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We have previously shown a fraction of stromal fibroblasts/myofibroblasts (Fibs) from leukemic bone marrow cells expresses leukemia-specific transcripts along with hematopoietic and Fib-related markers. Normal bone marrow-derived Fibs (nFibs) do not express CD34 or CD45; however, nFibs may express hematopoietic markers with some specific stimulations. CD34 expression was detected in nFib cultures following the addition of a culture supernatant of blood mononuclear cells stimulated with phytohemagglutinin (PHA)-P. To identify the molecules responsible for inducing CD34 expression in nFibs, cDNA clones were isolated using functional expression cloning with a library constructed from PHA-P-stimulated human blood mononuclear cells. Positive clones inducing CD34 transcription in nFibs were selected. We confirmed that an isolated positive cDNA clone encoded human interleukin (IL)-1 beta (β). CD34 expression was observed in the nFib cultures with recombinant human (rh) IL-1β protein. And CD34 transcription was suppressed when a rhIL-1β neutralizing antibody was added to the IL-1β-stimulated nFib cultures. nFibs expressed gp130 and IL-6 receptors, and CD45 expression was detected in nFibs cultured with rhIL-1β and rhIL-6. Chronic myelogenous leukemia (CML) cells reportedly respond well to IL-1β. When CML-derived Fibs were cultured with rhIL-1β and rhIL-6, CD45-positive cells increased in number. Cell fate may be influenced by an external specific stimulation without gene introduction.

The role of the FMS-like tyrosine kinase 3 (FLT3) gene in acute myeloid leukemia (AML) has been well documented and the FLT3-internal tandem duplication (FLT3-ITD) mutation has been identified as a prognostic factor in AML. Due to its low incidence, the role of the FLT3 mutation remains unclear in myelodysplastic syndrome (MDS) patients. To investigate the impact of the FLT3-ITD status on the prognosis of MDS at diagnosis, we retrospectively analyzed 72 MDS patients admitted to Teikyo University Hospital. FLT3-ITD was examined by a reverse transcription-polymerase chain reaction using complementary DNA synthesized from mRNA extracted from bone marrow mononuclear cells at the diagnosis of MDS. Fifteen patients (20.8 %) were positive for FLT3-ITD and had significantly worse overall survival (OS) and progression-free survival (PFS) than patients who were negative (P0.001 and P0.001, respectively) in a multivariate analysis. We also investigated whether the Wilms' tumor gene-1 (WT-1) copy number was associated with the FLT3-ITD mutational status using data available on WT-1 from 57 patients. A WT-1 transcript copy number ≥50/μg total mRNA in peripheral blood was detected in 35 patients (61.4 %). All FLT3-ITD-positive patients showed WT-1 ≥50. The FLT3-ITD-positive group showed significantly higher WT-1 transcription levels than the negative group. These results indicate that the FLT3-ITD mutation has a prognostic impact at the diagnosis of MDS and is associated with a high level of WT-1.

We investigated the potential of nutritional and inflammatory parameters as prognostic factors for follicular lymphoma (FL), and also examined the predictive value of the early progression of disease within 24 months of first-line chemo-immunotherapy (POD24). We retrospectively analyzed 46 patients with FL admitted to Teikyo University Hospital and treated with chemo-immunotherapy between May 2009 and July 2019. Physical characteristics, blood parameters, and markers or scores for consumptive/inflammatory and nutritional conditions were used as variables. Nine parameters correlated with poor overall survival (OS) in univariate analysis: An Eastern Cooperative Oncology Group (ECOG) scale performance status (PS) ≥2, five or more involved nodal sites, positive bone marrow (BM) involvement, a serum albumin level3.5 g/dL, CRP0.5 mg/dL, lactate dehydrogenase (LD) higher than the upper normal limit (UNL), high-density lipoprotein cholesterol (HDL-C)40 mg/dL, modified Glasgow prognostic score of 1-2, and the geriatric nutritional risk index82. In multivariate analysis, ECOG PS ≥2, positive BM involvement, and a serum HDL-C level40 mg/dL remained significant for poor progression-free survival. One-year OS rate after receiving salvage chemotherapy was lower in the POD24 group (50%) and POD24 correlated with ECOG PS ≥2, positive BM involvement, a serum lactate dehydrogenaseUNL, and HDL-C40 mg/dL by Fisher's exact test. These results indicate that low serum HDL-C levels appear to be important for predicting the risk of POD24 and the worse prognosis of FL.

Rationale: Anaplastic lymphoma kinase (ALK) + anaplastic large cell lymphoma (ALCL) is considered as a good prognosis lymphoma. However, in an extremely rare subset of patients, ALK+ ALCL with leukemic presentations is known to be chemotherapy-resistant. Although several novel therapies have been tested, the standard therapy for relapsed/refractory ALK+ ALCL has not been established yet. Patient concerns: An 18-year-old female patient who had conventional chemotherapy- and Brentuximab Vedotin (BV)-resistant ALK+ ALCL with leukemic presentation. She was successfully treated with an ALK inhibitor, crizotinib. Crizotinib induced complete remission (CR) and bridged to allogeneic bone marrow transplantation (BMT). Diagnosis: However, her ALCL relapsed on day 60 after BMT and she developed high grade fever and lymphadenopathy. Intervention: Although crizotinib was given to the patient immediately after relapse, she developed grade 3 nausea and could not continue to take it. Then, we gave alectinib to the patient, which promptly induced sustained CR without any further chemotherapy. The patient received second stem cell transplantation using umbilical cord blood with myeloablative regimen in 2nd CR. Outcomes: The patient has been in CR under maintenance therapy of alectinib for more than 16 months. Lessons: Both ALK inhibitors demonstrated drastic efficacy for our patient who had chemotherapy- and BV-resistant ALK+ ALCL with leukemic presentation. Alectinib showed less gastro-intestinal toxicity than crizotinib and the patient was able to take it even at the relatively early phase of stem cell transplantation.

Rationale: Patients with relapsed acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) have poor prognosis. Many patients are not eligible for 2nd HSCT due to organ dysfunction or other complications that prevent them from tolerating conditioning chemotherapy. In those ineligible patients for 2nd HSCT with myeloablative conditioning regimen, reduced intensity conditioning (RIC) are often used. RIC regimens are less toxic but has a less direct anti-tumor efficacy so that RIC regimens are not suitable for the patients with high tumor burden. To overcome this dilemma, Gemtuzumab Ozogamicin (GO) has been used as a part of RIC regimens to add anti-tumor efficacy. We report here a relapsed AML patient who was treated with GO monotherapy followed by stem cell infusion. Patient concerns: A 25-year-old male with AML experienced relapse 9 months after allo-HSCT. Diagnosis: Since he had mild renal and cardiac dysfunction and his AML did not progress rapidly, we decided not to give him an intensive chemotherapy. However, after azacitidine (AZA) and donor lymphocyte infusion therapy, his leukemic blasts did not decrease. Interventions: Originally, we had planned to proceed with a 2nd allo-HSCT with RIC regimen that consisted of fludarabine, melphalan and fractionated GO (3 mg/m2/dose) on day -21, -18, and -15. However, the patient developed appendicitis after the last dose of GO when his neutrophil was 0 cells/μl. Based on his medical acuity, we terminated the rest of the patients conditioning regimen and the patient did not receive any further chemotherapeutics. The patient was still infused with peripheral blood stem cells from the donor on day 0. Outcomes: His appendicitis was resolved by antibiotics without surgery. His AML has been in CR more than 18 months under AZA maintenance therapy. Lessons: GO monotherapy could be a conditioning regimen of 2nd allo HSCT from the same donor as the first HSCT for relapsed AML patients.

Background and Aims: We previously reported that interleukin 1-beta (IL-1-b) stimulated bone-marrow stromal myofibroblasts from normal individuals to express CD34. And, when myofibroblasts were cultured with IL-1-b and IL-6, GATA-2 and CD45 expressed. We report here that bone marrow-derived myofibroblasts from acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) patients were also activated to express leukemia-specific molecules when IL-1-b and IL-6 were added to the cultures. Materials and Methods: Bone marrow samples were collected from informed 7 AML patients (2 cases of M2 with t(8;21), 3 of M0, M2, and M7 positive for FLT-ITD mutation, 2 of Philadelphia-positive bi-phenotypic acute leukemia (Ph-AL)), and 3 CML ones, whose mononuclear cells were separated, and were cultured, split, and re-cultured for one month to eliminate non-adherent cells, vascular cells, and monocytes/macrophages to prepare myofibroblasts. The obtained cells were further cultured for one more month to obtain myofibroblasts with stably growth, and then leukemia-specific molecules (FLT3-ITD, BCR-ABL1 fusion (major and minor), RUNX1-RUNX1T1 fusion) were analyzed at DNA and RNA levels. Cells were further cultured with IL-1-b and IL-6 for two weeks, and morphological changes and expressions of specific molecules were observed. Results: When bone marrow-derived myofibroblast obtained from myeloid leukemia patients were analyzed, leukemia-specific molecules were detected at DNA levels (FISH analysis detected 0.5-5% BCR-ABL1 fusion in CML- and Ph-AL-derived myofibroblast-cultures, 1-2% RUNX1- RUNX1T1 in AML with t(8;21)-cultures, and positive FLT3-ITD mutation at genomic PCR in AML-cultures); however, RT-PCR analyses revealed that leukemia-specific transcripts were not detected in all cells. When myofibroblasts were cultured with IL-1-b and IL-6, leukemia-specific transcripts were detected with RT-PCR analysis in all cases. Also, CD45, and GATA-2 expressed in these cultures. Discussion: We reported previously that a few populations of bone marrow-derived myofibroblasts obtained from AML and CML patients expressed leukemia-specific transcripts and proteins when cultured as a separated single colony; however, when myofibroblasts were cultured not as a fractionated single colony but a whole stromal cells for a long term, expression of leukemia-specific molecules weakened and no longer confirmed. Myofibroblasts with leukemic character seem to stop their proliferation and keep dormant. One interesting point is that normal stromal myofibroblasts derived from myeloid leukemia patients can inhibit the growth of myofibroblasts that have leukemic characters. Some mechanisms may work on this observation. Also, an important issue is that myofibroblasts reportedly have an ability of antigen presentation. If some myofibroblasts can express leukemia-specific molecules as well as HLA when cultured with IL-1-b and IL-6, they can act as a leukemia-specific antigen-presenting cell. We now attempt to research this theme to develop a new cell-mediated vaccine for myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cellsin vitro. To analyze a biological contribution of CML-derived myofibroblastsin vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimericBCR-ABLtranscript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferationin vivo.

Background and Aims We recently reported that graft-versus-leukemia (GVL) effect was observed in acute lymphocytic leukemia (ALL) with TCF3-PBX1 (53rd ASH, and 15th ASGCT). To understand better the mechanism of this GVL effect, we analyzed blood T cell receptors (TCRs) obtained from ALL patients with TCF3-PBX1. Materials and Methods The institutional ethical committee approved our study using clinical materials. Three informed ALL patients with TCF3-PBX1 were eligible. Leukemic blasts, blood lymphocytes after underwent allogeneic hematopoietic stem cell transplantation to achieve and keep complete remission, and cells from pathologically GVHD-diagnosed organs (liver, and skin) were collected. Lymphocytes were co-cultured with the patient-derived ALL blasts that were priory irradiated at 20 Gray (GVL model) or GVHD-related cells (GVHD model) for 14 days with recombinant human interleukin-2. Then, TCR Valpha (Va) and Vbeta (Vb) usages were analyzed with RT-PCR. When the preferential usage of Va and Vb was observed, T cells underwent single-cell culture, and the clones showing ALL blast-killing activity and not GVHD effect were selected, and their TCR nucleotide sequences were analyzed. Results In GVHD model and GVL one, TCR Va and Vb were oligoclonally used, in which Vb 6, 10, and 14 were preferentially used in GVL model but not in GVHD. On Va no selective usage was demonstrated between GVL model and GVHD one. Furthermore, the obtained single cells with GVL effect had specific TCR Vb nucleotide-sequences. Discussion In GVL model TCR Vbs were preferentially used. Currently we isolate tcf3-pbx1-specific oligopeptide-antigen recognized by the isolated TCRs, and observe cytotoxic GVL effect with the isolated TCRs in vitro. We also validate in vivo implications of the isolated GVL-specific T cells to treat leukemic non-obese diabetes/severe combined immunodeficiency mouse generated by the injection of ALL-blast with TCF3-PBX1. Disclosures: No relevant conflicts of interest to declare.

Background We previously reported that when adult human dermal fibroblasts were cultured with interleukin (IL)-1-b, vascular endothelial growth factor (VEGF)-A was produced significantly (54th ASH). And, when antihuman VEGF-A neutralizing antibody (VEGF-A Ab) was added to the cultures, CD138 (Syndecan-1) expressed significantly. CD138 is a member of cell-surface transmembrane haparan sulfate proteoglycans, and expresses in plasma cells from multiple myeloma (MM) cases. Membrane-anchoring CD138 shows a better prognosis in an immunodeficiency murine transplantation model in vivo; however, when extra-domain of CD138 is digested by heparanase to be shed from the cell-surface, MM cells invade to various kinds of tissues, and the patients show poor prognosis. Aims To validate a biological implication of inhibition of VEGF-A-signaling in MM cells, we observed effects of VEGF-A Ab to bone marrow cells from MM patients. Cell-proliferations as well as morphological changes were also observed time-dependently. Materials and Methods Institutional ethical committee approved our study, and bone marrow cells were obtained from the informed MM patients as well as normal individuals. Cells were separated with gravity-sedimentation method, and the prepared mononuclear cells were cultured with or without VEGF-A Ab, and the expression of specific genes was analyzed. Results Twenty MM patients were eligible, in which three showed significant poor prognosis, and worsened after underwent intensive chemotherapy or allogeneic hematopoietic stem cells transplantation. Thirteen out of twenty expressed CD138, and when cells were cultured with VEGF-A Ab for four days, CD138-expression increased significantly in all cases. Four did not express CD138; however, CD138-expression was observed after 4 day’s culture with VEGF-A Ab. In three progressed cases CD138-expression decreased in accordance with the disease-progression; however, when VEGF-A Ab was added to the cell-cultures, CD138 was induced to express. Heparanase-expression was observed in 10 cases out of 20, which were down-regulated when VEGF-A Ab was added to the cultures. In contrast, in bone marrow cells from seven normal individuals CD138-expression was very low, which was down-regulated with the addition of VEGF-A Ab. Heparanase-expression was not observed in these normal cells, and were induced to be observed in four out of seven when VEGF-A Ab was added to the cultures. Discussion Expression of CD138 is induced in fibroblast by the addition of fibroblast growth factor-2, and in keratinocytes by epidermal growth factor and keratinocyte growth factor; however, an induction of CD138 by the VEGF-A Ab has not been reported. Several cytokines including VEGF-A influence plasma cell-proliferation; however, little is reported on cytokine-suppression therapy. Inhibition of the signaling of VEGF receptors by the chemicals including solafenib is not specific for VEGF-A. Currently we validate the efficacy of the inhibition of VEGF-A-signaling to MM cells and their environmental cells using RNA interference. Disclosures: No relevant conflicts of interest to declare.

9053 Background: Risk stratification of patients with febrile neutropenia (FN) is important to select the suitable therapeutic option. Although the MASCC scoring system is used as a reliable risk-discriminator, the objective evaluation of “burden of illness” has been difficult to be pointed-out. The latest IDSA Guidelines on FN proposes a set of risk factors based on the expert’s recommendations. The present study demonstrates usefulness of newly proposed 3D-index on predicting mortality of chemotherapy-induced FN in hematologic malignancies (HM). Methods: Patients with HM admitted to our hospital between Jan 2008 and Dec 2011 were enrolled, and data from 282 FN episodes in 129 FN patients including 20 infection-associated deaths were retrospectively analyzed to determine useful prognostic factors on the mortality of FN. Correlation between mortality and 59 characteristics including 3D-index were examined by univariate analysis and receiver operating characteristic curve analysis. 3D-index is a duration and severity of neutropenia and degree of fever. Total duration of days during neutropenia was not calculate at the time of risk evaluation. 3D-indexes after three and five days from the start of FN were also analyzed. Results: 32 out of 59 characteristics were significantly correlated with mortality by univariate analysis. Among them, 3D-index and 3D-indexes after three and five days were demonstrated as more useful factors (3D-index, p=0.0015; 3D-index after three days, p=0.0030; 3D-index after five days, p=0.0044). And 3D-index after three days over 4000 indicates patients' mortality above 20%, index 2600-4000 15-20% , index 900-2600 10-15%, and index below 900 below 10%, respectively. Conclusions: 3D-indexes after three days were suggested as useful predictors for infection-related mortality during FN. It was suggested that FN patients were categorized into four risk groups according to the value.

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