bluuebunny/crossref_metadata_2025 huggingface dataset:
Gvhd Increases Apoptosis of CD8+, but Not CD4+, T Cells Expanded by Vaccines but Is Not Dependent on Fas Ligand
Authors: Capitini Christian M. , Guimond Martin , Fry Terry J
Published: October 2019

Abstract 2098

Donor lymphocyte infusions (DLIs) administered following allogeneic blood and marrow transplantation (alloBMT) can enhance T cell reconstitution through homeostatic peripheral expansion (HPE), but can also cause graft-versus-host-disease (GVHD). Vaccines are a strategy to skew T cells toward a “third-party” tumor antigen, but it is not clear if the effects of GVHD on vaccine-responding T cells is equivalent to the effects on alloantigen-reactive polyclonal T cells. Previous work by others has demonstrated “bystander” effects of GVHD causing activation-induced cell death through the Fas pathway on polyclonal T cells; however, the impact of GVHD on T cells expanded by a vaccine expressing a third-party nonalloantigen remains unclear. T cell depleted (TCD) minor histocompatibility antigen (mHA)-mismatched alloBMT (CD45.1+ B6 –> CD45.1+/CD45.2+ B6 × C3H.SW) was followed by a CD45.1+ B6 DLI on day +14 to induce GVHD. CD45.2+ B6 Rag2-/- T cell receptor transgenic (TCRTg) CD4+ or CD8+ T cells specific for epitopes derived from the male histocompatibility antigen complex (HY) were administered on day +28 with an HY-expressing male dendritic cell vaccine to determine the impact of the allogeneic environment on the persistence, proliferation and survival of vaccine-responding T cells. Despite the fact that syngeneic and allogeneic recipients had similar degrees of lymphopenia, the absolute number of CD4+ and CD8+ TCRTg T cells was decreased 5 and 7 days respectively after adoptive transfer and vaccination in alloBMT recipients compared to syngeneic recipients (p < 0.05). To determine the mechanism of decreased persistence of the TCRTg T cells, proliferation was measured by assessing dilution of CFSE and apoptosis was measured by FACS analysis for Annexin V. Importantly, since both the CD4+ and CD8+ TCRTg T cells do not undergo HPE, all proliferation has to be antigen-driven. Neither CD4+ nor CD8+ TCRTg T cells proliferated in the absence of HY vaccine following alloBMT, confirming lack of bystander proliferation. Interestingly, both CD4+ (p < 0.01) and CD8+ (p < 0.05) TCRTg T cells undergo less vaccine-induced proliferation after alloBMT compared to syngeneic recipients. The amount of CD8+ TCRTg T cell apoptosis following vaccination was higher in allogeneic recipients (p < 0.05), but there was no difference in CD4+ TCRTg T cell apoptosis. Surprisingly, using a DLI from Fas ligand-deficient B6 (gld) donors on day +14 was sufficient to induce GVHD but did not prevent CD8+ TCRTg T cell apoptosis. Thus, diminished vaccine responses during GVHD appear to result in part from impaired CD8+ and CD4+ T cell vaccine-driven proliferation, but there is an additional contribution on CD8+ T cell apoptosis that is not dependent on recognition of alloantigen or the Fas-Fas ligand pathway.

Disclosures:

No relevant conflicts of interest to declare.
Urokinase Receptor (uPAR)-Dependent Integrin Redistribution Represents a Central Mechanism for Growth Factor Induced Endothelial Cell Migration
Authors: Novotny Rene , Unseld Matthias , Poettler Marina , Zielinski Christoph , Binder Bernd , Prager Gerald
Published: October 2019

Abstract 2119

Tumor angiogenesis is induced when the net balance of pro- and antiangiogenic molecules is tipped in favor of angiogenesis, the so called ‘angiogenic switch’. Recently, we described a mechanism how VEGF induces pro-urokinase (pro-uPA) activation, which led to uPAR-complex formation and internalization of beta-1 integrins into the endosomal compartment via LDLR-proteins such as ApoER2 or VLDLR. Thereby, uPAR plays a central role for VEGF-induced endothelial cell migration.

Here, we describe that uPAR-induced integrin internalization and redistribution to the leading edge is not only limited to VEGF-induced endothelial cell migration, but plays a central role for others angiogenic growth factors such as fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF). Furthermore, we found that a hitherto undescribed binding site on domain 3 of uPAR for direct LDLR-protein interaction is required and sufficient for uPAR-dependent integrin redistribution. Interference with the uPAR/integrin internalization either by the Receptor Associated Protein (RAP) or a specific LDLR-binding site mimicking peptide (P1), the migratory response of endothelial cells towards the growth factors VEGF, HGF, FGF-2, or EGF was almost blocked (20.24% ± 4.56%). Consistently, expression of a mutated uPAR lacking interaction site for LDLR-proteins in uPAR-/- endothelial cells via a retroviral construct led to reduced invasive response towards angiogenic growth factors in vitro as well as in a Matrigel plug in vivo assay.

From these data we conclude that uPAR/LDLR-protein interaction represents a central molecule in growth factor-induced endothelial cell behavior, which might open a new avenue for therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
Association of HLA-G Haplotype Risk Model with Overall Survival In Acute Myeloid Leukemia Patients
Authors: Volbracht Anja , Novotny Juergen R , Ochsenfarth Crista , Switala Magdalena , Frey Ulrich , Horn Peter A , Duehrsen Ulrich , Rebmann Vera , Nuckel Holger
Published: October 2019

Abstract 2169

Introduction:

The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution and exerts multiple immunosuppressive functions. Thus, HLA-G expression in tumor cells favors tumor immune escape and tumor progression. The HLA-G gene polymorphism is extremely restricted in comparison to the classical HLA antigens. However, specific HLA-G polymorphisms in exons 3, 4, 5, 7 and parts of the 3′UTR encode for different haplotypes/alleles. Because of the prognostic role of HLA-G in cancer, especially in B-CLL, we investigated here the role of the HLA-G haplotypes in acute myeloid leukemia (AML).

Methods:

Genotyping was performed on the basis of examining exons 3, 4, 5, 7 and parts of the 3′UTR by pyrosequencing in 166 patients with AML. In total, we found 17 different haplotypes in the cohort.

Results:

The haplotype distributions between 166 AML patients and 190 healthy controls were significantly different in two alleles, arguing that these two haplotypes of HLA-G on the one hand increases (*01:06, p=0.042) and on the other hand decreases (*01:01:12 variante, p=0.0014) the susceptibility for AML. Next a risk model was generated for overall survival (OS) adapted to specific haplotypes/alleles of the HLA-G gene. The favorable group (A) consisted of the alleles *01:06 and *01:01:20 variant at codon 57 G (12 patients), the intermediate group (B) of *01:01:20, *01:04:10, *01:01:30 and *01:01:41 variant at codon 57 A (113 patients) and the unfavorable group (C) of the remaining 11 alleles (41 patients). The OS for patients in group C was significantly (p=0.02) shorter (median OS 613 days) than for those in group B (median OS 961 days) and A (median OS not reached). Multivariate analysis shows a trend that the risk HLA-G model (Hazard ratio (HR) 1.4, p=0.084) was an independent predictor next to the established prognostic factors cytogenetics (HR 2.1, p=0.001), age (HR 1.8, p=0.009), leucocytes (HR 1.6, p=0.026), ECOG stage (HR 1.5, p=0.006) and platelets (HR 2.1, p=0.015). Moreover, the new risk model was able to further subdivide patients with intermediate cytogenetic risk profile (median OS: group A not reached; group B 987 days; group C 624 days; p=0.10).

Conclusion:

Our study is the first study demonstrating that the combination of different alleles of the HLA-G gene is associated with the overall survival in AML patients. This fact emphasizes the extensive role of this gene by the tumor escape mechanism and could be responsible for progress in other cancers.

Disclosures:

No relevant conflicts of interest to declare.
SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells
Authors: Mak Duncan H , Manton Christa , Andreeff Michael , Carter Bing Z
Published: October 2019

Abstract 2177

The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL.

Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy.

Disclosures:

No relevant conflicts of interest to declare.
Prolonged Administration of the Telomerase Vaccine GRNVAC1 Is Well Tolerated and Appears to Be Associated with Favorable Outcomes In High-Risk Acute Myeloid Leukemia (AML)
Authors: Khoury H. Jean , Collins Robert H. , Blum William , Maness Lori , Stiff Patrick , Kelsey Stephen M. , Reddy Anita , Smith Jennifer A. , DiPersio John F.
Published: October 2019

Abstract 2190

Background:

Telomerase activity in leukemic blasts is frequently increased in high-risk AML patients, and the major protein catalytic subunit of telomerase (hTERT) includes immunogenic epitopes which can stimulate generation of hTERT-specific cytotoxic T cells.

Methods:

We evaluated the feasibility, safety, and immunologic effects of telomerase catalytic subunit (hTERT)-expressing autologous dendritic cells (DC) in adult AML patients. GRNVAC1 (VAC1) was produced at a central facility from patient-specific leukapheresis harvests. Immature DCs from monocyte-enriched PBMCs were electroporated with an mRNA construct encoding hTERT and a lysosomal targeting sequence (LAMP-1). Further culture with cytokines induced a mature DC phenotype. AML patients were eligible if in CR1 and with intermediate or high risk cytogenetics or in CR2. PBMCs were harvested after induction, and before or after completion of planned consolidation cycles. Patients in ongoing CR or early relapse (<20% marrow blasts) were vaccinated. Vials of VAC1 containing 1 × 107 cells were administered by intra-dermal injections weekly × 6, followed by 4 weeks off, then as boost vaccinations every other week × 6. In cases where additional vials were available, injections could then continue on a q28 day extended boost schedule. This abstract summarizes safety of administering VAC1 up through the extended boost schedule.

Results:

Between September 2007 and December 2009, 33 patients with a median age 61yrs (range, 30–75) were enrolled and leukapheresed. 21 patients received VAC1 vaccinations, 19 in CR (16 in CR1 and 3 in CR2) and 2 in early relapse. Eight of 16 in CR1 were at intermediate risk of relapse and 8 were at high risk of relapse. A median of 17 (6-32) vaccinations were administered to those 19 patients. VAC1 was well tolerated with no toxicities with the exception of one patient who developed ITP after 6 vaccinations. Local transient erythema at the site of injection was common in all patients.

With a median follow-up of 10.5 months (range, 1.2–27.2) months, 4/19 relapsed and 7 continue to receive VAC1. The Kaplan-Meier estimate of disease free survival (DFS) at 12 months, measured from beginning with the first vaccination of VAC1, was 79% for the 19 CR patients (95% CI: 52%-91%), 75% (31%-93%) for the eight patients in the intermediate risk group, and 81% for the 11 patients in the high risk group. Median DFS was not reached.

Conclusions:

Prolonged administration of VAC1, up to 32 vaccinations (the maximum number administered so far), is well tolerated in patients with AML, and associated with favorable DFS, especially in high-risk patients.

Disclosures:

DiPersio: Genzyme: Honoraria.
A Functional Variation In the NKG2D Gene Regulates NKG2D Receptor Expression and Is Associated with Better Transplant Outcomes After Fully-HLA-Matched Unrelated Bone Marrow Transplantation
Authors: Espinoza Luis J. , Takami Akiyoshi , Yoshioka Katsuji , Nakata Katsuya , Nakao Shinji
Published: October 2019

Abstract 221

A functional variation in the NKG2D gene regulates NKG2D receptor expression and is associated with better transplant outcomes after fully-HLA-matched unrelated bone marrow transplantation

NKG2D, an activating receptor expressed by NK cells and some T cells, plays important roles in both immunity to microbial infections and cancer immune surveillance. A single nucleotide polymorphism (SNP rs1049174) in the 3′-untranslated region (3′UTR) of the NKG2D gene determines two genotypes, HNK1 genotype with high NK cytotoxicity, and the LNK1 genotype with low NK cytotoxicity. Individuals with the HNK1 genotype have reduced risk of cancer (Cancer Res 2006; 66(1):63-70). In the present study, we analyzed the impact of SNP rs1049174 on transplant outcomes after HLA-matched unrelated bone marrow transplantation. The NKG2D genotypes were retrospectively analyzed in a cohort of 360 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program. The donor HNK1 genotype was associated with lower transplant related mortality (TRM) in patients with standard risk (hazard ratio [HR], 0.58; 95% confidence interval [CI], 0.35–0.97; P=0.04) (Fig.1). The donor HNK1 genotype was also associated with a trend toward better OS (HR, 0.69; 95% CI, 0.46–1.04; P=0.08). The host NKG2D genotypes did not significantly influence the transplant outcomes. NK cells from HNK1 donors expressed higher levels of NKG2D on the cell surface, and also expressed higher levels of NKG2D mRNA. Luciferase reporter gene constructs transfected into NK cells showed that the LNK1 construct more effectively attenuated gene expression compared with the HNK1 construct (60% vs. 35%). Bioinformatics tools predicted an interaction between the rs1049174 SNP motif with the micro-RNA-1245 (miR1245). Next, in an mRNA/miRNA binding assay, the LNK allele showed higher affinity to the miR1245 than the HNK1 allele. Moreover transfection of NK cells possessing the LNK1 allele with a miR1245 mimic resulted in 25 fold decrease in mRNA NKG2D level versus only 2 fold decrease in mRNA NKG2D expression in HNK1 allele derived NK cells, thus suggesting that the 3′UTR SNP may regulate the expression of NKG2D through its binding to the putative repressor miR1245. These findings provide evidence that the NKG2D 3′UTR polymorphism is functional, and that the NK cells from donors with the HNK1 genotype exert higher cytotoxicity through their increased expression of NKG2D, thereby resulting in an improved outcome for bone marrow transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Impaired Ribosome Maturation In Human Cells Depleted of Shwachman-Diamond Syndrome Protein SBDS
Authors: Sezgin Gulay , Nihrane Abdallah , Henson Adrianna , Wattenberg Max , Ellis Steven , Liu Johnson M.
Published: October 2019

Abstract 2242

Background:

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm.

Methods and Results:

To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1).

Conclusions:

Disclosures:

No relevant conflicts of interest to declare.
Distinct Impact of Imatinib on Growth In Prepubertal and Pubertal Children with Chronic Myeloid Leukemia
Authors: Shima Haruko , Tokuyama Mika , Tanizawa Akihiko , Tono Chikako , Hamamoto Kazuko , Muramatsu Hideki , Watanabe Akihiro , Hotta Noriko , Ito Masaki , Kurosawa Hidemitsu , Kato Koji , Tsurusawa Masahito , Horibe Keizo , Shimada Hiroyuki
Published: October 2019

Abstract 2277

Imatinib is now widely used for treating chronic-phase chronic myeloid leukemia (CML) in children as well as in adults, and long-term adverse effects of imatinib therapy in children are now gaining attention. One of its adverse effects is the negative impact on growth in children, suggested by 3 recently published case reports. However, the incidence or prospect of growth impairment resulting from imatinib treatment has not been fully elucidated.

In this study, we retrospectively analyzed the clinical records of 48 children with chronic-phase CML who were treated with imatinib as a first-line therapy between 2001 and 2006. The median age at diagnosis was 9 years (2 to 15 years). Cumulative change in height while on imatinib was assessed using the height standard deviations score (height-SDS), the converted height data from age- and sex-adjusted Japanese norms. Our data indicated that growth impairment (decrease in height-SDS) was observed in 72.9% of the patients, with median maximum reduction in height-SDS of 0.61 during imatinib treatment. Growth impairment was noticeable in children who were prepubertal at the commencement of imatinib treatment, while only mild or no growth impairment (with no decrease of height-SDS) was observed in most patients who were pubertal at the commencement of imatinib treatment. Furthermore, in prepubertal children with growth impairment, growth velocity tended to recuperate concomitant with pubertal maturation, suggesting that imatinib has little impact on growth during puberty. To our knowledge, this is the first report to describe and compare the distinct inhibitory effect of imatinib on growth in prepubertal and pubertal children with CML.

Although the introduction of imatinib was a breakthrough in CML therapy, the possibility of continuous remission after discontinuation of imatinib remains uncertain. Thus, the possibility of adverse effects of long-term exposure to imatinib has become a huge issue, especially when treating children. We consider that it is important to promote awareness of growth deceleration in children, especially in young children who started imatinib treatment before puberty and are inevitably going to be subject to prolonged exposure.

Disclosures:

No relevant conflicts of interest to declare.
Methylation and Silencing of miRNA-124 In Hematopoietic Cells by EVI1
Authors: Senyuk Vitalyi , Premanand Kavitha , Xu Peng , Cattaneo Francesca , Dickstein Jerome , Nucifora Giuseppina
Published: October 2019

Abstract 2476

The inappropriate expression of EVI1 is associated with aggressive myelodysplastic syndromes (MDS) and acute myeloid leukemia. We previously showed that in vivo EVI1 causes an MDS-like disease in mice and that in vitro it alters cell cycling, self-renewal, and differentiation pathways of primary bone marrow (BM) cells. It is likely that these alterations contribute to the oncogenic potential of EVI1. MicroRNAs (miRNA) are small noncoding RNAs that are 21 to 25 nt in length that up- and down-regulate gene expression during cell development, proliferation, differentiation, and apoptosis. MiRNAs play a critical role in solid and hematopoietic cancers. We found that in hematopoietic cells the expression of several miRNAs is significantly altered by EVI1. Among them, miRNA-124 is strongly downregulated in EVI1-expressing primary BM and 32Dcl3 cells. To evaluate the role of miRNA-124 in primary murine BM cells, we isolated Lin- cells and infected the cells with the empty vector, EVI1, or both EVI1 and miRNA-124. We confirmed that EVI1 alters cellular pathways regulating self-renewal, S1 phase entry, and differentiation. More importantly, however, we found that when miRNA-124 is co-expressed with EVI1, the expression of proteins such as Cyclin D3 and BMI-1, that control some of these pathways, is normalized. These results suggest that the effects of EVI1 are mediated by miRNA-124 repression. We hypothesized that the down-regulation of miRNA-124 could be associated with inappropriate DNA methylation of the miRNA. Both in man and mouse, there are three known alleles of miRNA-124. In the mouse, the alleles are located on chromosome bands 2H4, 3A1, and 14D1. Analysis of the three alleles indicates that miRNA-124-3 harbors the larger number of CpG dinucleotides, including 24 CpG within a stretch of 190 bp upstream of and overlapping the stem-loop start-site. By using the bisulfite DNA sequencing method, we found that in BM cells the number of methylated CpG dinucleotides in this region is significantly increased after EVI1 expression. The DNA methylation is mostly centered in two clusters, which we named cluster I and cluster II. These data clearly show that miRNA-124 is a critical target of EVI1 in human diseases.

Disclosures:

No relevant conflicts of interest to declare.
Adoptive Transfer of Primed CD62L+ T Cells Mediate Selective GVL In a Murine Model of Pediatric ALL
Authors: Qin Haying , Meadors Joanna L. , Fry Terry J.
Published: October 2019

Abstract 2535

Background:

While current upfront therapy will cure over 80% of patients with pediatric ALL, treating recurrence remains a major challenge. Blood or marrow transplantation (BMT) offers a curative option but approximately 50% of patients undergoing BMT will relapse. Donor lymphocyte infusions (DLIs) have demonstrated efficacy in myeloid leukemia but are much less effective for ALL. Strategies to improve the potency of DLI can result in GVHD and may compromise outcome. Thus, we explored strategies to improve on the potency and specificity of the graft versus leukemia response in a murine model of pediatric ALL.

Methods:

A transplantable cell line was generated from E2a-PBX1 transgenic mice (that express a recurring translocation t(1;19) present in approximately 5% of all pediatric ALL). Lethally irradiated C57Bl/6 (H2b) mice received T-cell-depleted C3H.SW (H2b, minor histocompatibility antigen miHA mismatch allogeneic) or C57Bl/6 (syngeneic) bone marrow followed by E2aPBX1 IV on day +6. A purified T cell DLI was given on day +7. DLI donors were primed with irradiated cells as indicated intraperitoneally 14 days prior to harvest. In some experiments T cell subsets were purified using magnetic beads.

Results:

E2a-PBX1 is a pre-B cell ALL that expresses B220, BP-1, CD43 and the alpha chain of the IL-7 receptor. The distribution of leukemia is similar to that observed in humans with bone marrow, lymph node, liver, spleen and central nervous system involvement confirmed by necropsy and imaging following injection of luciferase-expressing cells. Administration of naive T cells (6×106) following syngeneic or allogeneic BMT was not effective at treating E2a-PBX1 indicating that the presence of miHA did not increase the efficacy of naïve T cells consistent with the poor response rate to DLI in patients with ALL. However, primed T cell DLI (6×10ee6) from a donor immunized by irradiated E2a-PBX1 resulted in a statistically significant prolongation of survival in syngeneic (and cure in allogeneic BMT models suggesting the presence of tumor-specific antigens as well as a contribution from miHA. However, mice receiving sufficient primed allogeneic T cells for cure developed GVHD-associated weight loss and 40% late mortality (after 30 days). While purified, primed CD4 or CD8 T cells prevented GVHD associated mortality, 40–60% of mice developed leukemia. Remarkably, mice receiving magnetic-bead purified CD62L+ T cells from primed allogeneic donors were cured of leukemia with no GVHD-associated weight loss or mortality.

Conclusion:

Naïve T cells (analogous to DLI given post-relapse in the clinic) were ineffective at treating ALL our model. However, priming of allogeneic DLI donors with whole tumor cells results in cure but with the development of GVHD. CD62L+ expressing T cells from primed donors mediate curative GVL without GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Weight Loss: An Important but Often Overlooked Clue for the Diagnosis of Cancer
Authors: Peddi Prashanth , Chitneni Shalini , Noel Mary , Schwartz Kenneth A.
Published: October 2019

Abstract 2552

Background:

One of the primary goals of care for cancer patients is to detect growth and or recurrence as early as possible. We retrospectively evaluated unexplained weight loss of 5% or more as an indicator of cancer growth or recurrence in patients who were followed in a hematology/oncology clinic. In addition, we noted the frequency of physician recognition of the weight loss as well as the time delay from the onset of weight loss to detection of new cancer/metastasis.

Methods:

A search of the data base in the electronic medical record identified 90 cancer patients whose weight had decreased by 5 % or more over a maximal time period of 3 years. Weight loss in 30 of the patients could be explained as intentional (diet, exercise, gastric bypass) or because of advanced cancer or diuresis or documentation error. These 30 patients were excluded from the analysis.

Results:

The 60 included patients were evaluated on serial clinic visits, average 5.5 visits per patient. Weight loss of 5% or more that was not addressed on the first two visits was categorized as a missed clue. This occurred in 52 of the 60 (86%) patients. Among the 52 patients 11 (21%) had new cancers with weight loss documented in the medical record, but overlooked by their physician. Of the remaining 8 patients whose weight loss was addressed 3 (37%) had new cancers. The reasons for the weight loss in the remaining patients without new cancers were due to side effects of chemotherapy, dysphagia, recent hospitalization, infection and unknown. In patients whose weight loss was not noted after 2 clinic visits the time lag between the onset of weight loss and diagnosis of advancing cancer varied from 0.5 months to 29 months with a mean delay of 8.9 months. This compares with a mean delay of 0.9 months in those 8 patients whose evaluation was initiated as soon as their weight loss was documented in the chart (p=0.03).

Conclusion:

1. Weight loss in 23% of hematology/oncology patients was associated with cancer growth or recurrence. 2. The importance of weight loss as a clue to the diagnosis of a new cancer or to cancer progression is frequently not appreciated resulting in a significant delay in initiating diagnostic investigations.

Disclosures:

No relevant conflicts of interest to declare.
A Potential Epigenetic Bookmarking Function for the Hematopoietic Transcription Factor GATA-1.
Authors: Kadauke Stephan , Lamonica Janine M , Lau Alan , Chou Margaret , Blobel Gerd
Published: October 2019

Abstract 2601

Hematopoietic lineage choice decisions are stably maintained through many cell divisions. For example, erythroid precursor cells undergo several rounds of cell division during their maturation. During each mitosis, most transcription factors separate from chromatin causing transcription to cease globally. Mitosis therefore poses a challenge for transcription factors to re-associate with the appropriate target sites in chromatin of newborn cells. The epigenetic mechanisms that cement lineage stability and resist cell reprogramming during mitosis are poorly understood, although recent evidence supports the idea that “bookmarking” factors that remain associated with mitotic chromatin may play a role in this process. We therefore investigated whether the hematopoietic transcription factor GATA-1 might be retained at specific sites during mitosis. GATA-1 controls the expression of essentially all erythroid-specific genes and might therefore play a role in maintaining erythroid gene expression programs throughout the cell cycle. Surprisingly, we found that while a substantial fraction of GATA-1 dissociates from chromatin in mitosis, foci of high GATA-1 density are present within mitotic chromatin. To determine the exact locations of GATA-1 binding during mitosis, we developed a method to highly purify mitotic erythroid cells in sufficient quantities for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq). These experiments revealed that a subset of sites bound by GATA-1 during interphase is occupied continuously throughout mitosis. Importantly, continuously GATA-1-occupied sites are enriched at promoters and cis-regulatory elements of genes coding for key developmental regulators of hematopoiesis (e.g., Fog1/Zfpm1, Gata2, Lyl1) but are notably absent at erythroid physiological and structural genes (e.g., Hba, Hbb, Epb4.9). To examine the importance of mitotic chromatin binding by GATA-1, we engineered a version of GATA-1 bearing a mitosis-specific degron that targets GATA-1 for degradation during mitosis but not interphase. Preliminary results show that mitotically degraded GATA-1 fails to induce differentiation when expressed in GATA-1-null erythroblasts. This suggests an important mitotic function for GATA-1. Current work focuses on delineating the mechanism by which continuous chromatin occupancy of GATA-1 throughout mitosis ensures proper erythroid differentiation. The results will be presented and discussed at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
Estimated GFR Derived From Plasma Creatinine by Mass Spectrometry (MSMS) Is A Reliable Measure of Renal Function During the Hyperfiltration Phase In Sickle Cell Disease (SCD)
Authors: Inusa Baba , Booth Caroline , Turner Charles , Appleby Helen , Padmore-Penniston Giselle , Dalton R. Neil
Published: October 2019

Abstract 2649

Aim:

The hypothesis is that a height based eGFR formula using creatinine measured by mass spectrometry is a reliable measure of GFR in children with SCD.

Background:

The kidney is a particularly sensitive to hypoxia and renal failure is a major cause of morbidity and mortality in SCD. Children with SCD commonly hyper filtrate, thus making it difficult to establish a reliable Glomerular Filtration Rate (GFR) measure. It is essential to correctly assess the GFR, in order to identify when it begins to fall back towards normal and then starts to decline. Reporting of estimated glomerular filtration rate (eGFR) derived from plasma creatinine (pCr) measurements is now recommended for the classification and monitoring of patients with chronic kidney disease. The new MDRD formula requires the use of pCr methods traceable to stable isotope dilution mass spectrometry (MS). In children the recommendation is to use one of the formulae based on height (Ht), e.g. Schwartz et al1, Morris et al2, both of which were derived using traditional Jaffe creatinine methodology; eGFR = 40 × Ht (cm)/pCr (μmol/l). The introduction of MS traceable creatinine methods requires a review of the k factor. This correlation has never been validated in hyper filtration/or children with SCD.

Methods:

43 HbSS patients, age range 5–20yrs, attending the Evelina Children's Hospital were studied, including transfused and non-transfused patients. The plasma samples were also used for the measurement of plasma creatinine by stable isotope dilution mass spectrometry. eGFR was calculated using k=31.

Results:

Inutest GFR ranged from 70–175 ml/min/1.73m2. There was significant correlation between eGFR and inutest GFR (P<0.01, r=0.68).

Conclusions:

These preliminary data suggest that eGFR should prove valuable in monitoring GFR in children with SCD, even during hyper filtration, providing the constant is appropriately adjusted for the analytical method of creatinine.

Disclosures:

Inusa: Novartis: Research Funding.
ROR1 Is Expressed on the Surface of Non-Neoplastic Human B-Lymphocyte Precursors (Hematogones) and on a Subset of B Acute Lymphoblastic Leukemia
Authors: Broome H. Elizabeth , Rassenti Laura Z , Wang Huan-You , Go William Y. , Meyer Lilly , Kipps Thomas J.
Published: October 2019

Abstract 2735

ROR1 is a putative receptor tyrosine kinase that is expressed during embryogenesis, on chronic lymphocytic leukemia (CLL) cells, but not on normal mature lymphocytes, including normal CD5+ B cells (Fukuda et al PNAS 2008; 105:3047). ROR1 binds Wnt5a, and this ligand enhances the survival of CLL cells in vitro, suggesting that ROR1 functionally contributes to leukemogenesis. Additional evidence for the importance of ROR1 function in leukemia is the noted decrease in survival of B acute lymphoblastic leukemia (B-ALL) cells in vitro after RNAi targeting of ROR1 (Tyner et al. PNAS 2009; 106:8695). To further explore the significance of ROR1 in B-ALL, we analyzed 30 cases of B-ALL for surface expression of ROR1 via multiparameter flow cytometry using a fluorochrome-conjugated mAb specific for ROR1. Only two of 30 cases of B-ALL showed uniform surface ROR1 staining with a mean fluorescence intensity (MFI) that was 6.2 or 3.6 times greater than that of B-ALL cells stained with a control mAb. The other 28 B-ALL cases showed a mean MFI ratio of 1.4 (S.D. = 0.4). The two ROR1+ cases shared distinctive immunophenotypic and cytogenetic changes consistent with a more mature B-ALL subtype. We also evaluated marrow from patients recovering from chemotherapy or hematopoietic stem-cell transplantation and found that non-neoplastic B-lymphocyte precursors (BLP, hematogones) also expressed surface ROR1. These BLP were positive for CD10, CD19, CD38, and dim CD45 with variable expression of CD20, CD34, TdT, and cytoplasmic immunoglobulin. While the surface antigen phenotype of B-ALL is similar to BLP, flow cytometry can distinguish B-ALL from BLP because B-ALL shows more uniform antigen expression, lacking the continuum of maturation stages present in BLP. Also, B-ALL usually has aberrant antigen-expression intensity or aberrant antigen co-expression such as terminal deoxynucleotidyl transferase (TdT) plus CD20 on the same cell. Of 22 marrow samples tested with 1–20% hematogones, all had CD10+, CD19+, dim CD45+ cells that expressed ROR1 with a mean MFI ratio of 4.9 (S.D. 2.0). Further study found ROR1 was restricted to a subset of CD10, CD19, and low-level CD45 cells that lack expression of CD34 or TdT. This subset is consistent with an intermediate stage of B-cell maturation, possibly associated with B-lymphoid positive and negative selection. In contrast, CD10+, CD19+ germinal center B cells from reactive lymph nodes were negative for surface ROR1 (N=4). This pattern of ROR1 expression on BLP and a subset of distinctive B-ALL suggests that surface expression of ROR1 occurs during a discrete stage of B-cell maturation in the marrow that may be expanded during recovery from myeloablative therapy and/or hematopoietic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Treatment In Pediatric Patients with Immunohematologic Diseases Refractory to Convertional Therapy
Authors: Trucco Matteo , O'Riordan Mariann , Berman Brian W.
Published: October 2019

Abstract 2764

Objective:

To evaluate the effectiveness and safety of rituximab in the treatment of antibody mediated hematologic disorders refractory to conventional therapy.

Study Design:

Retrospective chart review of 16 patients treated at Rainbow Babies and Children's Hospital with rituximab for antibody mediated hematologic disorders. Treatment effects and adverse reactions potentially attributed to rituximab therapy were recorded.

Results:

Patients (age range 4 months- 17 years) with the following disorders were included: autoimmune hemolytic anemia (4); chronic immune thrombocytopenia (5); autoimmune pancytopenia (4); hemophilia with inhibitor (2); acquired Factor VIII inhibitor (1). All patients received rituximab at 375mg/m2/dose, after failure to obtain a durable response to conventional therapies. The majority of patients were treated with 4 weekly dose “cycles”. One patient only received 2 doses, and two patients were maintained on prolonged weekly therapy. Two patients showed no clinical or laboratory improvement with rituximab therapy. Two patients initially responded to rituximab therapy, but relapsed 2 months after therapy. Of the remaining patients, 7 had no recurrence of disease by the end of the study period, median follow-up 14months (4mo-72mo). The remaining patients relapsed requiring repeat cycles of rituximab. Median time to relapse was 15 months (13-18mo). The most common adverse reactions were serum sickness (n=3) and hypogammaglobulinemia (n=2). Of the patients who developed hypogammaglobulinemia, one improved after a single infusion of IVIG, the other patient required chronic IVIG infusions. One patient developed chronic sinusitis and one developed acute demylinating encephalomyelitis after receiving rituximab. It is unclear if these illnesses were due to the rituximab or secondary to underlying immune dysfunction.

Conclusions:

Rituximab appears to be effective in a variety of antibody mediated hematologic disorders refractory to more conventional therapies in children and adolescents. The observed adverse reactions were generally mild and self-limited.

Disclosures:

Off Label Use: Rituximab. B cell immununosuppresion.
Isopentenyl Pyrophosphate (IPP), a Metabolite produced in Myeloma Cells Induces the Chemotaxis of γδT Cells
Authors: Ashihara Eishi , Munaka Tatsuya , Kimura Shinya , Kanai Masaki , Abe Hirohisa , Hirai Hideyo , Shoji Shuichi , Maekawa Taira
Published: October 2019

Abstract 2766

γδT cells, which control the innate immune system, are classified into three subtypes on the basis of Vγ chain. Of these subtypes, Vγ2Vδ9 T cells display anti-tumor immunity. We have demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands Vγ2Vδ9 T cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex-unrestricted manner (Sato, Int J Cancer, 2005; Uchida, Biochem Biophys Res Commun, 2007; Sato, Cancer Immunol Immunother, 2008.). N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for Vγ2Vδ9T cells. In the present study, we investigated the chemotactic factors for Vγ2Vδ9T cells by using a micro total analysis system-based microfluidic cellular analysis device (Kanai, Sens Actuators A, 2004; Munaka, Analyst, 2007.). This microchip possesses a minute-volume (240 nL) chamber integrated with a micro-sample injector that permits the injection of a small amount (several nL) of a solute (Figure 1). Because of the minute size of this chamber, a concentration gradient can be maintained free from the influence of fluid convection and stirring, and the solute can consequently spread in a diffusion-dependent manner. Therefore, administration of a humoral factor via the sample injector mimics its release from the cell surface. We first investigated whether the supernatant of RPMI8226 multiple myeloma (MM) cells treated with zoledronic acid (ZOL) induced chemotaxis of γδT cells. We treated RPMI8226 MM cells with ZOL (1 mM) overnight and collected the supernatant. Human γδT cells were obtained by the culture of peripheral blood mononuclear cells as previously reported (Uchida, Biochem Biophys Res Commun, 2007.), and these cells were cultured in the microchip. After the injection of supernatant, γδT cells migration was observed under a microscope and continuous time-lapse recording was performed for 30 min. γδT cells migrated toward the injector, indicating that the supernatant of ZOL-treated RPMI8226 cells includes a chemoattractant factor for γδT cells. We next applied soluble MICA (sMICA), sICAM-1, sVCAM-1, and IPP and examined the migration of γδT cells. Among them, sMICA and IPP were chemoattractive for γδT cells, and the velocity of γδT cell migration was increased by the injection of IPP compared to the solvent control (Figure 2). These observations indicate that IPP, a metabolite of the mevalonate pathway in MM cells, or sMICA is a chemotactic factor for γδT cells when the target MM ells are treated with ZOL.

Disclosures:

Munaka: Shimadzu Corporation: Employment. Kanai:Shimadzu Corporation: Employment. Abe:Shimadzu Corporation: Employment.
Resminostat In Relapsed or Refractory Hodgkin Lymphoma: Initial Results of the SAPHIRE Phase II Trial with a Novel Oral Histone Deacetylase (HDAC) Inhibitor.
Authors: Walewski Jan , Paszkiewicz-Kozik Ewa , Borsaru Gabriela , Moicean Andreea , Warszewska Agnieszka , Strobel Klaus , Biggi Alberto , Hauns Bernhard , Mais Anna , Henning Stefan W. , Hentsch Bernd
Published: October 2019

Abstract 2811

Resminostat (4SC-201) is a novel pan-HDAC inhibitor that has shown anti-tumor activity in a broad panel of preclinical models and revealed a very favorable safety and promising efficacy profile in patients with various solid tumor types in an initial Phase I trial. Resminostat is currently under investigation in a number of Phase II clinical trials. The SAPHIRE study evaluates the therapeutic activity of resminostat in relapsed/refractory Hodgkin Lymphoma (HL) patients after high dose chemotherapy and autologous hematopoietic stem cell transplantation. The trial is designed as an open-label, single-arm international trial with a Simon Minimax design. Resminostat is administered orally at a once daily dose of 600 mg in 2-week cycles consisting of 5 consecutive days treatment followed by a 9 day treatment free period. Dose delay and reduction is allowed for management of adverse events. Patients undergo assessment of disease status by computed tomography in combination with positron emission tomography (PET/CT), as recommended by the International Working Group (IWG) criteria for the evaluation of HL. PET/CT scans are conducted after cycle 3 and cycle 6 and thereafter every 4th cycle during an optional follow-up treatment period in which patients may remain on treatment until disease progression or occurrence of intolerance to protocol therapy. The primary endpoint of the study is the estimation of overall objective response rate (OOR), secondary endpoints include time to response (TTR), duration of response (DOR), safety and tolerability and the study of drug regulated biomarkers in this patient cohort. As of July 2010, 19 patients with advanced HL have been enrolled in the 1st Simon stage of the study. Treatment was well tolerated with common related grade 2 –3 AEs being anemia and thrombocytopenia that were well manageable by dose modification or symptomatic treatment. Analysis of PET/CT data through an independent central assessment committee confirmed the achievement of the clinical activity requirements for the advancement of the trial into the 2nd Simon recruitment phase. Updated clinical data of this ongoing study on the efficacy and safety of the HDAC inhibitor resminostat in relapsed/refractory HL patients will be presented at the meeting.

Disclosures:

Walewski: 4SC AG: Consultancy. Warszewska:4SC AG: Consultancy. Strobel:4SC AG: Consultancy. Biggi:4SC AG: Consultancy. Hauns:4SC AG: Employment. Mais:4SC AG: Employment. Henning: 4SC AG: Employment. Hentsch:4SC AG: Employment.