bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the Syk-mTOR Pathway in Follicular Lymphoma Cell Invasion and Angiogenesis,
Authors: Bezombes Christine , Fruchon Séverine , Kheirallah Samar , AlSaati Talal , Ysebaert Loic , Laurent Camille , Fournié Jean-Jacques , Laurent Guy
Published: October 2019

Abstract 3656

Follicular lymphoma is the second most common non-Hodgkin's lymphoma. The disease affects the lymph nodes and 50% of patients present with bone marrow infiltration, however the mechanisms involved in dissemination of the disease are not yet known. We previously reported that follicular lymphoma cells display an over-expression of Syk, a tyrosine kinase involved in many cellular processes including cell migration. Therefore we sought to explore its role in the invasive process. Here we show that follicular lymphoma patients display higher MMP-9 and VEGF levels than healthy donors. Moreover, using Syk siRNA and the Syk inhibitor R406, we demonstrate that, in follicular lymphoma cells, Syk is involved in the regulation of MMP-9 and VEGF expression, and that invasion and angiogenesis is mediated through a PI3K-mTOR module. Finally, using a follicular lymphoma xenograft mouse model we observe that R406, the active metabolite of fostamatinib (R788), inhibits MMP-9 expression and angiogenesis in vivo. Altogether, this study provides strong evidence that Syk represents an encouraging therapeutic target in follicular lymphoma and suggests the potential use of fostamatinib as an anti-invasive and anti-angiogenic drug.

Disclosures:

No relevant conflicts of interest to declare.
Incremental Value in Outcome Prediction with Molecular Signatures in Diffuse Large B-Cell Lymphoma,
Authors: Hong Fangxin , Kahl Brad S. , Gray Robert
Published: October 2019

Abstract 3687

INTRODUCTION:

Multiple gene expression-based biomarkers have been identified in diffuse large B-cell lymphoma (DLBCL) that are predictive for survival outcomes. Most studies assess predictive significance based on p-value from multivariate Cox regression; few investigations have evaluated the incremental usefulness of these biomarkers in risk prediction. Using the recently developed concordance measures (e.g., C-statistics) on censored survival data, we assessed the usefulness of two published gene-based risk signatures and compared them to the known clinical prognostic factors; with an overall goal of investigating the added value.

METHOD:

The added value of biomarkers was assessed by C-statistic and the Integrated Discrimination Improvement (IDI). The overall C-statistic is an estimated concordance between prediction and observation (event vs. non-event) - the probability that predicted risk score is higher for subject with earlier time of event. The IDI measures overall improvement in sensitivity and specificity. The signatures we selected are a six-gene predictor by Lossos et al. (2004) and a three-component signature (∼400 genes) by Lenz et al. (2008). We used the Lenz dataset which include 233 patients with DLBCL who received R-CHOP therapy (median follow-up=2.81 yr), and focused on predicting 3-year survival outcome (42% censored). Clinical prognostic factors evaluated are the traditional IPI components (stage, performance status, age, LDH, and number of extra nodal sites).

RESULTS:

The C-statistic was 0.60 and 0.721 for six-gene predictor and three-component signature, suggesting good discrimination ability by molecular signature when used alone. However, the performance is inferior to IPI risk factors, with a C-statistic of 0.733. When integrating gene signatures with IPI risk factors, the C-statistic was increased to 0.744 and 0.762, an improvement of only 0.011 (95% CI, -0.049 to 0.071) and 0.029 (95% CI, -0.033 to 0.091) for six-gene predictor and three-component signature, respectively. Furthermore, assessment by IDI reveals an added value of only 0.011 (95% CI, -0.008 to 0.081) and 0.076 (95% CI, 0.013 to 0.16) for the two molecular signatures. Kaplan-Meier survival curves for the four quartile groups based on the predictor scores confirms the marginal benefit in risk prediction using molecular signatures. (Figure 1).

CONCLUSIONS:

These results indicate that molecular biomarkers are inferior to clinical factors for risk assessment in DLBCL and provide little added value in risk prediction. These calculations suggest we will need to consider more than gene expression to develop highly discriminatory risk prediction models. However, the study of gene expression and clinical outcomes retains considerable potential to enhance understanding of disease mechanisms and uncover new therapeutic targets.

Disclosures:

No relevant conflicts of interest to declare.
Development of a Bruton's Tyrosine Kinase (Btk) Inhibitor, ONO-WG-307: Efficacy in ABC-DLBCL Xenograft Model – Potential Treatment for B-Cell Malignancies,
Authors: Kozaki Ryohei , Yoshizawa Toshio , Tohda Shuji , Yasuhiro Tomoko , Hotta Shingo , Ariza Yuko , Ueda Yoshiko , Narita Masami , Kawabata Kazuhito
Published: October 2019

Abstract 3731

Purpose:

ONO-WG-307 is a small molecule inhibitor that covalently binds to Btk. Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and new therapies, preferably chemo-sparing therapies, or as add-on to existing treatment regimens are required to help treat patients with ABC-DLBCL. Therefore, Btk constitutes an interesting therapeutic target, thus the activity of ONO-WG-307 was evaluated in an ABC-DLBCL xenograft model.

Methods:

Tumor cells (TMD-8) were implanted subcutaneously into female SCID mice. Tumors were allowed to grow to a volume of 100–200 mm3 before the mice were randomized into groups based on tumor size. ONO-WG-307 was administered orally at doses up to 10 mg/kg bid. Tumors were measured two or three times weekly after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3 or after a maximum period of 2 months. In parallel, an exploratory pharmacodynamic marker of Btk inhibition (Phosphorylated-Btk [P-Btk]) was also investigated in vivo.

Results:

Treatment with ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth in a TMD-8 xenograft model. Furthermore, parallel analysis of a pharmacodynamic marker, P-Btk, supported that Btk was inhibited and the level of P-Btk inhibition was correlated with the decreased tumor volumes observed in the TMD-8 model.

Conclusion:

ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in the ABC-DLBCL xenograft model, with Btk inhibition further supported using a PD marker. Given the need to treat and overcome disease resistance especially in ABC-DLBCL, the use of a Btk inhibitor is a novel, mechanistic approach to treating B cell malignancies. Additional work is underway, combining ONO-WG-307 with chemotherapy and other targeted agents.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of AKT Signaling Potently Inhibits the Growth of JAK and MPL-Mutant Cells in the Myeloproliferative Neoplasms In Vitro and In Vivo,
Authors: Khan Irum , Huang Zan , Wen Qiang Jeremy , Koppikar Priya , Levine Ross L , Tefferi Ayalew , Crispino John
Published: October 2019

Abstract 3862

Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs).

Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion.

Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms.

Disclosures:

No relevant conflicts of interest to declare.
High Expression of NEK2 Induces Drug Resistance by Inhibiting Premature Senescence Through Activation of the PI3K/Akt Pathway in Myeloma and Other Cancers,
Authors: Zhan Fenghuang , Xiao Fang , Zangari Maurizio , Xu Hongwei , Tricot Guido J.
Published: October 2019

Abstract 3923

Background:

Autologous transplantation in combination with the newer drugs has significantly prolonged survival of patients with myeloma (MM). However, the majority of patients still relapses. Our previous study has shown that NEK2, a chromosomal instability (CIN) gene, is up-regulated in many types of cancer cells and is highly related to drug resistance. Recently, premature senescence has been determined to be an indicator of the efficacy of anti-cancer drugs. In this study, we investigate the correlation between the inhibition of premature senescence and NEK2 induced drug resistance.

Materials and Methods:

Four MM cell lines (Δ47, KMS28PE, ARP1 and U266) and 2 other cancer cell lines (MCF7, H1299) over- and under-expressing NEK2 using a lentiviral delivery system were studied. NEK2 induced drug resistance was evaluated by colonogenic and standard apoptotic assays. The phenotypic changes of premature senescence were determined by using SA β-galactosidase (SA β-gal) staining. The mechanisms of NEK2-induced anti-senescence activity were studied in vitro and in the 5TGM1 murine MM mouse model.

Results:

Over-expression of NEK2 stimulated cancer cells into increased S and G2/M phase, resulting in polyploidy. All NEK2 over-expressing cancer cells were able to override doxorubicin-induced premature senescence and to maintain their proliferative potential. Mechanistic studies revealed that NEK2 activated the PI3K/Akt pathway. This was confirmed by the use of a specific PI3K/Akt inhibitor, KP372-1, which abrogated NEK2-induced drug resistance by decreasing clonogenicity of NEK2 over-expressing cells and by decreasing tumor burden and extending mouse survival in the 5TGM1 myeloma mouse model even better than bortezomib. In contrast, knockdown NEK2 by shRNA also induced premature senescence. We determined that NEK2 activates PI3K/Akt pathway by directly interacting with pp1. And the Akt down-stream targets GSK-3, MDM2, NF-kB and the tumor suppressor genes p53, p21, p27, the pro-apoptotic and pro-survival genes of the Bcl-2 family as well as the ABC transporters that related to drug resistance were identified to contribute to the inhibition of premature senescence resulting in drug resistance.

Conclusion:

NEK2 inhibits doxorubicin-induced premature senescence, resulting in increased drug resistance by activating the PI3K/Akt pathway. Alternatively, down regulation of NEK2 by shRNA inhibits cell proliferation and decreases drug resistance. Thus, targeting PI3K/Akt pathway should eliminate NEK2 induced drug resistance by re-establishing drug-induced premature senescence.

Disclosures:

No relevant conflicts of interest to declare.
TNF-α/Fas-RIP-1-Induced Cell Death Signaling Separates Hematopoietic Stem Cells/Progenitors Into Two Distinct Populations
Authors: Xiao Yechen , Volk Andrew , Zhang Shubin , Wei Wei , Breslin Peter , Zhang Jiwang
Published: October 2019

Abstract 393

Tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) have been found to induce a negative regulatory effects on hematopoiesis and have been implicated in the pathogenesis of human bone marrow failure (BMF) syndromes. However, the molecular mechanism by which these factors inhibit hematopoiesis is still not completely known. We previously reported that Tak1-knockout mice (Tak1−/−) develop BMF due to the mass apoptosis of hematopoietic cells, including hematopoietic stem cells and progenitors (HSC/Ps). Taking advantage of this mouse model, we studied the effects of TNF-α and Fas-induced death signaling on HSC/Ps by examining their contributions to the development of BMF syndromes in Tak1−/− mice. To do so, TNF-α and Fas-induced signaling were genetically inactivated in Tak1−/− HSC/Ps in order to examine to what degree both the apoptosis of HSC/Ps and BMF in vivo can be prevented. We found that complete inactivation of TNF-α signaling by the deletion of both Tnfr1 and Tnfr2 (TNF receptors 1 and 2) is able to protect up to 30–40% of Tak1−/− HSC/Ps from apoptosis. In vitro studies suggested that Fas signaling also contributes to less than 10% of Tak−/− HSC/P death. However, since Fas works on the same population of cells as TNF-α, and because TNF-α signaling is dominant in vivo, inactivation of Fas signaling failed to inhibit the apoptosis of HSC/Ps and BM damage in Tak1−/− mice. In addition, inhibition of RIP-1 (Receptor-Interacting Protein-1) activity by the specific inhibitor Nec-1 (Necrostatin-1) but not inhibition of FADD/caspase-8 signaling was able to protect the same percentage of the Tak−/− HSC/Ps from death as complete inactivation of TNF-α signaling did, but was unable to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSC/Ps (Tak1, Tnfr1 and r2 compound mutant). This suggests that TNF-α, acting through RIP-1, induces death in 30 to 40% of HSC/Ps. To investigate the causes of apoptosis in the remainder of cells, we looked for factors which either protect Tak1−/−Tnfr1−/−r2−/− HSC/Ps from death or further induce such death. We found that the expression of major pro-survival genes is significantly down-regulated in Tak1−/− HSC/Ps. The survival of the Tak1−/−Tnfr1−/−r2−/− HSC/Ps can be further improved by transducing the over-expression of dominant negative (DN)-caspase-9, as well as by Bcl-xl. Our studies suggest that there is heterogeneity in BM HSC/Ps. Only a portion of HSC/Ps is responsive to TNFα/Fas-RIP-1-induced cell death, whereas the death of the remaining HSC/Ps is induced by an intrinsic apoptotic mechanism. Tak1 is involved in mediating hematopoietic cytokine- and pro-inflammatory factor-induced survival signaling, protecting against both the TNF-α/Fas-RIP-1-dependent and independent death of HSC/Ps.

Disclosures:

No relevant conflicts of interest to declare.
Pomalidomide and Dexamethasone in Relapsed Myeloma: Results of 225 Patients Treated in Five Cohorts Over Three Years,
Authors: Lacy Martha Q , LaPlant Betsy R. , Laumann Kristina , Gertz Morie A , Hayman Suzanne R , Buadi Francis K , Dispenzieri Angela , Kumar Shaji , Lust John A. , Russell Stephen , Dingli David , Zeldenrust Steven R , Greipp Philip R. , Fonseca Rafael , Bergsagel P.Leif , Roy Vivek , Stewart Keith , Reeder Craig B. , Hall Robert L , Rajkumar S. Vincent , Mikhael Joseph R.
Published: October 2019

Abstract 3963

Background:

Pomalidomide at doses of 2 or 4 mg/d has demonstrated excellent activity in patients with relapsed multiple myeloma (MM). Between November 2007 and November 2010, we opened 5 sequential phase 2 trials using the pomalidomide at differing doses with weekly dexamethasone (Pom/dex) regimen to study the efficacy of this regimen.

Methods:

The five cohorts consisted of: Cohort 1 (N=60): relapsed MM with 1–3 prior regimens, 2 mg dose; Cohort 2 (N=34): lenalidomide refractory, 2 mg dose; Cohort 3 (N=35): bortezomib/lenalidomide refractory, 2 mg dose; Cohort 4 (N=35): bortezomib/lenalidomide refractory, 4 mg dose; and Cohort 5 (N=60) lenalidomide refractory, 1–3 prior regimens, 4 mg dose. Pomalidomide was given orally 2 mg daily or 4mg daily on days 1–28 of a 28-day cycle with oral dexamethasone given 40 mg daily on days 1, 8, 15 and 22. Response was assessed by the International Myeloma Working Group Uniform Response criteria. All patients received aspirin 325 mg daily for DVT prophylaxis or full dose anticoagulation.

Results:

A total of 225 patients were enrolled across all 5 cohorts. One patient was ineligible and excluded from analysis. The median age was 63 years (32.0–88.0). The median time since diagnosis was 53 months. Forty percent had high-risk molecular markers. Eighty-nine percent had received previous IMIDs including thalidomide (53%) and lenalidomide (81%). Sixty-two percent had previous bortezomib and 73% had prior transplant. The median follow-up is 12.6 months, but varies from 9.4 months for the most recent cohort to 30 months for the first cohort. Sixty-nine percent are alive and 30% remain progression free. Toxicities ≥ grade 3 are shown in table 1 and patient outcomes are shown in Table 2.

Conclusions:

Pom/dex is remarkably active and well tolerated even in heavily pretreated patients. Responses are durable. Response rates and toxicity are similar between the 2 mg and 4 mg doses.

Disclosures:

Lacy: Celgene: Research Funding. Fonseca:Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.
Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,
Authors: Shima Haruko , Shino Mika , Yamagata Kazutsune , Aikawa Yukiko , Koseki Haruhiko , Kitabayashi Issay
Published: October 2019

Abstract 3998

Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells.

Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency.

To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B.

Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Lung Endothelial Injury Induced by HNA-3a Antibodies in TRALI
Authors: Bayat Behnaz , Tjahjono Yudy , Akylbek Sydykov , Hippenstiel Stephan , Weissmann Nobert , Sachs Ulrich J , Santoso Sentot
Published: October 2019

Abstract 40

Transfusion related acute lung injury (TRALI) is a severe side-effect of blood transfusion characterized by the acute onset of non-cardiogenic pulmonary edema. Meanwhile, TRALI turned out to be the leading cause of transfusion related fatalities. Accumulated evidence demonstrates that antibodies against human neutrophil antigens (HNAs) play a major role in the pathomechanism of TRALI. Recent studies show that antibodies against the allelic isoform of choline transporter like protein 2 (CTL-2 Arg154 isoform; also known as HNA-3a) are associated with high TRALI mortality. The mechanism underlying this fatal reaction, however, is not clear. In this study, we aimed to identify the mechanism of fatal TRALI induced by HNA-3 antibody under in vitro as well as in vivo conditions.

Analysis of mRNA by real-time PCR in different blood cells and endothelial cells (EC) revealed abundant copies of CTL-2 transcripts in ECs in comparison to neutrophils and platelets. This result was confirmed by immunochemical analysis using rabbit antibody specific for CTL-2 as well human antibodies against HNA-3a. In contrast to treatment of neutrophils, treatment of EC with HNA-3a antibodies, but not with antibodies against the antithetical allelic CTL-2 isoform (Gln154 isoform; HNA-3b), leads to significant production of reactive oxygen species (ROS). When HNA-3aa EC monolayers were treated with human anti-HNA-3a antibodies, a significant increase in albumin-FITC influx in transwell system was observed when compared to controls. In line with this observation, a strong reduction of transendothelial electrical resistance was measured. Microscopically, drastic stress fiber formation and gap formation was visible. Immunoblotting analysis of HNA-3a treated ECs showed a significant degradation of VE-cadherin. This observation indicates that anti-HNA-3a antibodies induce EC barrier disturbance via ROS-mediated destabilization of VE-cadherin in cell junctions. Accordingly, this antibody-mediated barrier leakage can be ameliorated by the vasoactive peptide adrenomedulin (ADM), which prevents cell destruction in response to oxidative stress.

In an in vivo murine model of TRALI, with lipopolysaccharide primed C57BL/6 mice upon injection of HNA-3a antibodies a significant increase in lung weight and elevated concentration of albumin and number of neutrophils in the bronchoalveolar lavage was observed, indicating the formation of lung edema in these mice. Neutrophil depletion mitigated this effect in mice, but failed to prevent TRALI.

Our data demonstrate the direct influence of transfused HNA-3a antibodies on endothelial barrier integrity in vitro as well as in vivo. This novel mechanism of TRALI may be helpful in defining prevention and treatment strategies in order to decrease transfusion mortality.

Disclosures:

No relevant conflicts of interest to declare.
Excess Heparan Sulphate Inhibits CXCL12-Mediated Hematopoietic Cell Migration and Engraftment After Bone Marrow Transplant in Mice with Mucopolysaccharidosis Type I,
Authors: Watson H. Angharad , Holley Rebecca J , Langford-Smith Kia J , Wilkinson Fiona L , van Kuppevelt Toin H , Wynn Robert , Wraith J. Ed , Merry Catherine L R , Bigger Brian W
Published: October 2019

Abstract 4010

The primary axis of migration for transplanted hematopoietic stem and progenitor cells (HSPC) is CXCL12/CXCR4. Heparan sulphate (HS) is required for CXCL12 presentation and receptor binding, but the functional role of HS is poorly defined. The alpha-L-iduronidase knockout mouse (Idua−/−) accumulates HS and dermatan sulphate, recapitulating the neurodegenerative lysosomal storage disease Mucopolysaccharidosis I Hurler (MPSIH). MPSIH is primarily treated with HSPC transplant, but clinical experience suggests a historical engraftment defect in patients.

We show significantly reduced HSPC migration in Idua−/− recipients and under limiting engraftment conditions we show a significant haematopoietic engraftment defect in Idua−/− recipients. No significant donor cell effect was observed. Bone marrow but not peripheral blood CXCL12 levels are slightly elevated in Idua−/− mice. CFU frequency in BM is unchanged between genotypes but reduced significantly in peripheral blood of Idua−/− mice. In whole bone marrow, and on mesenchymal stem cells from Idua−/− mice, HS is present in significant excess, particularly in extracellular matrix, and cell surface locations, with significant increases in all sulphation modifications, especially 2-O-sulphation. Finally we show that excess HS, and particularly HS with increased 2-O -sulphation, functionally inhibit haematopoietic progenitor cell migration in vitro. These data provide novel insight into the influence of highly sulphated HS in CXCL12 mediated haematopoietic progenitor cell migration and help to explain why HSCT engraftment has been historically low in MPSIH.

Disclosures:

No relevant conflicts of interest to declare.
MyD88-Dependent Expansion of An Immature GR-1+CD11b+ Population From Donor Bone Marrow Reduces Early Mortality After Allogeneic Hematopoietic Stem Cell Transplantation,
Authors: Lim Ji-Young , Park Gyeongsin , Youn Hyewon , Choi Eun-Young , Jeong Dae-Chul , Min Chang-Ki
Published: October 2019

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.
Impaired Donor T Cell Egress From Recipient Lymphoid Tissue Profoundly Diminishes Acute GvHD,
Authors: Fulton LeShara M , Coghill James , West Michelle L. , Foger Niko , Bear James , Serody Jonathan S
Published: October 2019

Abstract 4022

The migration of donor T cells into and out of host lymphoid tissue is critical for the induction of acute Graft-versus-Host Disease (GvHD). A number of groups including ours have found that the lack of lymphoid tissue or impaired migration into that tissue prevented acute GvHD. However, it is not clear if preventing egress of effector T cells from lymphoid tissue can inhibit GvHD. Coronin 1A (Coro 1A) is an actin binding protein involved in cell migration and motility, and has previously been shown to contribute to the ability of T cells to migrate within lymph nodes. To elucidate a role for Coro 1A in GvHD pathogenesis we used mice deficient in Coro 1A (Coro 1A−/−). Methods : A haplotype-matched Hematopoietic Stem Cell Transplant (HSCT) model was used. C57BL/6 (H-2b, “B6”) mice were used as donors while C57BL/6 x DBA2 F1 (H-2bxd, “B6D2”) functioned as recipients. Na•ve conventional T cells (Tns) were isolated from the spleens of wild type B6 (WT), enhanced green fluorescent protein expressing B6 (WT eGFP), Coro 1A−/−B6, and Coro 1A−/−eGFP mice. B6D2 mice were lethally irradiated to 900 rads on day −1 and injected with 4 × 106 Tns from WT, WT eGFP, Coro 1A−/−, or Coro 1A−/−eGFP mice supplemented with 3 × 106 WT B6 T cell depleted bone marrow cells on day 0. Recipients were followed for survival and assessed for GVHD twice weekly using a validated scoring system. In those mice given WT eGFP or Coro 1A−/− eGFP Tns, recipient organs were imaged by stereofluorescence microscopy on days 3 and 14 post transplantation. Results : Mice that received Coro 1A−/− Tns displayed significantly attenuated GvHD with greater than 90% surviving to transplant day 60. In contrast, those animals receiving WT Tns demonstrated a 100% mortality rate by the end of the study period (Fig 1A, p<0.05 by Fisher's exact test). Stereomicroscopy performed 3 days after transplantation demonstrated a lower eGFP signal within the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) of those mice given Coro 1A−/−eGFP T cells versus those receiving WT eGFP cells. By 14 days post transplantation, however, the converse was observed, with more cells found within the MLNs and PPs of recipient mice receiving Coro 1A−/−eGFP cells (Fig 1B). Total T cells numbers in the liver and colon, however, were decreased at this time point in Coro 1A−/−eGFP recipients, indicating an impaired ability for these cells to exit host lymphatic tissue and traffic to GvHD target organs. Conclusion : These data indicate that impaired egress from lymphoid tissue, by blocking the function of Coro 1A, leads to a profound decrease in GvHD pathology and donor T cells in GvHD target organs. These data suggest that blocking the migration of donor T cells out of lymphoid tissue is a new viable approach to prevent acute GvHD.

Disclosures:

No relevant conflicts of interest to declare.
ABT-737 Sensitizes B Cell Tumors for Killing by CD19-Retargeted T Cells,
Authors: Karlsson Hannah S C , Lindqvist Camilla , Paul-Wetterberg Gabriella , Wiklund Helena Jernberg , Nilsson Kenneth , Simonsson Bengt , Frisk Per , Loskog Angelica
Published: October 2019

Abstract 4032

Introduction:

T cells expressing tumor-targeting chimeric antigen receptors are showing promise in clinical trials for patients with B cell leukemia and lymphoma. However, increased levels of anti-apoptotic proteins, a common trait among B-cell tumors, may hamper treatment efficacy. ABT-737 is a small molecule inhibitor of anti-apoptotic proteins such as BCL-2, BCL-xL, BCL-w, and MCL-1, which induces apoptosis via the intrinsic apoptosis pathway in contrast to T-cells that utilize the extrinsic pathway controlled by death receptors and their ligands. ABT-737 has been shown to efficiently promote apoptosis in B-cell tumors as exemplified in models of pre-B-ALL. Recently, ABT-737 was shown to synergize with TRAIL to induce apoptosis. This prompted us to investigate if ABT-737 could be combined with T-cell therapy to enhance tumor cell death.

Methods:

PBMCs from healthy donors and patients with pre-B-ALL was genetically engineered with a second generation chimeric antigen receptor (CAR) targeting CD19 on B-cells. The T-cells and ABT-737 were tested both individually, and in combination, for their cytotoxic capacity in in vitro assays such as flow cytometry and the Caspase-Glo® 3/7 assay. The effects were studied in a panel of B-cell tumor cell lines (Daudi, U698, Karpas422, DG75, Nall-1) since they may exhibit different apoptosis resistance profiles. The expression of anti-apoptosis molecules in these cell lines was investigated by PCR.

Results:

PCR confirmed expression of BCL family proteins in the cell lines tested. CD19-targeting T-cells specifically induced apoptosis in CD19+ tumor cells. Similarly, but less efficiently, ABT-737 as single agent increased apoptosis in the various tumor cell lines. When combining T-cell and ABT-737 therapy, the tumor cell death was significantly increased to that of single agent treatment. The effect varied from additive to synergistic effects. The tumor cell lines did not change the level of antigen presenting molecules (MHC I and II), death receptors (Fas) or adhesion or costimulatory molecules (ICAM-I, CD80, CD86) upon ABT-737 treatment. Hence, the effect did not likely represent increased killing by enhanced physical interaction between T-cells and tumors but rather simultaneous engagement of both intrinsic and extrinsic apoptosis pathways.

Conclusion:

The apoptosis inducer ABT-737 is potently enhancing CD19-targeting T-cell therapy. By triggering both intrinsic and extrinsic apoptosis pathways also resistant tumors may succumb to treatment.

Disclosures:

Simonsson: Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria.
Neutropenic Fever During Stem Cell Mobilization Is Associated with Decreased CD34+ Cell Collection and Poor Survival Following Autologous Stem Cell Transplantation,
Authors: Duong Hien , Bolwell Brian J. , Rybicki Lisa , Sweetenham John William , Pohlman Brad , Dean Robert , Andresen Steven , Kalaycio Matt , Sobecks Ronald , Koo Anna , Copelan Edward A.
Published: October 2019

Abstract 4053

High-dose chemotherapy followed by autologous stem cell transplant can improve long-term outcome of patients with relapsed non-Hodgkin and Hodgkin lymphoma. Stem cell mobilization with chemotherapy in addition to G-CSF improves stem cell collection, however it is also associated with increased risk of neutropenic fever. We analyze whether hospitalization for neutropenic fever (NF) affects outcomes after transplantation.

From 1/1998 to 12/2008, 599 patients underwent chemotherapy priming with etoposide + G-CSF (27 patients also received rituximab). Forty five (8%) did not proceed to transplant. Of the 554 (92%) patients who were transplanted, the median age was 51 years, and 348 (63%) were male. The diagnoses were 422 non-Hodgkin lymphoma (63%) and 132 Hodgkin lymphoma (24%). A majority of these patients (92%) had received 3 or less prior chemotherapy regimens. Only 6% had been exposed to fludarabine therapy. Most patients had chemosensitive disease (88% were in CR or PR) at time of transplant. For preparative regimen: 517 (93%) received busulfan/etoposide/cyclophosphamide, 32 (5.8%) received melphalan, 5 (0.9%) received other chemotherapy.

Of the transplanted patients, 121 (21.8%) were admitted for NF during stem cell mobilization. The median duration of admission for NF was 5 days (range 1–20). Six (5%) patients had bacteremia, 1 (<1%) had parainfluenza virus pneumonia, and the remainder 114 (94%) did not have any infection source or organism identified. The patients hospitalized for NF had significantly lower CD34+ cell collection (7.49 × 10^6 CD34 cells vs. 9.36 × 10^6, P=0.029) and lower CD34+ cell dose (7.34 × 10^6 CD34 cells/kg vs. 8.60 × 10^6 CD34+ cells/kg, P=0.047) despite more apheresis days required for collection (4 days vs. 3 days, P=0.01). Following transplant hospitalization, the 30-day readmission rate was higher in NF patients (16.8% compared to 8.8%, P=0.012). Hospitalization for NF was also associated with worse survival as shown below:

In conclusion, admission for neutropenic fever during stem cell mobilization is associated with significantly increased number of apheresis days required for adequate stem cell collection, lower CD34+ cell collection, higher 30-day re-admission rates, and poorer survival.

Disclosures:

No relevant conflicts of interest to declare.
Matched Pair Analysis of Cryopreserved Versus Fresh Allogeneic Peripheral Blood Stem Cell Transplantation
Authors: Trummer Arne , Krauter Juergen , Stadler Michael , Ganser Arnold , Buchholz Stefanie
Published: October 2019

Abstract 4529

Published data about outcome after cryopreserved allogeneic peripheral blood stem cell transplantation are scarce and, so far, have only been compared to historic cohorts of fresh graft recipients. We have performed a matched pair analysis of 66 patients receiving either cryopreserved or fresh grafts from matched related donors at our institution between January 2005 and June 2011 with a median follow-up time of 576 days (range: 18–2080 days).

For matching patients we calculated a propensity score including patient age, sex, diagnosis, performance status, remission status before transplantation (first remission vs. other), conditioning therapy (standard vs. reduced intensity), GvHD prophylaxis (with or without methotrexate) and CD34 cell count in the graft. Consequently, there were no significant differences between both groups for these parameters: median patient and donor age were 53 years (range: 18–68 and 23–76 years, respectively) for 34 male and 32 female patients with a performance status of ECOG 0 (n=60) or ECOG 1 (n=6). Diagnoses were AML (n=47), ALL (8), lymphoma (10) and CMPN (1). 31 patients were in first remission before transplantation. Reduced intensity conditioning therapy (n=52) and GvHD prophylaxis without methotrexate (n=41) were more frequent than standard conditioning (n=14) and prophylaxis with methotrexate (n=25), respectively.

Median cell counts were also almost equal in fresh and cryopreserved grafts for CD34 cells (5.7×106/kg (range: 3.1–11.4×106/kg) vs. 5.1×106/kg (2.6–12.3×106/kg), respectively), total nuclear cells (10.0×108/kg (4.9–21.4×108/kg) vs. 9.6×108/kg (5.0–19.1×108/kg) and CD3 lymphocytes (3.8×108/kg (2.1–6.8×108/kg vs. 3.4×108/kg (0.6–4.5×108/kg)).

All patients engrafted. Median neutrophil engraftment with an ANC >0.5×109/l was reached after 16 days (range: 10–21) vs. 15 days (10–31) (p=.15 by paired t-test) and platelet engraftment >20×109/l (for 3 consecutive days without requiring transfusion) occurred after 13 days (8–33) vs. 14 days (9–45) (p=.27).

Median follow-up time was similar between both groups (566 vs. 586 days, p=.894) and mean overall survival time, as calculated by Kaplan-Meyer analysis, was 1113 days for patients receiving fresh compared to 1258 days for patients receiving cryopreserved grafts (p=.582 by log rank test). Relapse or progression occurred in 13 vs. 14 patients, giving a mean disease/progression-free survival time of 922 vs. 1114 days (p=.467).

In summary, we did not observe any relevant outcome differences between patients receiving fresh or cryopreserved peripheral stem cell grafts of matched related donors. Therefore, the use of cryopreserved grafts can be considered safe and may even allow a more flexible transplant scheduling compared to fresh allografts.

Disclosures:

No relevant conflicts of interest to declare.
Oral Loading of Tacrolimus and Sub-Therapeutic Levels on the Day of Transplant Do Not Predict the Incidence of Acute Graft Versus Disease or Survival After Allogeneic Stem Cell Transplant
Authors: MacPherson Jill , Bachier Carlos , LeMaistre Charles F. , Sowell Hannah , Garcia Luis , Curel-Sanchez Alessandra , Cain Wilma , Caga Ursula , Means Jeremy , Shaughnessy Paul
Published: October 2019

Abstract 4543

Tacrolimus is proven to be effective in the prophylaxis of acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (ASCT). Published studies that initiate tacrolimus loading intravenously indicate that levels should be between 5 and 20ug/L on the day of transplant. Our institution often performs ASCT in the outpatient setting and therefore we developed a process for oral loading of tacrolimus. We retrospectively reviewed our experience in 94 consecutive patients (pts) who received ASCT from 10/10 HLA matched donors, 47 were matched sibling donors (MSD) and 47 were matched unrelated donors (MUD) with a median age of 50 and 52 respectively. Preparative regimens were IV busulfan based in 68 pts (BuFlu n=44, BuCY n=24), TBI based in 19pts, and other in 7 pts. All pts received GVHD prophylaxis with tacrolimus started orally between day -3 and -1 at a dose of 0.06 mg/kg/day divided twice daily. Standard short course methotrexate (MTX) was prescribed for all pts (D1 dose 15mg/m2, D3, 6, and 11 dose 10mg/m2). Pts who had sub-therapeutic tacrolimus levels (less than 5ug/L) on the day of transplant (Day 0) were compared to pts who had therapeutic tacrolimus levels (greater than 5ug/L) for incidence of any aGVHD and overall survival. Sixty nine patients had tacrolimus levels less than 5 ug/L on day 0 and 25 patients had tacrolimus levels of 5 ug/L or greater. There was no difference in incidence of any GVHD or survival between the groups (Table 1). Thirty one pts did not receive the entire MTX dose prescribed (most commonly because of withholding day 11 MTX dose), but there was no significant difference in the incidence of aGVHD in these pts despite the level of tacrolimus on D0. With a median follow up of 441 days 54/94 pts (57%) survive; 40/69 pts (58%) with day 0 tacrolimus levels that were less than 5 ug/L, and 14/25 pts (56%) with day 0 tacrolimus levels that were greater than or equal to 5 ug/L. In conclusion, oral loading of tacrolimus prior to ASCT at our institution results in sub-therapeutic levels on the day of transplant in the majority of patients, however, this does not appear to impact the overall incidence of aGVHD or survival. This study was limited by its retrospective nature, small size, and single center experience. Prospective randomized studies will be needed to determine if oral loading of tacrolimus impacts outcomes of ASCT.

Tacro; tacrolimus, OS; overall survival, MUD; matched unrelated donor, SIB; sibling. Survival determined in mean months from date of transplant.

Disclosures:

Shaughnessy: Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Otsuka: Honoraria, Speakers Bureau.
Sequential Therapy with Activated Prothrombin Complex Concentrates and Recombinant Factor VIIa in the Treatment of Unresponsive Bleeding in Patients with Hemophilia and Inhibitors
Authors: Park Young shil , Choi Yong-Mook , Park Ji Kyoung
Published: October 2019

Abstract 4664

The development of inhibitors to factor VIII or IX is the most common, severe, challenging and expensive complication of the treatment of patients with hemophilia. Hemophilia patients with inhibitors can develop bleeding episodes, which are refractory to monotherapy with either recombinant factor VIIa (rFVIIa) or activated prothrombin complex concentrates (APCC). Management of such bleeds is often difficult.

This report describes the results of a retrospective chart review of four hospitalized patients with severe hemophilia and inhibitors who have been treated with sequential doses of APCC and rFVIIa for refractory bleeding.

Sequential therapy was defined as the administration of both rFVIIa and APCC within 12 h. In 4 cases of patients with inhibitor, bleeding was not controlled by initial bypassing treatment. One patient had historical peak inhibitor titer increased to 1126 BU, and during peripheral inserted central catheter insertion for immune tolerance induction therapy, bleeding was not controlled. After APCC and rFVIIa were administered sequentially, bleeding was controlled. The second and third patients had bleeding problem after total knee replacement for hemophilic arthropathy, and two agents were administered sequentially. The last patient had small bowel resection because of intestinal obstruction, and post-operation bleeding was not controlled well. DIC was developed. Two agents were administered sequentially for bleeding problem and bleeding was controlled. Sequential therapy administration alternated one APCC dose to 1–2 rFVIIa doses: dosing intervals ranged between 3 and 6 h; APCC (75–100 U kg) was given every 12 h; rFVIIa (90 μg/ kg) was given every 3–6 h. Bleeding control was achieved in 12–24 h in all patients. Sequential therapy was discontinued after 2–5 days. No clinical adverse events were observed.

Sequential therapy with APCC and rFVIIa was efficacious without adverse events. A prospective clinical trial is needed to provide further evidence.

Disclosures:

No relevant conflicts of interest to declare.