ALBERT

Description: Signal regulatory protein-α (SIRPA) is an immunoglobulin superfamily transmembrane protein with intracellular docking sites for two Src homology domain containing tyrosine phosphatases, and most abundantly expressed in neurons and myeloid cells. SIRPA is a critical immune inhibitory receptor on macrophages. CD47 is a ligand for SIRPA, and CD47 interaction with SIRPA serves as a ‘self-recognition’ that prevents phagocytosis of the cells expressing CD47. Recently, we (Nature immunology 2007) identified polymorphism in murine Sirpa as a critical modulator of engraftment in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation, and non-obese diabetic (NOD) mouse Sirpa polymorphism has far greater reactivity with human CD47 than that of the respective alleles of other strains resulting in effective engraftment of human hematopoietic cells. In addition, as well as the mouse, human SIRPA is highly polymorphic in the IgV domain. Then, we examined whether polymorphism in SIRPA induced the difference for suppressive effect of human macrophage on hematopoiesis. First, we examined sequence alignment of SIRPA IgV domain (exon 3 of SIRPA) of healthy control 18 people. We identified two major variants; 6 people with variant1 (V1) and 12 people with variant2 (V2). The SIRPA amino acid sequences of V1 and V2 are different in 13 residues, and its residues in orthologous position between species that is polymorphic between NOD and other strains as well as between V2 and V1. Next, we examined that whether there is a difference in the effect on hematopoiesis between V1 macrophage and V2 macrophage by long term culture-initiating cells (LTC-IC) assay on MS-5 stromal cells. For macrophage preparation, peripheral mononuclear cells from healthy donors were purified by positive selection using MACS CD14 Micro Beads (Miltenyi Biotec), and they were incubated with M-CSF for 3 days. For LTC-IC assay, 5×102 differentiated macrophages were seeded onto established MS-5 stroma in 96-well tissue culture plates. The next day, human hematopoietic CD34+ cells were seeded at doses of 102 to 5×103 cells per well and cultured for 4 weeks. At the end of the culture, cells were detached and plated into methylcellulose progenitor assays. Macrophage had a suppressive effect on the number of LTC-IC in all cultures. There was tendency that V1 macrophage had a greater suppression compared to V2 macrophage, however the differences between V1 and V2 were marginal (p=0.03). These data suggests that human macrophages have suppressive effect on hematopoiesis, and human SIRPA polymorphism modulates macrophage-mediated suppression of hematopoiesis in allogenic model, likewise in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation. Moreover, SIRPA polymorphism might be related to graft failure in the allogenic hematopoietic stem cell transplantation.

Description: Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.

Description: We investigated the expression of an apoptosis-associated antigen (Fas) (CD95) on hematopoietic progenitor cells in the presence or absence of interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF- alpha). CD34+ cells freshly isolated from bone marrow did not express Fas. However, IFN-gamma and/or TNF-alpha induced the expression of both the mRNA of Fas and Fas itself in a dose-dependent fashion on the surface of CD34+ cells after 48 hours of serum-free culture. IFN-gamma and TNF-alpha had a synergistic effect on the induction of Fas, when both cytokines were added to the culture. The TNF-alpha-induced Fas expression is mediated by p55 TNF-alpha receptor. CD34+ cells cultured in medium alone or with stem cell factor (SCF) showed some slight expression of Fas. When anti-Fas antibody (IgM) was added to CD34+ cells after the induction of Fas expression, CD34+ cells underwent apoptosis, as shown by a decrease in the number of viable cells, morphologic changes, the induction of DNA fragmentation, and a decrease in the number of colony-forming cells (CFC) including colony-forming unit granulocytes/macrophages (CFU-GM) and burst-forming unit erythroids (BFU-E). These observations indicate that IFN-gamma and/or TNF-alpha, well known as negative hematopoietic regulators, induce functional Fas on hematopoietic progenitor cells. The suppression of hematopoiesis by negative hematopoietic regulators may be mediated in part by Fas induction.

Description: An acquired JAK2 V617F mutation has been detected in up to 90% of patients with polycythemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, how a single mutation may be responsible for such different clinical phenotypes is unknown. Mice transplanted with bone marrow cells in which V617F JAK2 was retrovirally expressed developed PV-like features, but not ET or IMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated transgenic mice expressing V617F JAK2 driven by the murine H2Kb promoter. We established two lines. The expression of V617F JAK2 mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines. One line showed leukocytosis after 4 months of age, with a predominance of granulocytes. Among 43 mice, examined after 3 months of age, 8 (19%) showed polycythemia and 14 (33%) showed thrombocythemia. Two polycythemia cases also showed thromobocytosis. The other line showed extreme leukocytosis and thromobocytosis at one month of age. The leukocytosis progressed as the animals aged, but the thrombocytosis tended to resolve at 8 months. They showed anemia that means Hb value from 9 to 10 g/dL at one month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly with myeloid cell and megakaryocyte invasion was observed. We conclude that in vivo expression of V617F JAK2 results in ET-like, IMF-like, and PV-like disease.

Description: Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved outcome of patients with chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) with lesser incidence of serious adverse events. Recently, cases with fatal pulmonary hypertension (PH) have been sporadically documented in association with dasatinib treatment. French group reported incidence of PH diagnosed by cardiac catheterization as 0.45% (13 of 2900 patients) in the symptomatic patients treated with dasatinib. In contrast, a Korean group prospectively evaluated PH by non-invasive echocardiography in CML patients treated with dasatinib, and demonstrated 8 of 66 patients (12.1%) exhibited a significant increase in right ventricular systolic pressure, indicating subclinical PH might be more common event in dasatinib-treated patients. In this study, we prospectively examined the patients treated with three TKIs by echocardiography to clarify incidence of clinical and subclinical PH as well as factors associated with PH. Patients and Methods: Total of 108 patients (99 with CML, 9 with Ph+ ALL) receiving TKIs in our institutions were enrolled to this study. Forty-one patients have been on treatment with dasatinib (38%), 37 with imatinib (34%) and 30 with nilotinib (28%). Patients underwent echocardiography to evaluate values of tricuspid regurgitation pressure gradient (TRPG), which relates to severity of PH. Patients with higher values of TRPG than the upper limit (30mmHg) was suspected of PH by European Society of Cardiology criteria. Results: Among 108 patients, median age was 63 years old, and median duration of TKIs treatment was 26.5 months. Median daily dosage was 100 mg for dasatinib, 300 mg for imatinib, 600 mg for nilotinib groups, respectively (Table 1). In imatinib group, patients' age was significantly higher, and duration of treatment was also longer than those of the 2nd generation TKIs. Echocardiography revealed mean values of TRPG as 23.2, 23.2 and 23.1 mmHg in dasatinib, imatinib and nilotinib groups, respectively. There was no significant difference in TRPG values among 3 groups (p=0.99). We also found no relationship between TRPG values and duration of TKIs treatment in each group. Interestingly, we detected a significant inverse correlation between daily dosage of imatinib and TRPG values (p=0.012, Figure 1), while such relationship was not observed in dasatinib and nilotinib groups (p=0.68 and p=0.49). TRPG values higher than 30 mmHg were documented in 13 of 108 patients (12.0%); 5 of 41 (12.2%) in dasatinib group, 4 of 37 (10.8%) in imatinib group, and 4 of 30 (13.3%) in nilotinib group (p=0.95). Discussion: PH is characterized by proliferation of pulmonary vascular smooth muscle cells (SMCs). Recent reports showed that imatinib suppresses abnormal proliferation of SMCs through inhibiting platelet-derived growth factor receptors (PDGFR), resulting in improvement of PH in animal models. Clinical studies in symptomatic PH patients reported that imatinib considerably improved pulmonary hemodynamics. Of note, in our study, dosage of imatinib was significantly correlated with lower values of TRPG, suggesting imatinib possibly decreases TRPG values in a dose-dependent manner. This finding strongly supports the reports indicating imatinib as a therapeutic agent of PH. In contrast, in vitro studies have shown that dasatinib has stronger potential to inhibit PDGFR compared to imatinib; nevertheless, onsets of PH have been reported in dasanitib-treated patients, but not with imatinib nor nilotinib. Our study demonstrated the incidence of TRPG elevation as 12.0% in dasatinib group, which was consistent with Korean report (12.1%). However, there was no significant difference in TRPG values among 3 groups, indicating no apparent evidence which dasatinib treatment might be specifically associated with occurrence of PH. These results suggested that subclinical PH might be more common than expected. Careful follow-ups with echocardiography are necessary for the patients under any TKI treatments. Table 1. Patient characteristics Dasatinib group (n=41) Imatinib group (n=37) Nilotinib group (n=30) p Median age 55(17-77) 68(22-92) 62.5(24-85) 0.0004 Median dosage (mg/day) 100(18-100) 300(100-600) 600(300-800) Mean months from start of TKI 68.5(2-287) 104.8(2-228) 61.3(3-153) 0.007 Mean TRPG(mmHg) 23.2(9-40) 23.2(8-46) 23.1(7-35) 0.995 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1901 Poster Board I-924 Leukocyte alkaline phosphatase (LAP) is considered as a neutrophil activation marker. The level of LAP is quantitated as the LAP score. It is well known that patients with chronic myelogenous leukemia (CML) usually have low LAP scores, whereas those with BCR-ABL negative chronic myeloproliferative disorders (MPD) have elevated LAP scores. In CML patients, the premature release of granulocytes from the bone marrow into the peripheral blood is considered as the cause of low LAP scores. However, the reason for elevated LAP scores in BCR-ABL negative MPD patients has been unclear. An acquired JAK2V617F mutation is observed in most patients with BCR-ABL negative MPD. It has been shown that the JAK2V617F mutation induces constitutive activation of its downstream signaling pathways such as STAT3/STAT5, Ras/MAPK and PI3K pathways. We speculated that an elevated LAP score might be due to the activation of Jak2 downstream pathways through the JAK2V617F mutation. We analyzed LAP expression in BCR-ABL negative MPD patients. JAK2V617F homozygous patients had higher LAP expression than JAK2V617F heterozygous or negative patients. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of JAK2V617F positive patients. The myeloid cell line NB4 was transfected with the JAK2V617F mutation and a wild-type Jak2 using lentivirus vectors. It was observed that the JAK2V617F mutation, but not wild-type Jak2, enhanced cell proliferation. Then the LAP expression in NB4 cells was evaluated after these cells were differentiated by all-trans retinoic acid and granulocyte colony-stimulating factor. It was observed that the JAK2V617F mutation, but not wild-type Jak2, increased LAP expression. Next, we examined which of the Jak2 downstream pathways played a major role in increasing LAP expression and prompting cell proliferation. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, STAT3 siRNA and STAT5 siRNA, we demonstrated that the JAK2V617F mutation primarily used the STAT3 pathway to increase LAP expression. On the other hand, the JAK2V617F mutation used the STAT5, the Ras/MAPK and the PI3K pathways, but not the STAT3 pathway, to prompt proliferation of NB4 cells. In conclusion, we obtained direct evidence that the JAK2V617F mutation induced elevation of LAP scores via the STAT3 pathway, and prompted proliferation of NB4 cells via the STAT5, the Ras/MAPK and the PI3K pathways. Our findings showed the possibility that the JAK2V617F mutation might use specific downstream pathways depending on various phenotypic manifestations of BCR-ABL negative MPD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2231 Poster Board II-208 Primary B cell development from hematopoietic stem cells occurs in the bone marrow (BM). During their development, B cell precursors progress through a series of distinct developmental stages; early-B cells, pro-B cells, and pre-B cells. The number of these B cell precursors is gradually declined with aging. Benign B cell precursors, originally termed hematogones (HG), can be observed in the BM during hematopoietic recovery phase in some patients who received chemotherapy or allogeneic bone marrow transplantation (allo-BMT). However, little is known about HG following cord blood stem cell transplantation (CBT). We retrospectively analyzed populations of B cell precursors of BM samples from 115 patients who received allogeneic stem cell transplantation (SCT). Patients included 49 women and 66 men, with a median age of 49 years (19-66 years). The underlying diseases of 115 patients varied; 43 acute myelogenous leukemia, 18 acute lymphocytic leukemia, 4 chronic myelogenous leukemia, 15 myelodysplastic syndrome, 17 malignant lymphoma, and 18 others. 65 patients underwent allo-BMT and 50 underwent CBT. Mean number of the infused cells amounted of 2.80 × 108 cells/kg (0.92-4.02 × 108) for allo-BMT and 2.78 × 107 cells/kg (1.77-5.00 × 107) for CBT. 68 patients received myeloablative conditioning regimen and 47 received reduced intensity conditioning regimen. GVHD prophylaxis included 3 cyclosporine (CSP) alone, 16 CSP and mycophenolate mofetil, 33 CSP and methotrexate (MTX), 5 tacrolimus (Tc) alone, and 58 Tc and MTX. Median time between day 0 of SCT and days performed on evaluation of HG by BM aspiration was 38 days (14-140 days). At that time, engraftment of donor cells was confirmed by chimerism analysis, indicating HG were proven to be donor-origins. HG were detected averagely in 1.56% of BM cells (0.01-12.27%) for allo-BMT recipients and 7.04% (0.03-55.56%) for CBT recipients, respectively. These results indicated CBT recipients presented much higher frequency and prominent reconstitution of HG compared to allo-BMT recipients (p

Description: Abstract 2301 To evaluate stem cell potential of human cells in xenotransplant models, a variety of immunodeficient mouse lines have been developed. Depletion of lymphoid cells including T, B and NK cells by introducing with Il2rgnull, and SCID or RAGnull mutations is necessary to avoid rejection of human cells in these models. Interestingly, in mice having these immunodeficiencies, the NOD strain shows even better engraftment of human cells as compared to C57BL/6 or Balb/c strains. Recently, we found that in xenograft rejection, the innate phagocytic reaction of mouse macrophages could occur because murine signal regulatory protein alpha (mSIRPA) on macrophages cannot bind to human CD47 (hCD47). However, NOD-specific polymorphism of mSIRPA allows NOD-type SIRPA to bind hCD47, resulting in inhibition of phagocytic reaction against human cells in this strain at least in vitro. Here, we have established a new immunodeficient BRGS mouse line, C57BL/6.Rag2nullIl2rgnull mice with NOD-type SIRPA. To test the reconstitution activity of human hematopoiesis in vivo, we transplanted 5 × 103 CD34+CD38− human cord blood cells intrafemorally into C57BL/6.Rag2nullIl2rgnull (C57BL/6-RG), BRGS or NOD.Rag1nullIl2rgnull (NOD-RG) mice. At 8 weeks after transplantation, human CD45+ cells were not detectable in C57BL/6-RG mice in the bone marrow. Both BRGS and NOD-RG showed successful reconstitution, and their frequency of human CD45+ cells in the bone marrow was 59.9 % and 55.8% in average, respectively. The frequency of human CD45+ cells was maintained at least until 24 weeks after transplantation. Percentages of CD19+ B cells, CD33+ myeloid cells and CD34+CD38− cells that contain the majority of human hematopoietic stem cells (HSCs) were almost equal between the BRGS and the NOD-RG strains. In the spleen, the majority of human cells were CD19+ B cells expressed surface immunoglobulin light chain λ/κ, reflecting their normal maturation. In the thymus, CD4+CD8+ thymic precursors, and CD4+ and CD8+ single positive T cells were present, and they expressed surface T cell receptor (TCR)-ab or TCR-gd. These data show that replacement of the C57BL/6-Sirpa with the NOD-Sirpa is sufficient for the C57BL/6-RG strain to gain the human cell engraftment ability equal to the NOD-RG strain. In addition, the BRGS strain has normal complement activity, in contrast to the NOD strain that does not have C5, a component necessary for complement-dependent cytotoxic (CDC) activity. We injected 8 × 105 cells of Raji cells into BRGS or NOD-RG mice, and administered rituximab, an anti-CD20 antibody that has both CDC and ADCC activities, to test the in vivo efficacy of rituximab on transplanted Raji cells. After injection of rituximab, percentages of human CD45+ Raji cells were significantly decreased in BRGS mice (15.1 %), whereas percentages of Raji cells in NOD-RG mice were only slightly reduced by rituximab treatment (79.2 %). These data clearly show that the CDC activity of antibodies can operate in the BRGS strain. Collectively, this study formally proves that the excellent transplantability of human grafts in the NOD strain is explained simply by a single gene mutation, NOD-specific polymorphism of SIRPA, and that the BRGS strain should be very useful in future xenotransplant experiments of human stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Immunodeficient mice are used to reconstitute human hematopoiesis and analyze human hematopoietic function in vivo by xenotransplantation of hematopoietic stem cells (HSCs). It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD is critical for efficient human cell engraftment in xenotransplantation models. We have reported that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows the mouse SIRPA to bind human CD47, preventing activation of recipient macrophages to engulf human hematopoietic cells (Takenaka et al., Nature Immunol., 2006). We then developed a C57BL/6.Rag2nullIl2rgnull mouse line harboring the NOD-type Sirpa, named the BRGS mouse. The engraftment efficiency of human HSCs in BRGS mice was equal to that of NOD-Rag2nullIl2rgnull mice. This BRGS strain should be useful for further genetic modification because its background is simplified (Yamauchi et al., Blood, 2013). One of the persistent problems with the current xenograft models is that reconstituted human hematopoiesis is B-lymphoid dominant, whereas the engraftment levels of human myeloid cells, including megakaryocyte/erythrocyte lineage cells are quite low. In mouse syngeneic and allogeneic transplantation, recipients bearing mutation in the receptor tyrosine kinase Kit can accept donor HSCs without irradiation preconditioning. These data suggest that the Kit mutation impairs endogenous murine HSC self-renewal and allows donor HSCs efficiently to access to niche space. To develop a more efficient xenograft model, we further introduced a loss-of-function KitWv into the BRGS strain to deteriorate self-renewal and reconstitution capability of host HSCs. Here we show that this new mouse strain, termed the BRGSK mouse, showed very efficient human cell engraftment including myeloid, erythroid and megakaryocyte lineage cells. CD34 + cells obtained from human umbilical cord blood were transplanted into sublethally-irradiated BRGSK and BRGS mice at the age of 6-8 weeks by intrafemoral injection. At 8-12 weeks after transplantation, the average human hematopoietic chimerism defined by human CD45+ cells was more than 90% in the BM and this chimerism was maintained over 24 weeks in homozygous BRGSK recipients. Furthermore, the chimerism of human CD33+ myeloid cells in the BM was significantly improved (~5.7% in BRGS vs. ~32.5% in BRGSK). Surprisingly, CD71- CD235a+ mature erythrocytes and CD41+ platelets were detected in the BM. All human erythroid series including mature erythrocytes, CD71+ CD235a- early erythroblasts and CD71+ CD235a+ late erythroblasts were clearly seen (~0.1% in BRGS vs. ~42.2% in BRGSK). Immunohistochemical staining showed that these human erythroid cells were positive for human hemoglobin subunit alpha. Confocal immunofluorescence imaging revealed that human erythroid cells formed large erythroid islands, independently from mouse erythroid islands in the sternal marrow of recipient BRGSK mice. A number of CD41+ megakaryocytes were scattered in the BM. The BRGSK mice also well supported terminal differentiation of myeloid cells such as neutrophils, eosinophils, basophils, mast cells, monocytes, and dendritic cells. Thus, reconstitution and maintenance of human myelo-erythropoiesis is significantly improved in the new BRGSK strain. The new BRGSK xenograft model should be a strong tool to investigate normal and malignant human hematopoiesis. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: Background: Human herpes virus-6 (HHV6)-associated limbic encephalitis/myelitis is rare but life-threatening nervous system complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). When the allo-HSCT recipients present encephalitis-associated manifestations such as short-term memory dysfunction, disorientation, consciousness disturbance and seizures, it is essential to immediately perform polymerase chain reaction (PCR) analysis for detection of HHV6 viral DNA in the cerebrospinal fluid (CSF) for the definite diagnosis. However, if reactivation of HHV6 is confined to the spinal cord but not cerebrum, these patients lack encephalitis-like symptoms but manifest only sensory nerves-related symptoms such as severe unexplained pain, dysesthesia and prutitus, leading to the delayed examination of HHV6 DNA detection in CSF. As a result, the patients with HHV6-associated myelitis can be sometimes misdiagnosed as having calcineurin inhibitor (CI)-induced pain syndrome (CIPS), since such manifestations are commonly observed in these two syndromes. In this study, we evaluate incidence and clinical features of HHV6 encephalitis/myelitis and CIPS after allo-HSCT. Method: We retrospectively reviewed the medical records of 435 patients who underwent allo-HSCT between 2002 and 2014 in our facility. HHV6-associated encephalitis/myelitis was directly proved when HHV6 DNA was detected by PCR in CSF. For those patients who were unable to undergo lumbar puncture because of severe thrombocytopenia or a deteriorated general condition, we diagnosed HHV6-associated encephalitis/myelitis if they satisfied more than 2 of the following 3 criteria: (1) typical clinical manifestations as described above; (2) detection of HHV6 DNA in peripheral blood; or (3) limbic encephalopathy based on the selective involvement of the medial temporal lobe on magnetic resonance imaging (MRI). CIPS was diagnosed using three factors: (a) typical clinical manifestations including unexplained severe pain and cutaneous pruritus without skin rash; and (b) not detection of HHV6 DNA in CSF; or (c) abnormal findings at X-ray, MRI, or bone scintigraphy at joint of knee and foot. Results: Twenty-five patients were diagnosed as having HHV-6 encephalitis/myelitis with a cumulative incidence of 5.7%. Median onset was on day 19 after transplantation. HHV-6 encephalitis/myelitis was documented in 15 of 99 cord blood transplant (CBT) recipients (15.2%) and 10 of 336 recipients (3.0%) transplanted with bone marrow or peripheral blood stem cells. This result suggests that a higher incidence of HHV-6 encephalitis/myelitis occurring in CBT recipients. Four patients manifested typical symptoms at the onset of HHV6-associated encephalitis. However, 11 patients presented with dysesthesia and pruritus, described as typical manifestations of patients with CIPS, and the remaining 10 showed both symptoms. Six of 11 patients with CIPS-like symptom also exhibited dysautonomia (bladder and rectal disturbance and sinus tachycardia) and/or abdominal pain. Positive results for brain MRI scans (limbic encephalitis) were observed in 9 of 14 patients (64.3%) who developed encephalitis-type symptoms, which were not found in the 11 patients who had CIPS-like symptoms; none presenting with CIPS-like symptoms had positive results for spinal MRI as well. On the other hand, 8 patients (1.8%) were diagnosed as having CIPS on the 5 to 91 days (madian 22 days) posttransplant. For graft-versus-host disease (GVHD) prophylaxis, 2 patients received cyclosporine, and 6 received tacrolimus. CI concentrations in these patients were maintained within the target ranges at onset of the pain. Abnormal findings at X-ray and MRI were not observed in all patients, but only one patient showed abnormal findings at joint of hand, finger and ankle in bone scintigraphy. For the treatment of CIPS, in 7 patients dosage of CI was reduced, whereas in one CI was switched into another one. Clinical symptoms in all of these patients were improved and exacerbation of GVHD was not seen. Conclusion: Detection of HHV6 DNA in CSF is crucial to make a differential diagnosis of HHV6 myelitis and CIPS. Transplantation physicians should be aware that CIPS-like dysesthesia and pruritus might be early manifestations to suggest the reactivation of HHV6, especially for patients who develop myelitis. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.