestinal bleeding is a common complication of hematological malignancies, including MM. A variety of factors are involved in the gastrointestinal bleeding observed in MM patients [2]. First, paraneoprotein impairs the function of platelets and coagulation factors, leading to increased bleeding tendency. Treatments for MM may also increase the risk for bleeding, including from chemotherapy-induced thrombocytopenia, steroid-induced gastric ulcers, or the administration of aspirin or anticoagulants to prevent the thromboembolic complications associated with lenalidomide or thalidomide. Additionally, the deposition of amyloid protein in the gastrointestinal system can cause severe bleeding. In the present case, we found substantial involvement of myeloma cells in the gastric mucosa. Although the incidence is rare, previous case reports have indicated that gastrointestinal involvement of multiple myeloma, including severe gastrointestinal bleeding, may occur [2–5]. The prognosis of these patients is often extremely poor [2, 3]. A 62-year-old woman was hospitalized because of melena. She had been diagnosed with Bence Jones κ-type multiple myeloma (MM) 7 years prior. At the diagnosis of multiple myeloma, chromosome analysis of bone marrow cells revealed the karyotype abnormality 46, XX, t(7;8) (q32;q13) in one of 20 cells analyzed. Following the initial diagnosis of MM, she was treated with the combination of vincristine, doxorubicin, and dexamethasone, followed by high-dose melphalan chemotherapy with autologous stem cell transplantation (ASCT) [1]. She achieved only a partial response to these treatments, and was subsequently treated with thalidomide, and then bortezomib, for 3 years. During the clinical course, she developed multiple subcutaneous plasmacytomas. She underwent palliative radiation therapy, and treatment with lenalidomide was started with prophylactic anticoagulants. Three days before the hospitalization for melena, she experienced epigastric pain. At the time of hospitalization, the patient showed marked anemia (Hb 4.8 g/dL); however, her platelet counts were only modestly decreased (141 × 10/L; normal range 155– 345 × 10/L). Emergent esophagogastroduodenoscopy was performed, and massive bleeding was observed in the

Carcinoembryonic antigen (CEA), an oncofetal glycoprotein overexpressed in most gastrointestinal and lung cancers, is a candidate molecule for cancer immunotherapy. Recently, a CEA-derived 9-mer peptide, CEA652 (TYACFVSNL), has been identified as the epitope of cytotoxic T lymphocytes restricted with human leukocyte antigen (HLA)-A24, which is present in 60% of the Japanese population and in some Caucasians. The authors performed a clinical study of a vaccine using autologous dendritic cells (DCs) pulsed with CEA652 and adjuvant cytokines, natural human interferon alpha (nhuIFN-alpha), and natural human tumor necrosis factor alpha (nhuTNF-alpha), for the treatment of patients with CEA-expressing advanced metastatic malignancies. Ten HLA-A24 patients with advanced digestive tract or lung cancer were enrolled in the study to assess toxicity, tolerability and immune responses to the vaccine. DCs were generated from plastic adherent monocytes of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMCs) in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Generated DCs showing an immature phenotype were loaded with CEA652 and injected into patients intradermally and subcutaneously with 50% of the dose administered by each route every 2 weeks for a total of ten vaccinations. The total dose of administered DCs ranged from 2.7x10(7)cells to 1.6x10(8)cells. Adjuvant cytokines, i.e., 1x10(6) U/body of nhuIFN-alpha and nhuTNF-alpha, were administered to patients twice a week during the vaccination period. No severe toxicity directly attributable to the treatment was observed, and the vaccine was well tolerated. In the delayed-type hypersensitivity (DTH) skin test, two patients showed a positive skin response to peptide-pulsed DCs after vaccination, although none of the patients tested positive prior to vaccination. In the two patients who showed a positive skin response disease remained stable for 6 and 9 months respectively. These results suggest that active immunization using DCs pulsed with CEA652 peptide in combination with the administration of adjuvant cytokines is a safe and feasible treatment procedure.

Bendamustine is one of the new key drugs for patients with indolent lymphoma. Bendamustine, together with rituximab, significantly improves the treatment outcomes of these patients. In addition, previous clinical studies have shown the complication rate of severe infection in bendamustine-containing regimens to be relatively low as compared to those of conventional chemotherapeutic regimens such as CHOP. However, some clinical case reports have raised the possibility that bendamustine may abrogate the immune responses of patients and trigger opportunistic infections including cytomegalovirus reactivation. Herein, we report three indolent lymphoma cases becoming positive on cytomegalovirus antigenemia assay during bendamustine monotherapy. All events occurred after more than three courses of treatment with bendamustine. One patient showed decreased CD4 positive T lymphocytes before the development of cytomegalovirus antigenemia. All three patients were successfully treated with valganciclovir. Although the precise risk is unknown, it should be noted that bendamustine can potentially cause reactivation of/infection with cytomegalovirus and physicians should pay attention to the possibility of this infection during treatment with bendamustine-containing regimens.

The hypoxic microenvironment of the bone marrow, known as the hypoxic niche, supports hematopoietic stem cell quiescence and maintains long-term repopulation activity. Hypoxia also affects the expansion of progenitor cells and enhances erythropoiesis and megakaryopoiesis. In contrast to the known effects of hypoxia on normal hematopoiesis, the effects of the hypoxic environment of the bone marrow on the pathogenesis of myeloproliferative neoplasms (MPNs) have not been well studied. In the present study, we investigated the role of the hypoxic environment in the pathophysiology of MPNs, focusing on JAK2V617F, a major driver of mutation in Philadelphia-negative MPNs. We found that the activity of JAK2V617F was suppressed in hypoxic conditions not only in JAK2V617F-positive leukemia cells, but also in primary peripheral blood mononuclear cells from patients with polycythemia vera. Concomitant with the inhibition of JAK2V617F activity, hypoxia increased the expression of p27/KIP1, the primary negative regulator of the cell cycle, and inhibited cell cycle progression in JAK2V617F-positive leukemia cell lines. The spontaneous erythroid colony formation of primary cells from polycythemia vera patients was also suppressed under hypoxic conditions. We also revealed that the hypoxia-induced overproduction of reactive oxygen species played a crucial role in the inhibition of JAK2V617F through the oxidation and inhibition of SHP-2, a protein tyrosine phosphatase that contains SH-2, which is required for JAK2 activation. In conclusion, a hypoxic environment may modulate JAK2-positive MPN cell fate and disease progression through the suppression of SHP-2 function and the subsequent suppression of JAK2V617F activity.

The design of an effective procedure to sensitize and expand antigen-specific cytotoxic T lymphocytes (CTL) in vitro is essential for the development of effective adoptive cellular immunotherapy protocols for cancer. We have analyzed the capacity of tissue culture-derived dendritic cells (DC) to present specific peptide epitopes to CTL precursors. Our results demonstrate that peptide-pulsed DC were efficient in generating CTL responses specific for various viral and tumor epitopes. Furthermore, IL-7 and IL-10 potentiated the ability of the peptide-pulsed DC to trigger antigen-specific CTL responses. The CTL generated using this procedure efficiently recognized the naturally processed antigens and could be expanded approximately 100- to 1000-fold in tissue culture in 10 to 15 days without a loss of activity and specificity. The results and procedures described herein may facilitate the development of effective CTL-based adoptive immunotherapy for chronic viral diseases and cancer.

One approach to development of specific cancer immunotherapy relies on the induction of cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens (TAA). Induction of TAA-specific CTL could be used towards the eradication of established tumors, or to prevent their dissemination or recurrence after primary treatment. The present study identifies a set of CTL epitopes from TAA frequently found on solid epithelial tumors such as breast, lung and gastro-intestinal tumors. Specifically, HLA-A2.1 binding peptides from the MAGE2, MAGE3, HER-2/neu and CEA antigens were tested for their capacity to elicit in vitro anti-tumor CTL using lymphocytes from normal volunteers and autologous dendritic cells as antigen-presenting cells. A total of 6 new epitopes (MAGE2[10(157)], MAGE3[9(112)], CEA[9(691)], CEA[9(24)], HER2[9(435)] and HER2[9(5)]) were identified which were capable of specifically recognizing tumor cell lines lines expressing HLA-A2.1 and the corresponding TAA. In one case (CEA[9(24)]), induction of vigorous anti-tumor CTL responses required epitope engineering to increase HLA-A2.1 binding affinity. Finally, most of the newly identified epitopes (5 out of 6) were found to be highly crossreactive with other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating their potential in providing broad and non-ethnically biased population coverage. The results are discussed in the context of the development of multi-epitope-based therapies with broad applicability for patients suffering from commonly found tumors.

To study the clinical presentation of lung cancer patients at our palliative care unit (PCU).We examined the clinical presentation of lung cancer patients at our PCU and compared it with the clinical presentation of patients with malignant tumors besides those indicative of lung cancer.The PCU occupancy ratio of lung cancer patients to inpatients was 24%, which was dependent on the type of carcinoma. Lung cancer patients in need of oxygen inhalation and/or terminal sedation were more in number than those with other types of malignant tumors. On the other hand, few patients needed treatment. Dyspnea is the major reason for the terminal sedation of lung cancer patients.Severe dyspnea appeared to be the typical clinical presentation of terminal-phase lung cancer patients.

Rearrangements of the mixed lineage leukemia MLL gene at chromosome 11q23 are common chromosomal abnormalities in human leukemia. MLL fused with numerous partner genes causes different leukemia phenotypes that depend on the function of partner genes. MLLT3-MLL is generated by translocation t(9;11), which primarily induces acute myeloid leukemia in humans, whereas MLLT3-MLL induces ALL or biphenotypic leukemia in mice. The microenvironment that surrounds leukemia cells plays a central role in this process. We report a patient with mixed phenotype acute leukemia with MLLT3-MLL. This patient, a 44-year-old woman, initially exhibited extramedullary leukemia with multiple tumors and subsequently developed bone marrow disease. The leukemia cells exhibited myeloid (CD13 and MPO) and B cell (CD19 and CD79a) phenotypes. Chromosomal analysis and RT-PCR assay revealed tumor cells with the MLLT3-MLL fusion gene. We treated this patient with a drug regimen for AML (Ara-C plus anthracycline), and complete remission was obtained. This report describes the fourth case of mixed phenotypic leukemia with extramedullary disease. The extramedullary circumstance may underlie the biphenotypic features of these patients.

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

The introduction of rituximab, an anti-CD20 monoclonal antibody, has dramatically improved the treatment outcomes of patients with B cell lymphoma. Nevertheless, the clinical response to rituximab varies, and a subpopulation of patients does not respond well to this antibody. Although several molecular events have been shown to be involved in the mechanism of action of rituximab, recent studies have demonstrated that intracellular signaling pathways and the direct effects of rituximab on cell membrane components are responsible for the antilymphoma action of this drug. In the present study, we demonstrated that rituximab activated Syk and Akt, molecules with antiapoptotic functions, in several CD20-positive lymphoma cell lines. Notably, rituximab activated Syk and Akt in all the tested primary lymphoma samples from six patients. Our results show that the cholesterol levels in lymphoma cell membranes have a crucial role in the regulation of Syk and Akt. The depletion of cholesterol from the cell membrane completely blocked rituximab-induced Syk and Akt activation. Simvastatin, an inhibitor of cholesterol synthesis, also abrogated rituximab-mediated Syk and Akt activation. Finally, we report that rituximab inhibited the apoptosis induced by chemotherapeutic drugs, which was observed solely in Akt-activated cells. This work demonstrates for the first time that rituximab paradoxically works to suppress apoptosis under certain conditions in a manner that is dependent on the cell membrane cholesterol level. Our observations provide novel insights and suggest that the cell membrane cholesterol level represents a new biomarker for predicting patient response to rituximab. Furthermore, the modulation of lipid rafts could provide a new strategy for enhancing the antilymphoma action of rituximab.

Metformin, which is a member of the biguanide family, suppresses the growth of a variety of malignant hematological cells. It is widely accepted that metformin inhibits the growth of these cells primarily by suppressing the mTOR pathway. In contrast, we previously found that metformin directly suppresses the activation of JAK2V617F in HEL and SET-2 cells. In the present study, we investigated the precise molecular mechanisms through which metformin inhibits the auto-phosphorylation of JAK2V617F. Initially, we speculated that AMPK, which is one of the main downstream molecules activated by metformin, might be involved in the process. Compound C, an inhibitor for AMPK, resumed the JAK2 activity in cells treated with metformin. For the next step, we introduced siRNA against an α subunit of AMPK and analyzed the phosphorylation levels of JAK2V617F. We found that knock-down of AMPK enhanced the basal phosphorylation levels of JAK2V617F but that metformin-induced JAK2 inhibition was only partially diminished. These results suggested that although AMPK was responsible for the metformin-induced JAK2V617F inhibition, other mechanisms are also required to achieve the full inhibition of JAK2V617F activity.Recently, several groups have revealed that PP2A-activationg drugs suppress the activity of JAK2V617F. In addition, recent studies have demonstrated that metformin activates PP2A. These reports inspired us to investigate the possibility that PP2A might be responsible for the metformin-mediated suppression of JAK2V617F. PP2A consists of three different subunits: A, B and C. The A subunit works as a scaffold, and the C subunit has catalytic activity. The B subunit contains a variety of members, and the diversity of the B units determines the substrate specificity. Initially, we analyzed whether metformin activates PP2A in JAK2V617F-positive cells. We found that metformin decreases the phosphorylation of the C-subunit of PP2A at Tyr 308, which negatively regulates PP2A function. For further study, we prepared the A subunit of PP2A knock-down cells using siRNA. We expected that the knock-down of PP2A would restore the JAK2V617F activity; instead, JAK2V617F was suppressed in these cells. To explain the discrepancy, we focused on the B subunits and hypothesized that only a specific type of PP2A complex is involved in the de-phosphorylation of JAK2V617F. To this end, we introduced a panel of siRNA targeting different B subunits and analyzed the effects on JAK2V617F activity. Knock-down of the B56α subunit resulted in the activation of JAK2V617F. In contrast, the JAK2V617F phosphorylation levels were decreased in the B56γ knock-down cells. Interestingly, we found that B56γ knock-down cells have enhanced AMPK activity. These results indicate that among the PP2A complexes, B56α containing complex works as negative regulator for JAK2 but that the B56γ subunit-containing complex may positively regulate JAK2 by inhibiting AMPK.In conclusion, both AMPK and PP2A-containing B56α subunits are involved in the metformin-induced JAK2V617F inhibition. In contrast, the B56γ subunit-containing PP2A may diminish metformin-induced JAK2 inhibition via the negative regulation of AMPK. These results suggest that the specific inhibition of the B56γ subunit containing PP2A may enhance the anti-leukemic action of metformin. Disclosures Kirito: Novartis Pharma KK: Honoraria.

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P-labeled oligonucleotide probes that encoded parts of the partial amino-acid sequence for the Zn-containing Ec protein. Each DNA insert in 11 positives from a screen of 10(5) plaques encoded a 5' untranslated and a 3' untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the Ec protein. The three different mRNA sequences encoded in the cDNA probably correspond to single-copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P-labeled cDNA and gave a single positive in a screen of 5 x 10(5) plaques. A 3.1-kb genomic fragment (gf-3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf-3.1 sequence encodes an intronless mRNA for the Ec protein and contains appreciable amounts of 5' and 3' flanking sequences. In addition to a putative TATA box, two inverted-repeat sequences and one direct-repeat sequence, the 5' flank in gf-3.1 contains a sequence similar to the abscisic-acid-responsive element in other higher-plant genes but does not contain sequences similar to the metal-responsive elements in animal metallothionein genes. Consistent with these findings, RNA blotting shows that accumulation of Ec mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat Ec metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.

Interleukin-11 (IL-11) is a novel stroma-derived cytokine that acts on both hematopoietic progenitors and stromal cells. IL-11 was originally identified in a medium conditioned by the macaque bone marrow-derived stromal cell line PU-34 and cloned as a growth factor for the IL-6-dependent plasmacytoma cell line T1165. IL-11 stimulates T-cell dependent development of antibody-producing B cells and is synergistic with IL-3 to stimulate megakaryocyte colony formation. Adipogenesis inhibitory factor (AGIF) was cloned from the human bone marrow-derived stromal cell line KM-102. The AGIF cDNA sequence was revealed to be identical to that of the IL-11 cDNA. AGIF inhibits the process of adipogenesis of the bone marrow-derived preadipocyte cell line H-1 A . Other biological activities such as stimulation of stem-cell proliferation, erythropoiesis, lymphohematopoiesis and hepatic acute-phase response are also summarized. The human IL-11 gene consists of five exons and four introns, and was mapped on chromosome 19 at band 19q13.3-q13.4. A single class of high-affinity IL-11 receptor (IL-11R) of 151 kDa is present on 3T3-L1 preadipocytes. A protein-tyrosine kinase pathway may be involved in the initiation of the IL-11R-mediated signal transduction.

The homologue of the rat pancreatic elastase I gene was found in the human genome, but its transcription was completely suppressed in the adult human pancreas as we reported previously. In this study, we characterized the complete structure of the eight putative exons of the silent gene for human elastase I. A genotype analysis of the exon 1 DNA sequence revealed that at least two allelic elastase I genes are present in human genomes. A primate-specific repetitive DNA element (MER1) was identified in the 3'-flanking region of the human elastase I gene. The primary structure of human preproelastase I, deduced from the sequences of the eight exons, showed an 89% identity with that of porcine or rat pancreatic preproelastase I. The amino acid residues of the serine protease catalytic triad and the eight cysteine residues conserved in the elastase family were present at positions equivalent to those observed in porcine and rat elastase I, suggesting that the gene product may function as an elastolytic enzyme if this gene is expressed in any tissue.