ALBERT

Description: Intestinal TMA, a upcoming and serious problem after HSCT, usually manifests diarrhea, vomiting, GI bleeding and abdominal pain as initial clinical presentations similar to those of intestinal GVHD. But intestinal TMA should be distinguished from GVHD, because they needed to be treated in absolutely different way. Here we investigated the incidence of intestinal TMA and evaluated their clinical progressions in HSCT patients. We retrospectively reviewed 243 gastrointestinal mucosal biopsy slides obtained from HSCT patients having suffered from GI problems in the Catholic University Hospital, from 2000 to 2004. The collected cases were reviewed by two pathologists blindly and classified into TMA; showing microthrombi formation in the lumina of microvasculatures, GVHD; showing epithelial apoptosis and/or loss of glands, TMA+GVHD and non-specific inflammation. And next, their incidence and clinical outcomes were analyzed. On histopathological examination, 15 out of 243 cases (6.2%) showed luminal thrombi of microvasculature compatible with TMA, 58 cases (23.9%) showed findings compatible with GVHD, 3 cases (1.2%) showed both TMA and GVHD, and 173 cases (71.2%) showed nonspecific gastroenteritis. On peripheral blood smear examination, all 15 TMA cases showed less than 1% of fragmented RBC at the time of biopsy. But 2 out of 15 cases (13.3%) showed increase to 8% and 10% of fragmented RBC in follow-up, and they had been aggravated clinically and improved only after treatments including discontinuing immunosuppressant and plasma exchange. Among 58 GVHDs, 34 patients (75.9%) were improved with steroid pulse therapy, but 14 patients (24.1%) failed. These results show that intestinal TMA is not infrequent in HSCT patients and can be combined with intestinal GVHD. In conclusion, intestinal TMA should be recognized in pathological diagnosis of HSCT patients. It is important to differentiate intestinal TMA from GVHD in patients suffering from severe and refractory diarrhea after HSCT.

Description: Abstract 4194 Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known. We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice. In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells. We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients. Disclosures: No relevant conflicts of interest to declare.

Description: The bone marrow microenvironment (BMM) provides a protective niche that supports growth and survival of leukemic stem cells. Stromal-derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) plays pivotal roles in the cross-interactions between blasts and the BMM to prevent retention and mobilization of leukemic cells, as well as in normal hematopoiesis including the development of immune cells. Here, we show that the CXCR4 antagonist, Plerixafor, decreased the level of CXCR4 expression and inhibited SDF-1-induced migration of leukemic cells. Further, the inhibition of the interaction between leukemic cells and the BMM by the CXCR4 antagonist enhanced cytotoxic activity of immune cells as a result of increased susceptibility of leukemic cells to chemotherapeutic agents such as cytosine arabinoside (Ara-C) in a mouse model of acute myeloid leukemia (AML), suggesting biological effects of the BMM through immune cell activation. To examine the level of CXCR4 expressed by primary AML blasts and murine leukemic cells and the role of CXCR4 in migration at various concentration, 19 AML samples and murine C1498 and human Jurkat cells were subjected to FACS analysis and migration assay. CXCR4 expression in C1498 cells was about 31.29%, compared to 99.7% in the CXCR4+ Jurkat cell line and RNA expression was also confirmed. C57Bl/6J mice were used to construct a syngeneic AML animal model by infusion of 2x106 C1498 cells, and peripheral blood from a facial vein and samples from multiple organs from the sacrificed mice were obtained until day 30 post-injection. Total RNA was extracted from BM cells, liver, and spleen of mice at day 15 and 1ug RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit. All data were normalized to the amount of GAPDH expression, with samples run in triplicate. The quantitative real-time PCR assay for IFN-γ, perforin, and granzyme B, main cytokines produced by cytotoxic T and NK cells, was performed to examine the functional capacity of subsets. Data revealed that both normal human samples and AML mononuclear cells highly expressed CXCR4. CXCR4 expression in both groups was significantly decreased by CXCR4 inhibition, when treated with 5 µM Plerixafor. Our migration assay clearly showed the inhibitory effect of Plerixafor on SDF-1α-induced migration of primary AML and C1498 cells. Under SDF-1α conditions (100 ng/ml), C1498 cells were co-cultured with the CXCR4 antagonist at various concentrations. Migrations of both C1498 and primary AML cells were similarly inhibited. To test the direct role of the CXCR4 antagonist in apoptosis, C1498 cells were cultured with or without Ara-C. The Ara-C and Plerixafor dual-treated group (termed P+A group) displayed no significant difference in apoptosis when compared to the Ara-C only group, suggesting that apoptosis is exclusively controlled by Ara-C, but not Plerixafor, in vitro. However, leukemic blasts synergistically and significantly decreased in the P+A group, compared to those of the other groups, in a syngeneic leukemic mouse model experiments, suggesting an unexpected role for Plerixafor in leukemic blast suppression specifically in the AML niche. The frequency of CD4 and CD8 cells was maintained in the P+A group, compared to the Ara-C-only, Plerixafor treated only, and C1498 injected groups, implying that Plerixafor cannot independently alter the frequency of immune cells. Most of all, the expression levels of IFN-γ, perforin, and granzyme B in the spleens of the P+A group were significantly increased, compared to those of the control groups including the Plerixafor only group. This study demonstrates that the effects of CXCR4 inhibition on blast suppression and immune cell function in the tumor microenvironment and chemotherapy with Plerixafor represents an advanced therapeutic strategy of targeting the leukemic niche. Disclosures No relevant conflicts of interest to declare.

Description: Follicular dendritic cell neoplasm(FDCN), a rare tumor exhibiting a broad histopathological spectrum and unpredictable biologic behavior, has been classified into follicular dendritic cell tumor(FDCT) and follicular dendritic cell sarcoma(FDCS) regarded as borderline lesion and malignant tumor, respectively, by World Health Organization. But still no definite diagnostic criteria distinguishing FDCT from FDCS was established. Here in, as an approach to the differential diagnostic criteria, we performed clinicopathological analysis for 146 cases of FDCN, documented in English literature from January 1986 to May 2006. Through PubMED search with key words of FDC or dendritic cell tumor, we collected and reviewed 73 articles out of more than six thousands candidates, which contained 146 FDCNs with clinicopathological informations. We analyzed the carefully selected cases with parameters including age, gender, tumor location, tumor size, tumor margin, EBV association, mitosis, nuclear atypism, hemorrhage, necrosis and Castleman’s disease association. Recurrence, metastasis and death of disease were referred as event. Kaplan-Meier model was used for overall event-free survival analysis and the log-rank test was used to assess statistical significance. Mitotic Index (MI, ≥ 5/10HPF vs

Description: Abstract 229 Overexpression of MN1 (meningioma 1) is a negative prognostic factor in acute myeloid leukemia (AML) patients with normal cytogenetics, and induces a rapidly lethal AML in mice. We have shown previously that MN1, a transcription cofactor of retinoic acid receptor alpha (RARA), increases resistance to all-trans retinoic acid (ATRA) by greater than 3000-fold in an in-vitro differentiation model. We investigated the molecular mechanisms involved in the MN1-induced myeloid differentiation block by fusing potent transcriptional activation or repression domains to MN1, conducting a structure-function analysis of MN1, gene expression profiling, ChIP-on chip experiments, and functional validation of MN1 target genes. We found that (1) MN1 inhibits myeloid differentiation through transcriptional repression; (2) the C-terminal domain of MN1 is critical for induction of resistance to ATRA; (3) EGR2 is a putative direct target of MN1 and RARA that is repressed in MN1 leukemias; and (4) that constitutive upregulation of EGR2 in MN1 leukemias permits differentiation and prevents engraftment of transplanted cells. To investigate whether MN1 impacts on myeloid differentiation through transcriptional activation or repression we fused a strong transcriptional activation domain (VP16) or repression domain (M33) to MN1. MN1VP16 immortalized murine bone marrow cells, however, these cells could differentiate to mature granulocytes, and succumbed to cell cycle arrest upon treatment with ATRA. Mice receiving transplants of MN1VP16 cells had a median survival of 143 days (n=16) compared to 35 days in mice receiving MN1-transduced cells (n=18; p

Description: Background: Clamydophila psittaci (C. psittaci) has been proposed as an etiologic factor for extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in the ocular region. However, previous studies showed varied association rates ranging 0% to 87%, not constant even in the series of same geographical areas, which suggest that there could be some technical variance in detecting methods for C. psittaci. The authors validated five sets of primers for detecting C. psittaci DNA and investigated its association with ocular MALT lymphomagenesis. Material and Methods: Five sets of PCR primers including 4 previously reported and one newly designed were evaluated with positive control C. psittaci DNA (acquired from Korea Centers for Disease Control and Prevention). One hundred fifty cases of confirmed ocular MALT lymphomas were collected from pathology archives of our institutes from 2008 to 2014, and entered into this study. DNA was extracted from archival paraffin block sections with Qiagen DNA extraction kits. Quality of DNA was assessed with NanoDrop and beta-globin PCR. Standard PCR was performed together with negative (H2O and tonsil DNA) and positive control C. psittaci DNA. Results: In all five primer sets for C. psittaci DNA, each PCR product showed clear positive band and negative with appropriate positive and negative controls, respectively. All 150 ocular MALT lymphoma cases showed positive for Beta-globin DNA control, but negative for C. psittaci DNA in any of PCR product with five primer sets. Conclusion: These results suggest that possibility of the pathogenetic role of C. psittaci in ocular MALT lymphoma would have been overestimated so far, at least in Korean people. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4011 Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity. MyD88 is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the toll-like receptor (TLR) family. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs. We therefore investigated how the MyD88-dependent expansion of MDSCs from donor bone marrow (BM) contributes to protection of acute GVHD. To test this, we employed an intestinal GVHD murine model, C57BL/6 (H-2b) → B6D2F1 (H-2b/d), which differs at major and minor histocompatibility loci. Lethally irradiated recipient mice were transplanted with wild-type (WT) or MyD88 knock out (KO) mice T cell-depleted (TCD)-BM together with WT spleen T cells. Morbidity and mortality of GVHD was significantly worse in recipients of MyD88 KO TCD-BM with higher intestinal pathologic grading. Animals that underwent syngeneic HSCT did not show early mortality regardless of presence of MyD88 in BM, which ruled out myelosuppression-associated toxicity. The expression of Gr-1+CD11b+ in blood, mesenteric lymph nodes and liver on day 13 was significantly reduced in the recipients of MyD88 KO TCD-BM compared with those of WT TCD-BM while the percentage of donor T cells infiltrating colon and liver was significantly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of MyD88 KO TCD-BM. Injection of MDSCs from BM of non-tumor bearing donor markedly inhibited GVHD lethality in recipients of MyD88 KO TCD-BM. Moreover, in vivo administration of lipopolysaccharide (LPS), a TLR ligand, to donor mice expanded GR-1+CD11b+ in BM with enhanced expression of MyD88 mRNA. Recipients of TCD-BM from WT mice injected LPS showed attenuated GVHD severity as measured by weight loss and survival compared to those of TCD-BM from WT mice injected diluent. In summary, MyD88-dependent expansion of GR-1+CD11b+ population from donor TCD-BM appears to be critical for survival after allo-HSCT. Incomplete expansion of GR-1+CD11b+ population in target organs correlates with decreased apoptosis and increased infiltration of donor T cells into the target organs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5195 High-grade lymphomas are aggressive but largely curable, whereas low-grade lymphomas are indolent, but frequently recur to be incurable, paradoxically. Mesenchymal stromal cells (MSCs) have been known to participate for reconstituting microenvironment. Studies show that signalings between normal lymphoid cells and stromal cells were frequently altered in high grade lymphomas, but relatively conserved in low grade lymphomas. However, which cell and mechanism is responsible for lymphoma recurrence remains unclear. Here we hypothesized that the interaction with stroma may play a role for lymphoma cell growth and survival. For this, we investigated the effect of MSCs on lymphoma cell growth and chemo-resistance by using a coculture system with MSCs (derived from tonsil), as a stromal microenvironment for lymphoma cells. First, coculture of lymphoma cell line (Pfeiffer) and primary lymphoma cells with/without MSCs for 3 days showed that the MSC-cocultured cells grew more rapidly (1.4 times and 1.8 times for Pfeiffer and primary cells, respectively) than MSC-free lymphoma cells. To further investigate the underlying mechanism of promoting growth, lymphoma cells were cocultured with MSCs in the presence or absence of transwell filter. At day 4, the proliferation of lymphoma cells in the absence of transwell was 3.5 times higher than in the presence of transwell. Interestingly, the lymphoma cells cocultured with MSCs showed increased expression of CXCR4 compared with lymphoma cells cultured alone. When the cyto-protective effect of MSCs was examined by doxorubicin treatment (1ug/ml) in the presence or absence of MSCs, 91% of MSC-cocultured cells survived, whereas only 28% of stroma-free cultured cells survived. These results demonstrate that the direct contact interaction with stroma might be important for lymphoma cell growth and survival. In conclusion, our data suggest that the interaction with stromal microenvironment is an important factor for survival of lymphoma cells which cells might be responsible for chemoresistance, and raise the possibility that the stromal interaction could be a potential target for lymphoma treatment. Disclosures: No relevant conflicts of interest to declare. This research was supported by a grant (10172KFDA993) from Korea Food & Drug Administration in 2011. Disclosures: No relevant conflicts of interest to declare.