Masataka Hosoi

Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples.

Induced pluripotent stem cells (iPSCs) can be generated by the expression of defined transcription factors not only from normal tissue, but also from malignant cells. Cancer-derived iPSCs are expected to provide a novel experimental opportunity to establish the disease model. We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. Remarkably, the CML-iPSCs were resistant to imatinib although they consistently expressed BCR-ABL oncoprotein. In CML-iPSCs, the phosphorylation of ERK1/2, AKT, and JNK, which are essential for the maintenance of both BCR-ABL (+) leukemia cells and iPSCs, were unchanged after imatinib treatment, whereas the phosphorylation of signal transducer and activator of transcription (STAT)5 and CRKL was significantly decreased. These results suggest that the signaling for iPSCs maintenance compensates for the inhibition of BCR-ABL. CML-iPSC-derived hematopoietic cells recovered the sensitivity to imatinib although CD34(+)38(-)90(+)45(+) immature cells were resistant to imatinib, which recapitulated the pathophysiologic feature of the initial CML. CML-iPSCs provide us with a novel platform to investigate CML pathogenesis on the basis of patient-derived samples.

The effect of iron overload and chelation on erythroid differentiation

We investigated the mechanisms of hematopoietic disorders caused by iron overload and chelation, in particular, the inhibition of erythroblast differentiation. Murine c-kit+ progenitor cells or human CD34+ peripheral blood hematopoietic progenitors were differentiated in vitro to the erythroid lineage with free iron and/or an iron chelator. Under iron overload, formation of erythroid burst-forming unit colonies and differentiation to mature erythroblasts were significantly suppressed; these effects were canceled by iron chelation with deferoxamine (DFO). Moreover, excessive iron burden promoted apoptosis in immature erythroblasts by elevating intracellular reactive oxygen species (ROS). Interestingly, both DFO and a potent anti-oxidant agent reduced intracellular ROS levels and suppressed apoptosis, thus restoring differentiation to mature erythroblasts. Accordingly, intracellular ROS may represent a new therapeutic target in the treatment of iron overload.

Reversible posterior leukoencephalopathy syndrome following R-CHOP therapy for diffuse large B-cell lymphoma

Dear Editor, Reversible posterior leukoencephalopathy syndrome (RPLS) is rare and characterized by angioedema in the white matter of bilateral occipital lobes. It is usually related to hypertension or the use of immunosuppressive agents, and its development after chemotherapy is rare. We report a case of RPLS following severe paralytic ileus after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) chemotherapy. A 39-year-old female presented with dyspnea. Examination revealed bilateral neck and axial lymph node swelling. Computed tomography (CT) showed a superior mediastinal tumor, bilateral pleural effusion, cardiac effusion, general lymphadenopathy, and tumor infiltration of both kidneys and the liver. Excision biopsy of the neck lymph node confirmed diffuse large B-cell lymphoma. The clinical stage was IVa, and age-adjusted International Prognostic Index was high intermediate. Standard R-CHOP chemotherapy was started. Intrathecal therapy was not administered. A chest x-ray taken before the second cycle showed marked improvement in the pleural effusion and mediastinal shadow. On the ninth day from the second cycle, the patient developed severe constipation and abdominal pain. She was admitted to the hospital and diagnosed with paralytic ileus. Her symptoms gradually resolved with conservative treatment. Slight increase in blood pressure from 120–130/80– 90 to 150–170/90–110 mmHg was sometimes recorded a few days after hospital admission. On the 17th day from the second cycle, she experienced a generalized tonic-clonic seizure. Intravenous phenytoin infusion was started. Cranial CT revealed low-density areas in the white matter of both posterior lobes. On the next day, magnetic resonance imaging (MRI) revealed, at the same site, high signal changes on T2-weighted (Fig. 1a) and T2weighted fluid-attenuated inversion-recovery images, low signal changes and patchy enhancement on gadoliniumenhanced T1-weighted images, and low signal changes on diffusion-weighted images. Electroencephalogram revealed no abnormal findings. Cerebrospinal fluid examination showed evidence of neither lymphoma infiltration nor infection. The patient was diagnosed with RPLS, and we controlled her blood pressure. She recovered in a day without any neurological symptoms. MRI performed 2 weeks after the seizure revealed marked improvement of bilateral occipital lesions (Fig. 1b). As her lymphoma had regrown when the chemotherapy was interrupted, she received salvage therapy (cytarabine and etoposide) followed by autologous peripheral blood stem cell transplantation. She maintains complete response to date, and the seizure has not recurred for 1 year. The considering causes of RPLS include hypertension, eclampsia, renal failure, and cytotoxic or immunosuppressive medications [1]. Reports of the development of RPLS after CHOP or R-CHOP therapy are limited [2, 3] despite its wide use against lymphoma, and the risk factors of RPLS after chemotherapy remain unclear. In this case, the M. Hosoi :G. Yamamoto :Y. Imai :M. Kurokawa (*) Department of Hematology & Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Generation of induced pluripotent stem cells derived from primary and secondary myelofibrosis patient samples.

Induced pluripotent stem cells (iPS) derived from disease cells are expected to provide a new experimental material, especially for diseases from which samples are difficult to obtain. In this study, we generated iPS from samples from patients with primary and secondary myelofibrosis. The primary myelofibrosis cells had chromosome 13q deletions, and the secondary myelofibrosis (SMF) cells had JAK2V617F mutations. The myelofibrosis patient cell-derived iPS (MF-iPS) were confirmed as possessing these parental disease-specific genomic markers. The capacity to form three germ layers was confirmed by teratoma assay. By co-culture with specific feeder cells and cytokines, MF-iPS can re-differentiate into blood progenitor cells and finally into megakaryocytes. We found that mRNA levels of interleukin-8, one of the candidate cytokines related to the pathogenesis of myelofibrosis, was elevated predominantly in megakaryocytes derived from MF-iPS. Because megakaryocytes from myelofibrosis clones are considered to produce critical mediators to proliferate fibroblasts in the bone marrow and iPS can provide differentiated cells abundantly, the disease-specific iPS we established should be a good research tool for this intractable disease.

Biliary cast syndrome and benign biliary stricture as complications of allogeneic hematopoietic stem cell transplantation

Dear Editor, Biliary cast syndrome (BCS) and benign biliary stricture (BBS) are known as a complication of liver transplantation (LT) [1, 2]; however, it is uncommon in patients who received hematopoietic stem cell transplantation (HSCT). We report a case of BCS and BBS after bone marrow transplantation (BMT). A 36-year-old female with myelodysplastic syndrome received allogeneic BMT from ABO major mismatched donor (donor: A, recipient: O) after standard cyclophosphamide and total body irradiation (CY–TBI) conditioning. Graft-versus-host disease (GVHD)-prophylaxis was performed by cyclosporine A and methotrexate. Her early transplant course was complicated with systemic adenovirus infection, which caused multiple organ failure. She also had stage IV acute liver GVHD that showed good response to systemic administration of steroid and escalated dose of cyclosporine A. Despite improvement of acute GVHD, the levels of hepato-biliary enzymes continued to be elevated. Endoscopic ultrasonography revealed a large elongated object in the common bile duct. That object was removed endoscopically using an extraction balloon after balloon dilation of the papilla and was diagnosed as a biliary cast on day 168 (Fig. 1a, b) [3–7]. At this time, the cholangiogram revealed the appearance of a moderate stricture of the right hepatic duct. Apart from chronic GVHD, which was successfully treated by escalated dose of cyclosporine A, no further complication had been detected until jaundice developed suddenly on day 772. Magnetic resonance cholangiopancreatography demonstrated dilated ducts proximal to the stricture (Fig. 1c). She received endoscopic drainage with 7 French plastic stent after balloon dilation because of tight stricture (Fig. 1d). The cholangiogram of the stricture was smooth and there was no information suggesting malignancy by the intraductal ultrasonography and brushing cytology. Then, we kept the stent for 1 year to dilate the stricture with every 3 months exchange, and the stricture improved. BCS is a common complication of LT. However, there are only a few reports in non-LT [1, 2, 5, 8, 9], and there is no report after HSCT. BBS is also a rare complication after HSCT (0.12%) and occurs within 1 year after HSCT [10]. The risk factors of BCS in the non-liver transplant case is reported to include bile duct damage, ischemia, infection, fasting, parenteral nutrition, and abdominal surgery [11]. In post-liver transplantation settings, acute rejection is an important cause, which suggests that immune reaction can be a factor to develop BCS. The pathogenesis of post-HSCT BBS seemed to be associated with cholangitis, viral infection, and ischemia. BBS after LT may be related to alterations of immune response by several conditions such as M. Hosoi :Y. Nannya :H. I. Suzuki :K. Ueda : T. Takahashi : M. Kurokawa (*) Department of Hematology & Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Erdheim–Chester disease with an 18F-fluorodeoxyglucose-avid breast mass and BRAF V600E mutation

Abstract Erdheim–Chester disease (ECD) is a non-Langerhans cell histiocytosis. Herein we report a case of a 49-year-old woman who developed bilateral knee pain. Imaging procedures revealed multiple long bone lesions and a well-defined 18F-fluorodeoxyglucose-avid mass in the left breast. The breast mass was resected, and an open biopsy was performed on the right femoral lesion. Both specimens revealed involvement by histiocytic infiltrates with features suggestive of ECD. The BRAF V600E mutation was detected by DNA sequencing and immunohistochemistry.

A case of anaplastic large cell lymphoma, ALK positive, primary presented in the skin and relapsed with systemic involvement and leukocytosis after years of follow-up period

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) is one disease form of primary cutaneous T cell lymphoma (CTCL) that presents diffuse infiltration of large CD30 positive tumor cells. Its clinical course is usually favorable, with a 10-year disease-related survival of approximately 90% [1]. Unlike systemic anaplastic large cell lymphoma (ALCL), C-ALCL usually does not have translocations involving the anaplastic lymphoma kinase (ALK) gene. We report a case of ALK positive C-ALCL previously reported at the time of diagnosis [2] who developed systemic relapse with leukocytosis after years of the clinical course. A 54-year-old female first presented with progressive subcutaneous nodule on her left forehead. Excisional biopsy of the lesion revealed diffuse infiltration of anaplastic large lymphoma cells expressing CD30 and ALK, but in the previous report of this case at diagnosis, nucleophosmin (NPM)-ALK was not detected by reverse transcription-polymerase chain reaction (RT-PCR) [2]. She repeated local skin relapse thereafter, but was successfully treated by total excision or electron beam irradiation each time. Lymph node involvement occurred approximately two and a half years after her first presentation. By six courses of standard CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy, she achieved complete response (CR). After three years of another follow-up period, she presented repetitive vomiting after digestion and was admitted to our hospital. The laboratory data showed leukocytosis with atypical lymphocytes in the peripheral blood, thrombocytopenia, hyperbilirubinemia, and elevated serum inflammatory markers including C-reactive protein and soluble interleukin-2 receptor. Computed tomography (CT) revealed lymphadenopathy in the mediastinum and the abdomen, and hepatosplenomegaly and ascites that have not been evident one month before. Bone marrow biopsy revealed marked fibrosis. Flow cytometry of the peripheral blood detected CD30 and CD4 positive T cell population, which corresponded to the atypical lymphocytes initially involving the skin lesions, and she was diagnosed with relapse of ALCL. The cytogenetic analysis of the peripheral blood cells revealed a complex abnormal karyotype including t(2;5)(p23;q35), which is a typical chromosomal translocation resulting in the formation of the NPM-ALK gene often found in ALCL. Although temporary partial response (PR) was obtained after two courses of modified ESHAP therapy (methylprednisolone, etoposide, high dose cytarabine, and carboplatin), rapid tumor regrowth was evident after each course of chemotherapy. The disease became refractory after that, and the patient died five months after the systemic relapse. This case was previously reported as NPM-ALK fusion negative by RT-PCR. However, at the time of relapse, the peripheral blood cells revealed a complex abnormal karyotype including t(2;5)(p23;q35) suggesting NPM-ALK fusion. To obtain further information on the breakpoint of this translocation, fluorescent in situ hybridization (FISH) analysis using probes on the both sides of ALK gene at 2p23 [Vysis LSI ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc., Illinois, USA)] was performed and revealed 0 breaks per 200 tested cells at M. Hosoi M. Ichikawa Y. Imai M. Kurokawa (&) Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient’s age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient’s derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

Evans syndrome with cytomegalovirus infection followed by emerging peripheral T-cell lymphoma.

Dear Editor, Malignant lymphomas occasionally accompany autoimmune diseases. Autoimmune hemolytic anemia (AIHA) and idiopathic thrombocytopenic purpura (ITP) rarely occur in lymphoma. Here, we report the case of a patient with Evans syndrome who subsequently developed cutaneous Tcell lymphoma. An 80-year-old Japanese man visited a hospital with a chief compliant of petechiae. He was diagnosed with anemia and thrombocytopenia. At this hospital, cytomegalovirus (CMV) antigenemia was found to be positive, and he was administered gancyclovir. Although CMV antigenemia eventually became negative, anemia and thrombocytopenia persisted; therefore, he was transferred to our hospital. The initial results of laboratory investigations were as follows: white blood cell count, 7,900/μL; hemoglobin, 49 g/L; platelet count, 1×10/μL; reticulocytes, 4.3%; total bilirubin, 1.6 mg/dL; and lactate dehydrogenase, 598 IU/L. Coomb’s test was positive. Bone marrow aspiration showed no dysplasia and a modest increase in megakaryocytes. We diagnosed the patient with Evans syndrome. There were no symptoms suggestive of collagen disease, and antinuclear antibodies were absent. Computed tomography (CT) imaging of the chest and abdomen showed no abnormal findings. Because of a remarkable bleeding tendency, he was administered a high dose of gamma globulin (0.4 g/kg for 5 days) and prednisolone at a dose of 1 mg/kg. Four days after starting therapy, his platelet count began to increase. After 1 month, his blood cell counts became almost normal, and we started to taper the dose of prednisolone. Prednisolone administration was discontinued after 5 months. Three months after discontinuing prednisolone, the patient developed a progressive generalized itchy rash (Fig. 1). Examination of a skin biopsy specimen showed the presence of cutaneous T-cell lymphoma. CT imaging and bone marrow biopsy showed no other lymphoma lesions. He received a topical steroid and psoralen plus ultraviolet light therapy. One year after diagnosis of lymphoma, his blood cell counts remained normal. Immune cytopenias such as ITP and AIHA are rare complications of lymphoma [1]. In some cases, lymphoma cells produce autoantibodies against blood cells [2] In most cases, immune abnormalities associated with lymphoma are thought to cause immune cytopenia, and immune cytopenia occurs when the lymphoma is active. In such cases, antilymphoma treatment is more effective than conventional therapy with steroids [1]. Evans syndrome occurring in lymphoma is very rare [3–6], and our case is notable in the following three aspects. First, Evans syndrome preceded lymphoma by 8 months; most immune cytopenias with lymphoma are reported to occur when the lymphoma is active. Second, CMV infection coincided with Evans syndrome. Because CMV infection is an opportunistic infection, our patient was suspected to have an immune disorder in addition to Evans syndrome. Third, conventional steroid therapy was effective in our case, and Evans syndrome did not relapse after discontinuing administration of steroids. The findings of our case suggest that subclinical lymphoma can cause immune disorders that result in G. Yamamoto :M. Hosoi :M. Ichikawa :M. Kurokawa (*) Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp