三村 尚也

Immunosuppressive therapy (IST) with a combination of antithymocyte globulin (ATG) and cyclosporine (CsA) is an effective therapeutic modality for patients with aplastic anemia (AA) who are not eligible for allogeneic stem cell transplantation (Allo-SCT) from a human leukocyte antigen-identical sibling donor. However, there have been reports of some patients developing lymphoproliferative disorder (LPD) after IST for AA. We herein report a case of a 26-year-old man with severe AA (SAA) complicated by LPD after a single course of IST, who was successfully treated with Allo-SCT from an unrelated donor. Two months after starting IST for SAA, he developed LPD in the stomach. CsA was reduced, however, his neutrophil counts decreased, and CsA could not be discontinued. The patient was treated with rituximab monotherapy, and LPD resulted in complete remission. However, he failed IST for SAA and underwent Allo-SCT with reduced-intensity conditioning to recover his hematopoiesis. The patient has achieved complete hematopoietic recovery without the recurrence of LPD for five years after transplantation. This is the first report of successful Allo-SCT for SAA after the treatment of LPD caused by the use of rabbit ATG. This case provides useful information for the management of SAA with the development of LPD after IST.

Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.

We retrospectively reviewed 413 recipients with hematologic malignancies who underwent hematopoietic stem cell transplantation (HSCT) between June 1986 and March 2013. Recipients with antibody to hepatitis B core antigen (anti-HBc) and/or to hepatitis B surface antigen (anti-HBs) were regarded as experiencing previous hepatitis B virus (HBV) infection. Clinical data of these recipients were reviewed from medical records. We defined >= 1 log IU/mL increase in serum HBV DNA from nadir as HBV reactivation in hepatitis B surface antigen (HBsAg)-positive recipients, and also defined >= 1 log IU/mL increase or re-appearance of HBV DNA and/or HBsAg as HBV reactivation in HBsAg-negative recipients. In 5 HBsAg-positive recipients, 2 recipients initially not administered with nucleos(t) ide analogues (NUCs) experienced HBV reactivation, but finally all 5 were successfully controlled with NUCs. HBV reactivation was observed in 11 (2.7%) of 408 HBsAg-negative recipients; 8 of these were treated with NUCs, and fortunately none developed acute liver failure. In 5 (6.0%) of 83 anti-HBc and/or anti-HBs-positive recipients, HBV reactivation occurred. None of 157 (0%) recipients without HBsAg, anti-HBs or anti-HBc experienced HBV reactivation. In HSCT recipients, HBV reactivation is a common event in HBsAg-positive recipients, or in HBsAg-negative recipients with anti-HBc and/or anti-HBs. Further attention should be paid to HSCT recipients with previous exposure to HBV.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. (C)2014 AACR.

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Purpose: The efficacy of peptide vaccines may be enhanced by stimulating immune cells with multiple peptides derived from distinct tumor-associated antigens. We have evaluated the heteroclitic XBP1-US184-192 (YISPWILAV), heteroclitic XBP1-SP367-375 (YLFPQLISV), native CD138(260-268) (GLVGLIFAV), and native CS1(239-247) (SLFVLGLFL) peptides, which have strong HLA-A2 affinity and immunogenicity in combination, for their ability to elicit multiple myeloma antigen-specific responses. Experimental Design: Multipeptide-specific cytotoxic T lymphocytes (MP CTL) were generated by the stimulation of CD3(+) T lymphocytes from HLA-A2(+) individuals with either autologous mature dendritic cells or T2 cells pulsed with a cocktail of these four peptides. Results: The peptide cocktail did not compromise tumor antigen-specific activity of CTLs. MP-CTLs displayed increased total, effector memory (CCR7(-) CD45RO(+)), and activated (CD69(+)) CD3(+)CD8(+) T lymphocytes. In addition, MP-CTL showed IFN-gamma production, cell proliferation, and cytotoxicity against HLA-A2(+) multiple myeloma cells, including cells of HLA-A2(+) patients with multiple myeloma. Importantly, MP-CTLs showed specific responses in functional assays to each relevant peptide but not to an irrelevant HLA-A2-specific CMV pp65 (NLVPMVATV) peptide. Conclusions: These results highlight the potential therapeutic application of vaccination with a cocktail of HLA-A2-specific peptides to induce CTLs with a broad spectrum of immune responses against multiple myeloma antigens. Clin Cancer Res; 18(17); 4850-60. (C) 2012 AACR.

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock-in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild-type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild-type mice. The activation of glycogen synthase kinase 3b (GSK-3b) was associated with morphine tolerance, because an inhibitor of GSK-3 beta prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK-3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild-type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.

Chemoradiotherapy has improved the outcome of patients with esophageal cancer. Although a sufficiently long-time survival has resulted in the increase of several treatment-related late toxicities, little is still known about the incidence of secondary malignancies. In our hospital, 348 patients with esophageal cancer received chemotherapy consisting of nedaplatin and 5-fluorouracil and concurrent irradiation. Median and average follow-up durations were 8 and 21 months (1-92), respectively. Four patients developed leukemia after 19-48 months of follow-up. Two patients were diagnosed with overt leukemia from myelodysplastic syndrome presenting a complex karyotype, including the deletion of chromosome 5 or 7. Notably, one patient showed an additional chromosomal abnormality with t(9;22)(q34;q11). Other patients developed acute myeloid leukemia with t(9;22)(q34;q11) and Burkitt leukemia with t(8;14)(q24;q32). All patients eventually succumbed to leukemia. Platinum and fluorouracil have shown relatively lower risks for secondary malignancies in comparison with alkylating agents and topoisomerase II inhibitors. Especially, nedaplatin has never been described to introduce secondary neoplasms. Our report supports the idea that the concurrent administration of radiotherapy with these agents affects the risk of leukemia. Interestingly, rare balanced chromosomal abnormalities were observed in the present cases, thus providing new insights into the leukemogenesis of therapy-related leukemia.

The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferon gamma, and IL-6 regulator suppressor of cytokine signaling (SOCS) 1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing-MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM. (Blood. 2010;116(17):3227-3237)

Reactivation of hepatitis B virus (HBV) has been recognized as one of the most serious complications in patients receiving chemotherapy with rituximab. From October 2007 to December 2008, rituximab was administered to 123 B-cell lymphoma patients in our institute. Four patients with positive hepatitis B surface antigen (HBsAg) received preemptive entecavir, and none of them developed HBV reactivation. For 26 patients whose hepatitis B surface antibody (HBsAb) and/or hepatitis B core antibody (HBcAb) were positive, HBV-DNA was monitored for one year after completion of chemotherapy. During this period, HBV reactivation was observed in two patients. Hepatitis was prevented in one patient by the administration of entecavir at the time HBV-DNA turns positive. Another developed de novo hepatitis B due to failure of monitoring. Preemptive entecavir for HBsAg positive patients and HBV-DNA monitoring for HBsAb and/or HBcAb positive patients seem to be effective.

リツキシマブを投与したB細胞リンパ腫患者123例を対象に、B型肝炎ウイルス(HBV)再活性化予防について検討した。治療内訳はR-CHOP療法施行が97例、リンパ腫再発例でリツキシマブを併用したCHASE療法が13例、ESHAP療法が2例、MEVP療法が7例、その他の治療が4例であった。HBs抗原陽性は123例中4例に認め、いずれもHBV-DNA陽性で、化学療法開始時〜終了後最低1年間エンテカビル(ETV)の予防投与を行い、4〜12ヵ月経過して肝炎発症は認めていない。HBs抗体とHBc抗体のスクリーニングは92例で行い、HBs抗原陰性でHBs抗体とHBc抗体のいずれかが陽性は26例であった。化学療法開始時のHBV-DNA定量はいずれも測定感度以下で、開始後に感度以上となったのは2例であった。1例はETV投与により再度感度以下となり肝炎発症はなく、他の1例はde novo肝炎を発症したがETV投与で軽快した。なお、HBs抗体とHBc抗体を測定しなかった31例においては、肝炎発症は認めていない。

Chronic myeloid leukemia (CML) is generally diagnosed in the chronic phase. We have recently encountered two CML-blastic phase (BP) cases, a 71-year-old woman and a 74-year-old man, who resembled de novo acute leukemia. The complete blood count was normal at least 11 and 13 months before the presentation, respectively. The leukemic cells showed predominant lymphoid phenotype. The blasts and granulocytes were positive for BCR-ABL, indicative of CML-BP. Both patients were successfully treated with prednisone and vincristine, followed by Imatinib. Our cases indicate rare presentations of CML-BP with an extremely short chronic phase. Ph-positive de novo acute leukemia should be carefully distinguished from CML-BP.

Here we report three cases of hepatitis B virus (HBV) reactivation after cessation of preemptive lamivudine therapy in B-cell lymphoma patients treated with rituximab plus CHOP (R-CHOP). Two patients received eight cycles of R-CHOP, and one received two cycles of R-CHOP followed by two courses of rituximab. As all the patients were HBV surface antigen (HBsAg) positive, lamivudine was administered simultaneously with R-CHOP to prevent virus reactivation. All the patients developed hepatitis due to HBV reactivation 6, 8 and 13 months after completion of chemotherapy, and 4, 2 and 2 months after cessation of lamivudine, respectively. They were treated with either lamivudine or entecavir and all achieved full recovery. When HBV carriers undergo immunosuppressive anticancer treatment, prophylactic antiviral therapy is well recognized as effective. However, the optimal method of prophylaxis has not yet been established. Since the introduction of rituximab, new problems such as delayed HBV reactivation from HBsAg positive patients and de novo hepatitis B from HBsAg negative patients have emerged. Guidelines for prophylactic antiviral therapy in the era of rituximab need to be established.

リツキシマブ併用化学療法におけるラミブジン(LAM)予防投与終了後にB型肝炎ウイルス(HBV)再活性化を来したB細胞リンパ腫3例を報告する。3例ともにHBs抗原陽性のHBVキャリアであり,リツキシマブ併用CHOP療法にLAMによる肝炎予防を併用した。HBV再活性化の時期は,リツキシマブ併用化学療法終了後それぞれ6,8,13ヵ月後であった。また,LAMの内服期間は治療後それぞれ2,6,11ヵ月であった。2例はLAMの再開,1例はエンテカビル投与によって治療し軽快した。HBVキャリアに対する化学療法時の抗ウイルス薬予防投与は広く行われているが,その投与方法や期間は定まっていない。リツキシマブの登場以来,遅発性のHBV再活性化やHBs抗原陰性例でのde novo肝炎が問題となっており,肝炎発症予防のためのモニタリング法や抗ウイルス薬の適切な使用法を含めた早急な対策が必要である。(著者抄録)

We report a rare case of multiple myeloma that developed extramedullary plasmacytoma at the hilus of the liver causing obstructive jaundice. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) biopsy was quite useful in the diagnosis. A 71-year-old man was diagnosed with stage IIIA multiple myeloma in June 2007 based on osteolytic lesions, increased atypical plasma cells in the bone marrow, and monoclonal (M) protein of IgA-lambda, IgG-lambda, BJP-lambda type. M-protein was decreased by MP therapy following radiotherapy for the cervical lesion. However, in February 2008, M-protein started to increase again. The patient presented with obstructive jaundice in the middle of March. Abdominal ultrasound and MRI demonstrated a 12-mm mass at the hilus of the liver and the upper biliary tract dilatation, and a stent was placed across the bile duct stricture. EUS-FNA biopsy from the hepatic hilar mass showed multiple sheets of atypical plasma cells consistent with extramedullary plasmacytoma. The abdominal and intracranial mass did not respond to bortezomib therapy and gradually developed. Radiotherapy and high dose dexamethazone therapy were performed with little effect. The patient died in June 2008. To our knowledge, this is the first reported case of extramedullary plasmacytoma diagnosed by EUS-FNA.

71歳男性。患者は右頸部痛を主訴に、精査の結果、多発性骨髄腫(病期IIIA)と診断され、頸椎病変に放射線治療を施行後、MP療法を行い、M蛋白は減少した。しかし、その後、M蛋白の再増加をが認められ、左股関節〜大腿骨の新病変に対し放射線治療を行うとともVCAP療法を開始したが、発熱と閉塞性黄疸が出現した。腹部超音波および造影MRIでは中部総胆管から肝門部にかけて腫瘤性病変がみられ、EUS-FNAを行った結果、本症例は髄外形質細胞腫と診断され、治療として閉塞性黄疸に対する胆管ステント留置後、ボルテゾミブ療法を1コースが行なわれた。だが、腹腔内、頭蓋内の病変は増大し、放射線治療やデキサメタゾン療法を行うも効果が得られず、患者は最終的に骨髄腫の診断から12ヵ月後(黄疸出現後2.5ヵ月)に死亡となった。

Binding immunoglobulin protein (RIP) is an endoplasmic reticulum (ER) molecular chaperone that is central to ER function. We examined knock-in mice expressing a mutant BiP in order to elucidate physiological processes that are sensitive to BiP functions during development and adulthood. The mutant BiP lacked the retrieval sequence that normally functions to return BiP to the ER from the secretory pathway. This allowed us to examine the effects of a defect in ER function without completely eliminating BiP function. The homozygous mutant BiP neonates died after birth due to respiratory failure. Besides that, the mutant BiP mice displayed disordered layer formation in the cerebral cortex and cerebellum, a neurological phenotype of reeler mutant-like malformation. Consistent with the phenotype, Cajal-Retzius (CR) cells did not secrete reelin, and the expression of reelin was markedly reduced posttranscriptionally. Furthermore, the reduction in the size of the whole brain and the apparent scattering of CR cells throughout the cortex, which were distinct from the reeler phenotype, were also seen. These findings suggest that the maturation and secretion of reelin in CR cells and other factors related to neural migration may be sensitive to aberrant ER quality control, which may cause various neurological disorders.

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca2+ current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.