bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Granulocyte Colony Stimulating Factor (G-CSF) On Monosomy 7 Aneuploidy and T Cell Subsets in Healthy Donors.
Authors: Olnes Matthew J. , Leitman Susan , Biancotto Angelique , McCoy J. Philip , Miranda Susan , Tucker Zachary , Pfannes Loretta , Furnari Megan , Young Neal S , Sloand Elaine M
Published: October 2019

Abstract 3147

Poster Board III-84

Recent reports of chromosomal and immunological abnormalities in healthy donors receiving granulocyte colony stimulating factor (G-CSF) have raised concerns among hematologists that this popular cytokine may promote genomic instability or alter immune surveillance (Pampilon D et al Transfusion 2008; 48(7):1495-501). We previously reported that G-CSF altered the Th1:Th2 ratio in healthy individuals following short-term administration (Blood 2000 Apr 1;95(7):2269-74), but reported no new karyotypic abnormities after in vitro culture of bone marrow mononuclear cells with pharmacological doses of G-CSF (Proc Natl Acad Sci 2006 Sep 26;103(39):14483-8). There is no systematic study of the long-term effects of administering granulocyte colony stimulating factor (G-CSF) to healthy individuals. We examined CD34 cells of 10 healthy stem cell donors after they had received 10 mcg/kg G-CSF for 4 days; fluorescent in situ hybridization (FISH) was the method employed to monitor chromosomal changes. We also studied 37 healthy granulocyte donors who received G-CSF (5ug/Kg x 1 day) and dexamethosone for up to 42 times (median= 15; range 6-42) using FISH to examine chromosomes 7 and 8 and flow cytometry to define their T cell subsets. FISH did not detect chromosomal abnormalities in the CD34 cells of 10 donors mobilized with G-CSF; neither could monosomy 7 cells be isolated after culturing cells in media with 400 ng/mL G-CSF (previously shown to support outgrowth of monosomy 7 cells) for two weeks. Furthermore, FISH did not detect aneuploidy in the 37 regular granulocyte donors. Evaluation of T cell subsets by flow cytometry demonstrated similar percentages of CD4+ T cells in 18 granulocyte donors as compared to 23 untreated controls (57.5% vs 56.5%). However, donors had increased numbers of CD4+TNFαa+ Th1 T cells and decreased CD4+IL-6+ Th2 T cells (4.2% vs 1.6%, P= 0.0003 and 11% vs. 35%, P=0.04 respectively), while the donor Th2 subset expressed significantly more IL-6 per cell (P<0.01). CD4+CD25+FoxP3+ regulatory T cells (Tregs) were significantly increased in G-CSF-treated donors (10.1% vs 6.0%, P<0.0001), while Th17 T cells were not significantly different (2.4% vs 0.7%, P=0.423). G-CSF does not produce chromosomal abnormalities of monosomy 7 or trisomy 8 in healthy SCT donors or in serially treated granulocyte donors. However, there are significant changes in T cell subsets that modulate the immune response.

Disclosures

No relevant conflicts of interest to declare.
Two Alleles with RHCE*nt818C>T Change Encode the Low Prevalence Rh Antigen STEM.
Authors: Halter-Hipsky Christine , Hue-Roye Kim , Coghlan Gail , Lomas-Francis Christine , Reid Marion E.
Published: October 2019

Abstract 3159

Poster Board III-96

Background

According to the original (and only) report, the low prevalence Rh antigen, STEM, is associated with an altered e phenotype. Approximately 65% of hrS– and 30% of hrB– RBCs from South African donors are STEM+. STEM has a variable expression, which is an inherited characteristic. Anti-STEM has induced mild HDFN (Marais, et al., Transf Med 1993;3:35-41). The purpose of this study was to determine the molecular basis associated with STEM expression.

Material and Methods

Blood samples and reagents were from our collections. Hemagglutination and DNA extraction were performed by standard methods. Molecular testing included direct sequencing and cloning of cDNA, AS-PCR, PCR-FRLP, and sequencing specific exons of gDNA.

Results

Three STEM+ samples (including the original index case) had RHCE*ceBI [ce 48C (16C), 712G (238V), 818T (273V), 1132G (378V)] (Noizat-Pirenne, et al., Blood 2002;100:4223-31) and 6 had a new allele, which we name RHCE*ceSM (ce 48C, 712G, 818T). In contrast, 8 STEM– samples (which included hrS– and hrB– samples) did not have the RHCE*818C>T change. RBCs with the ceBI phenotype expressed STEM more strongly than those with the ceSM phenotype.

Conclusions

The previously reported allele RHCE*ceBI and a new allele, RHCE*ceSM, encode the STEM antigen. This study also revealed other new findings: (i) ceSM encodes a weaker expression of STEM than does ceBI, which explains the previously reported variable expression, (ii) provides an explanation for why not all hrS– and hrB– RBCs express STEM. RBCs with ceAR, ceMO, and ceEK, phenotypes are hrS– STEM–, and RBCs with ceS phenotypes type hrB–, STEM–, (iii) ceBI and ceSM do not express hrS but do express hrB. It is likely that anti-hrS made by hrS– STEM– people (ceAR, ceMO, ceEK) will be incompatible with hrS– STEM+ RBCs, and vice versa. Our findings provide a means to positively identify the STEM+ phenotypes, which, by hemagglutination, is virtually impossible due to the dearth of anti-STEM. Further, it provides a tool to provide suitable antigen-negative RBC products to a patient who has made an ‘e-like’ antibody.

Disclosures

No relevant conflicts of interest to declare.
Comparative Decay Rates of Human, Rhesus Macaque, Cynomolgus, and Porcine Activated Factor VIII.
Authors: Healey John F , Parker Ernest , Lollar John (Pete) S.
Published: October 2019

Abstract 3164

Poster Board III-104

The proteolytic conversation by thrombin of factor VIII (fVIII) to fVIIIa produces a A1/A2/A3-C1-C2 heterotrimer that spontaneously dissociates into inactive A1/A3-C1-C2 and A2 species. Human mutations that increase the rate of A2 subunit dissociation produce hemophilia A, indicating that A2 subunit dissociation is physiologically relevant and is an important regulatory feature of the blood coagulation mechanism. The A2 subunit dissociation rate from human fVIIIa is significantly faster than the corresponding dissociation rates from porcine or murine fVIIIa. The fast decay rate of human fVIIIa raises the question whether the f8 gene is under positive selection for this trait. To determine whether fast A2 dissociation occurs elsewhere in the primate lineage, we cloned cDNAs encoding B-domain deleted (BDD) fVIII from rhesus macaque and cynomolgus monkey liver. The deduced BDD amino acid sequences of rhesus and cynomolgus fVIII were 97.9 % and 98% identical to human fVIII, respectively, and were 99.9% identical to each other. The expression of rhesus and cynomolgus fVIII from baby hamster kidney-derived cells was similar to human fVIII and ten-fold lower than porcine fVIII. BDD human, rhesus, cynomolgus, and porcine fVIII molecules were purified to homogeneity by tandem ion-exchange chromatography. Concentrations of the purified constructs were calculated using a molar extinction coefficient at 280 nm based on their predicted tyrosine, tryptophan and cysteine compositions. Human, rhesus, and cynomolgus fVIII displayed similar specific coagulant activities by one-stage coagulation assay (6800, 4500, and 5200 units per mg, respectively). The kinetics of decay of human, rhesus, cynomolgus and porcine fVIIIa were measured following rapid activation of 1 nM fVIII by thrombin using a chromogenic substrate assay of purified intrinsic fXase complex under conditions in which fVIIIa was limiting. Decay curves were fit using nonlinear least-squares regression to a first-order model (Fig. 1). Decay rate constants for rhesus and cynomolgus fVIIIa were similar (0.31 and 0.27 min-1, respectively) and were slightly, but significantly lower than human fVIIIa (0.40 min-1). In contrast, the decay rate constant for porcine fVIIIa, 0.17 min-1, was 2.3-fold lower than human fVIIIa, consistent with previous observations. These results suggest that fast A2 subunit dissociation rates evolved before evolution of the primate lineage.

Disclosures

No relevant conflicts of interest to declare.
Kallikrein Inhibitors as Antiinflammatory Therapeutics.
Authors: Bryant John , Kolti Dhaval , Holsworth Daniel , Akbari Parmiss , Shariat-Madar Zia
Published: October 2019

Abstract 3179

Poster Board III-118

Plasma kallikrein is a novel target for antiinflammatory inhibitor design since it plays a role in the initiation of the intrinsic clotting, fibrinolysis, bradykinin-forming and neutrophil activation. The goal of this study was to synthesize a potent and selective inhibitor of kallikrein for the prevention and/or treatment of hyperfibrinolytic states, systemic inflammation, sepsis as well as cardiopulmonary bypass. Using a focus library of compounds, UK-097820, CP-013118 and PF-04886847 were found not to be cytotoxic and had the ability to abolish the hydrolysis of S2302 by kallikrein both in fluid phase and on the surface-mediated activation. In a competition study, PF-04886847 (3 mM) reduced the formation of a complex between C1-inhibitor and kallikrein by 40% following the activation of prekallikrein (kallikrein zymogen) on bound high molecular weight kininogen (HK) to human pulmonary artery endothelial cell (HPAEC). It was also mechanistically important to distinguish between direct kallikrein inhibition caused by the binding of kallikrein inhibitor to kallikrein versus the inhibitor's indirect effect on HK, prekallikrein or perturbation of the membrane function. The binding of PK to HK or HK to HPAEC surface was unaffected by the kallikrein inhibitors. To determine the selectivities of the kallikrein inhibitors, the effects of UK-097820, CP-013118 and PF-04886847-00 on factor XI and factor XII were examined. PF-04886847 was found to be a potent and selective inhibitor of kallikrein at a concentration ranging from 1 to 3 mM. BK stimulates cultured endothelial cells to release various mediators. The effects of the kallikrein inhibitors on the generation of bradykinin (BK), nitric oxide (NO) and prostacyclin and intracellular calcium signaling were determined. The generation of BK, NO formation and 6-keto-PGF1a release were blocked by PF-04886847, indicating that PF-04886847 might have potential applications for controlling unwanted inflammation provoked by BK-induced endothelium activation. In situ study, PF-04886847 blocked PK activation on intact aorta precontracted by phenylephrine. Taken together, we propose that PF-04886847 treatment might be beneficial in tissue injury and have potential clinical application.

Disclosures

No relevant conflicts of interest to declare.
Multiple Mapping of Peptide Sequences Recognized by Various Monoclonal Anti-ADAMTS13 Antibodies with Functional or Nonfunctional Effects On the Catalytic Activity.
Authors: Nakagawa Terumichi , Moriki Takanori , Yamaguchi Yusuke , Igari Atsuko , Soejima Kenji , Matsumoto Masanori , Fujimura Yoshihiro , Murata Mitsuru
Published: October 2019

Abstract 3182

Poster Board III-119

Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity.

Disclosures

No relevant conflicts of interest to declare.
HYC750: A Novel Stem Cell Mobilizer.
Authors: Schönekess Brett , Campion Rosalia De Necochea , Dardov Victoria , Sonjara Srdjan , Josephs Steven F. , Ramos Famela , Ichim Thomas , Minev Boris , Carrier Ewa
Published: October 2019

Abstract 3220

Poster Board III-157

Hyaluronic acid (HA) is a repeating disaccharide polymer having various biological activities depending on size: high molecular weight HA performs structural functions, whereas low molecular weight HA induces systemic cytokine-like effects. HYC750 is a Good Manufacturing Practices (GMP)-generated HA with a maximum molecular weight of 750 kDA that is currently in development as a hematopoietic stem cell mobilizer. Given the potential for proinflammatory activities of various very low MW HA fragments we sought to determine whether HYC750 may possess properties detrimental to clinical use. Dose-dependent administration of HYC750 to bone marrow stromal cells, the monocytic cell line RAW, and differentiated THP-1 cells did not result in alteration of proliferation or viability. Treatment of said cells with HYC750 did not elicit production of TNF-alpha as assessed by the L929 assay. In contrast, LPS treatment induced a dose dependent cytokine response. These data, combined with dose escalation studies in BALB/c mice without induction of inflammatory reactions or adverse effects suggested HYC-750 does not cause biological changes that are conventionally seen with low molecular weight HA administration. In order to quantify stem cell mobilization activity, 240 ug/mouse of HYC750 was administered for 5 consecutive days intraperitoneally and subsequently marrow, spleen and blood were sacrificed on day 6. A profound increase in cellularity of circulating cells was observed with the HYC750 treatment by forward and side scatter FACS profiles. A >2 fold increase in lineage negative, stem cell antigen (SCA-1) positive cells, c-kit positive cells was observed in the periphery in the treated animals. In contrast to G-CSF mobilization, HYC750 treatment was not associated with decreased marrow cellularity or local macrophage activation. These data suggest HYC750 is a novel mobilizing agent that does not elicit inflammatory responses. Recently, HYC750 received approval to be used in a Phase I, open-label, single-ascending-dose, safety study in healthy male volunteers, by the European Competent Authority in Seville, Spain.

Disclosures

Schönekess: Orcrist Bio: Employment, Equity Ownership.
Age Related Expression of hMLH1 but Not hMSH2 in Human Hematopoietic Stem Cells Predicts Genomic Instability.
Authors: Kenyon Jonathan , Saurastri Anshul , Gerson Stanton L.
Published: October 2019

Abstract 3235

Poster Board III-172

DNA damage repair pathways have been shown to be important for hematopoietic function and longevity. A number of studies focusing on the loss of DNA damage repair genes have reported age related loss of hematopoietic stem cells, reduced hematopoietic function, and increased rates of hematologic cancer. A poorly studied pathway with respect to its effect on hematopoietic function is the mismatch repair (MMR) pathway. Microsatellite instability (MSI), one marker of MMR failure, has been observed in acute lymphoblastic leukemia, myelodysplastic syndrome, and lymphocytes obtained from the peripheral blood of the elderly. Further, studies investigating MMR pathway disruption in mice show an increased incidence of age related hematologic disorders and failure to repopulate in competitive transplant experiments. These reports led to the formation of the hypothesis that age related loss of MMR in a subset hematopoietic stem/progenitor cells (HSC) leads to a progenitor population with a mutator phenotype potentially leading to cell death or leukemogenesis.

To test this hypothesis we clonally expanded HSC from cord blood, bone marrow, and bone core samples from various aged individuals most of whom had normal CBCs. These colony forming units (CFU) were then tested for MSI at five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250). These results showed a statistically significant increase in high grade instability (measured as MSI observed at >20% of loci tested) in CFU obtained from individuals over the age of 50 compared to those in the age range between 0 and 50 years (p=0.0034). Next we sought to determine if loss of gene expression in MMR genes could account for the observed MSI. Gene expression of two genes known to be transcriptionally downregulated in MMR failure, hMLH1 and hMSH2, were assessed by qRT-PCR in CFU obtained from pure CD34+ immunomagnetic separations of HSC from various aged donors. Further, individual CD34+ cells from these same donors were fixed to slides and immunostained for hMLH1 and hMSH2. We observed a significant loss of hMLH1 gene expression in a subset of HSC obtained from older donors above age 42, but no pattern of loss of hMSH2 gene expression. The age related loss of MMR in HSC suggests that MMR pathway proficiency is important for hematopoiesis. These results suggest that loss of MMR might precede myelodysplastic syndromes and leukemia in older individuals.

Disclosures

No relevant conflicts of interest to declare.
Association of Increased Thymidine Uptake Relative to Tumor Cell Proliferation in Low-Grade Follicular Lymphoma and DNA Repair.
Authors: Juweid Malik E. , Syrbu Sergei I. , Bleile Michelle , Buck Andreas , Wondergem Marielle , Vose Julie M.
Published: October 2019

Abstract 3236

Poster Board III-173

We have observed that the uptake of the thymidine analog 18F-fluorothymidine (FLT) is highly disproportional to cellular proliferation in untreated low-grade follicular lymphoma (FL). Since the uptake of thymidine/thymidine analogs is also increased with enhanced DNA repair synthesis, we investigated whether the “excess” uptake of FLT in FL may be related to DNA repair. We stained tumor samples from 20 patients each with grade 1 FL and DLBCL for Ki-67, a marker of cell proliferation and DNA replication but not DNA repair and 2 DNA replication and repair biomarkers: proliferating cell nuclear antigen (PCNA) and replication protein A (RPA). Median %Ki-67-positive cells (Ki-67 index) was 10% (range; 5-20%) in FL compared to 80% (range 60-90%) in DLBCL (P<4×10-20). In contrast, median %positive cells for PCNA and RPA were 90% and 100% in FL and 100% and 100% in DLBCL. In both FL and DLBCL, PCNA showed a characteristic staining pattern with 3+ or 4+ staining of the proliferating Ki-67-positive cells vs. 1+ to 2+ staining of quiescent cells. Similar results were obtained when staining for thymidine kinase I (TK1). Interestingly, similar staining pattern to that seen in lymphoma samples was seen in germinal center but not mantle zone cells of normal tonsils and reactive lymph nodes indicating that the DNA repair seen in both FL and DLBCL is likely related to somatic hypermutations (SHM) generated by error-prone DNA repair known to occur in FL, most DLBCLs and normal germinal center cells. Comparison of FLT uptake in FL and DLBCL indicated that > 70% of FLT uptake in FLs with a Ki-67 index of ≤10% was due to DNA repair. In contrast, contribution of DNA repair to overall FLT uptake in DLBCL with a Ki-67 index of ≥ 80% was <10% presumably due to the 4-fold lower fraction of quiescent compared with proliferating cells with high replicative DNA synthesis. This is the first demonstration of a high contribution of SHM/DNA repair to overall uptake of thymidine/thymidine analogs in low-grade FL. Our data further suggest that FLT use for assessing response to cytostatic therapy in low-grade FL will be confounded by this high contribution and highlights the pitfalls associated with the use of PCNA or RPA to assess proliferative activity in FL and DLBCL.

Disclosures

No relevant conflicts of interest to declare.
Cell Lines and “Knock-in” Mice Bearing FLT3/ITD Mutations Exhibit Elevated Repair Errors Generated through Alternative DNA Double Strand Break Repair Pathways: Implications for Genomic Instability.
Authors: Fan Jinshui , Li Li , Small Donald , Rassool Feyruz V.
Published: October 2019

Abstract 3237

Poster Board III-174

Activating mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor occur in approximately 30% of acute myeloid leukemia (AML) patients and, at least for internal tandem duplication (ITD) mutations, are associated with poor prognosis. We previously reported that FLT3/ITD mutations are associated with increased reactive oxygen species (ROS) production which leads to increased DNA double strand breaks (DSBs) and decreased repair fidelity, that may in part explain aggressive AML in FLT3/ITD patients. While the DNA-PK and Ku-dependent non homologous end-joining (NHEJ) pathway is one of the main routes for repairing DSBs in normal mammalian cells, recent reports have suggested that an alternative and less well defined NHEJ pathway (Alt NHEJ) involving DNA ligase IIIαa and PARP1, characterized by DNA sequence microhomology at the repair junctions, may play a role in the generation of deletions and translocations that can lead to cancer progression. Here, we report that in hematopoetic progenitor cells expressing FLT3/ITD (Ba/F3/ITD), the frequency of errors and the size of deletions during DSB repair is increased compared with the Ba/F3 control cells. Moreover, these data are confirmed in bone marrow mononuclear cells (BM MNCs) from FLT3/ITD “knock-in” mice, compared with WT control cells. Strikingly, we show that Ku proteins (Ku70 and Ku86), key components of the main NHEJ pathway, are down-regulated in FLT3/ITD-positive cell line and primary mouse BM MNCs. Concomitantly, DNA ligase IIIαa, a component of Alt NHEJ pathway is up-regulated in FLT3/ITD-expressing cells and plays a role in abnormal DSB repair. Importantly, after treatment with a FLT3 inhibitor, AML (MOLM-14, MV4-11) cells containing FLT3/ITD mutations demonstrate a reduction of misrepair and deletion size, and down-regulation of DNA ligase IIIαa, suggesting that FLT3 signaling regulates the pathways by which DSBs are repaired. Thus, therapy to inhibit FLT3/ITD may lead to repair that reduces repair errors and genomic instability.

Disclosures

No relevant conflicts of interest to declare.
A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.
Authors: Janssen Jeroen J.W.M. , Deenik Wendy , Smolders Karlijn G.M. , Terwijn Monique , Kelder Angele , Cornelissen J. J. , Schuurhuis Gerrit-Jan , Ossenkoppele Gert J.
Published: October 2019

Abstract 3245

Poster Board III-182

Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well.

Disclosures

No relevant conflicts of interest to declare.
High Prevalence of Dapsone-Induced Oxidant Hemolysis in North American Stem Cell Transplant Recipients without Glucose-6-Phosphate-Dehydrogenase Deficiency.
Authors: Olteanu Horatiu , Harrington Alexandra , George Ben , Bredeson Christopher , Hari Parameswaran N. , Kroft Steven
Published: October 2019

Abstract 3314

Poster Board III-202

Introduction

Dapsone (4-4'-diaminodiphenylsulfone) is commonly used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis in immunocompromised patients. Oxidant hemolysis is a known complication of dapsone, but its frequency in adult patients who have undergone stem cell transplantation (SCT) for hematologic malignancies is not well established. We report a high prevalence of hemolytic anemia associated with dapsone use in SCT patients in the absence of glucose-6-phosphate dehydrogenase (G6PDH) deficiency.

Patients and Methods

We retrospectively analyzed laboratory data from 30 SCT (29 Caucasian and 1 African-American; 15 male and 15 female) patients receiving dapsone (100 mg orally daily) for PCP prophylaxis to determine the frequency of oxidant hemolysis. All patients had undergone allogeneic (n = 27) or autologous SCT (n = 3) for hematological malignancy or aplastic anemia. Peripheral blood (PB) smears were reviewed in a blinded fashion for the presence of morphologic features of oxidant hemolysis (multiple bite cells in each high power field), with PB smears from the same patient group while not on dapsone serving as controls. Additional demographic and laboratory data, including G6PDH enzyme activity screening test, hemoglobin, reticulocyte count, lactate dehydrogenase and bilirubin levels, were collected from chart review.

Results

29 patients had normal G6PDH activity (results were not available for 1 patient). 38 PB smears were obtained while on dapsone (some patients had more than one bone marrow evaluation). 30 (79%) PB smears from 26 (87%) patients had morphologic evidence of overt oxidant hemolysis. This was significantly higher than in the controls, in which 5/22 (23%) PB smears had morphologic features of oxidant hemolysis (p<0.001, Fisher's exact test). Hemoglobin levels were lower in the dapsone group, compared to the controls (10.0 vs. 12.5 g/dL, p=0.012). Mean LDH (507 IU) and direct bilirubin (1.6 g/dL) were also increased in the patients on dapsone. None of the patients had laboratory signs of hemolysis while not on dapsone.

Conclusions

Dapsone-induced oxidant hemolysis in SCT patients is 20-fold higher than the reported rate in HIV-infected patients receiving this agent. This high prevalence in patients without G6PDH deficiency suggests the coexistence of other sources of oxidant stress specific to this patient cohort.

Disclosures

Bredeson: Esai: Honoraria.
Incidence and Risk Factors for Lower Alimentary Tract Mucositis (neutropenic enterocolitis) Following 1528 Courses of Chemotherapy in a Homogenous Population of Myeloma Patients: Clinical and Research Implications.
Authors: Krishna Somashekar G , Weizhi Zhao , Krishna Kavya , Grazziutti Monica , Barlogie Bart , Anaissie Elias
Published: October 2019

Abstract 3343

Poster Board III-231

Background:

lower alimentary tract mucositis (L-ATM), also known as neutropenic enterocolitis, is a complication of chemotherapy with high mortality rates. The incidence, risk factors and mortality of L-ATM remain unknown.

Purpose:

To determine the incidence, risk factors and mortality of L-ATM in a homogeneous population of patients with multiple myeloma.

Patients and Methods:

303 newly diagnosed myeloma patients (2004 - 2007) were enrolled in a protocol consisting of induction chemotherapy, tandem melphalan-based autologous stem-cell transplantation (ASCT) and consolidation. Lower -ATM was defined as grade II-IV enteritis/colitis occurring during neutropenia. All data were collected prospectively. The analysis of risk factors included body surface area (BSA), creatinine clearance (CrCl), serum albumin and other pretreatment variables. Multiple logistic regression model was used to compute adjusted odds ratio (OR) and 95% confidence intervals (CI).

Results:

47 patients (15.5%) developed L-ATM during 1528 chemotherapy cycles (including 536 ASCT). BSA < 2 m2 (OR 2.853; 95% CI 1.232, 6.607; p = 0.0144) and CrCl <60ml/min (OR 3.089; 95% CI 1.176, 8.114; p = 0.0221) increased the risk for L-ATM while higher serum albumin was protective (OR 0.695; 95% CI 0.517, 0.936; p = 0.0167). No L-ATM-related death was observed.

Conclusions:

Lower-ATM is not uncommon among myeloma patients undergoing multiple cycles of chemotherapy but does not contribute to mortality. The risk for L-ATM is determined by lower values of BSA, renal function and serum albumin. These findings have significant implications for the study and management of L-ATM in patients undergoing chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Follow-up of Recipients of Combined HLA-Matched Nonmyeloablative Bone Marrow and Kidney Transplantation for Multiple Myeloma with End-Stage Renal Disease.
Authors: Spitzer Thomas R , Sykes Megan , Tolkoff Rubin Nina , Kawai Tatsuo , McAfee Steven L , Dey Bimalangshu , Ballen Karen , Delmonico Francis , Saidman Susan , Sachs David H , Cosimi Benedict
Published: October 2019

Abstract 3368

Poster Board III-256

Specific tolerance following combined kidney and bone marrow transplantation (Kd/BMT)for patients with end stage renal disease (ESRD) with or without an underlying malignancy has been accomplished, as evidenced by prolonged normal renal function without ongoing immunosuppression (IS)and the demonstration of in vitro donor specific hyporesponsiveness (N Engl J Med 2008; 358:353-361; Am J Transplant 2006;6:2121-2133). In order to achieve potent anti-myeloma responses and induce tolerance through the induction of mixed chimerism (MC) for the renal allograft, 7 patients (median age 48 (34-55) yrs with multiple myeloma (MM) and ESRD received a combined HLA-matched Kd/BMT, with longest follow-up time of almost 11 years. An eighth pt developed cyclophosphamide (CY) cardiotoxicity on day -5 and did not receive a transplant. Preparative therapy for the transplants consisted of CY 60 mg/kg (days -5, -4) with hemodialysis 14 hrs after each CY dose, equine anti-thymocyte globulin, 15-20 mg/kg on days -1, +1, +3, and +5 and thymic irradiation (700 cGy) on day -1. Cyclosporine (CSP) was begun on day -1, with combined Kd/BMT on day 0. Nine additional donor lymphocyte infusions were given to 5 pts (5 for chimerism conversion, 4 for persistent/progressive disease (PD)). Acute (A) and chronic (C) GVHD developed in one patient following DLI for early PD, while chronic GVHD developed in two pts (one after a second stem cell transplant). Characteristics and outcomes of the 7 combined Kd/BMT recipients are as follows:

# after 2nd transplant (myeloablative) from the same donor ; FDC: full donor chimerism In summary, 5 of 7 pts are alive, 4 without evidence of MM from 2.7 to 10.9 yrs post-Kd /BMT. Three pts have normal renal function without IS, while two pts have normal renal function on IS for chronic GVHD. Sustained renal allograft tolerance and prolonged anti-myeloma responses are achievable following nonmyeloablative HLA-matched kidney and BMT and the induction of mixed chimerism. This study was supported by the Immune Tolerance Network, National Institute of Allergy and Infectious Diseases.

Disclosures:

Off Label Use: Equine anti-thymocyte globulin: in vivo T cell depletion Cyclophosphamide:conditioning therapy for transplantation.
Long-Term Complete Responses to Combination Therapies in Sézary Patients.
Authors: Daulat Seema , Talpur Rakhshandra , Hosing Chitra , Duvic Madeleine
Published: October 2019

Abstract 3369

Poster Board III-257

Introduction:

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma (CTCL), is defined as erythroderma and >1000 abnormal T-cells per mm3 in the peripheral blood, and is currently incurable. A recent study of erythrodermic CTCL conducted at our center reported a median overall survival of 5.1 years which is twice that in the literature [Vidulich K, et al. Int. J of Dermatol. 2009; 48: 243-252]. Although complete remissions (CR) may be achieved in earlier stages of CTCL, they are rarely achieved in SS patients.

Patients and Methods:

A retrospective analysis of a prospective database of 1550 CTCL patients seen at the MD Anderson Cancer Center since 1984 identified a subset of 145 SS patients (9%) whose long-term complete responses (>2 years) to various therapies is reported. The most commonly used combination therapies were topical steroids, psoralen with ultraviolet A (PUVA), oral retinoids (isotretinoin, acetretin, or bexarotene), alpha-interferon, and extracorporeal photopheresis (ECP). A combination of total skin electron beam radiation (TSEB) followed by induction chemotherapy and allogeneic stem-cell transplant was given to 23 CTCL patients. Chronic Staphylococcus aureus carriers identified by cultures were treated with antibiotics and preventive skin care.

Results:

Of the 145 Sézary Syndrome patients, 13 (8.96%) achieved CRs, including 6 females and 7 males. Their median age was 48 (20 to 85 years) at diagnosis. Nine were Caucasian, 3 were African American, and 1 was Hispanic. At initial diagnosis their stages were IB (n=1), stage III (n=3), stage IVA (n=4), and stage IVB (n=5). Six achieved a CR on front-line combination immunomodulatory therapy consisting of ECP with alpha-interferon and/or oral retinoids, antibiotics, and topical steroids. Seven SS patients failed therapy but were able to obtain CRs after TSEB and allogeneic peripheral blood stem cell transplantation (SCT) from matched related donors (n=6) or unrelated (n=1) donors. The conditioning regimen for SCT was fludarabine and melphalan. The median time between diagnosis and CR was 4.07 years (range 1 to 20 years). The median duration of CR is 5 years (mean 5.15 years, range 2 to 13 years).

Conclusion:

Sézary Syndrome is extremely symptomatic and usually refractory to current therapies. CRs may be achieved using immunomodulatory therapy with biological response modifiers and photopheresis or with total skin electron beam followed by non-ablative allogeneic stem cell transplant in appropriate patients and may have an impact on overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Results of Allogeneic Hematopoietic Cell Transplantation in Elderly Patients.
Authors: Stadler Michael , Dammann Elke , Buchholz Stefanie , Hertenstein Bernd , Krauter Juergen , Eder Matthias , Ganser Arnold
Published: October 2019

Abstract 3388

Poster Board III-276

BACKGROUND:

Allogeneic hematopoietic cell transplantation (alloHCT) has curative potential in many hematologic malignancies; however, substantial treatment related toxicities have restricted its use to younger patients, mostly below 55 years of age. With the development of reduced intensity conditioning regimens, alloHSCT is being applied more widely in elderly patients, too.

PATIENTS:

Here we report long term results of all consecutive alloHCT recipients above the age of 55 years at our institution between October 1996 and April 2009. Among a total of 649 patients allotransplanted, 172 were more than 55 years old: 86 patients in the age group over 55 to 60 years, 61 patients in the cohort over 60 to 65 years, and 25 patients older than 65 years.

RESULTS:

73 elderly patients were female and 99 male. Diagnoses included acute lymphoblastic leukemia (14 patients), acute myelogenous leukemia (AML, 57), myelodysplastic syndromes or secondary AML (71), chronic myelogenous leukemia (7), myelofibrosis (6), lymphoma (11), and multiple myeloma (6). Advanced disease was present in 131 patients (76%) at the time of transplant. 164 patients (95%) had a WHO performance score of 0 to 1. According to the HCT comorbidity index (HCTCI), 67 patients (39%) had no comorbid conditions, whereas 58 patients (34%) had 1 to 2, and 47 (27%) had 3 or more comorbidities (not significant). Conditioning regimens were of reduced intensity in 147 patients (85%, in the majority FLAMSA-based) and myeloablative in 25 patients (15%). 56 patients (33%) received a graft from a related donor and 116 (67%) from an unrelated donor. With a median follow-up of 11 months (range: less than 1 to 127), 55 patients have relapsed (32%). Non-relapse mortality was 27% (46 of 172 patients). At last follow-up, 80 of 172 patients (47%) were alive and 77 (45%) in complete remission. Probabilities of overall (OS) and disease-free survival (DFS) at four years were 40 and 35%, respectively. Interestingly, long term OS and DFS were similar among the patients aged 55 to 65 years when compared to the 477 patients below 55 years allotransplanted during the same period at our institution. Only the age cohort over 65 years had a significantly reduced outcome with 22% DFS at four years (p = 0.006). Comorbidities were distributed comparably in all three elderly age groups, but remained without statistical effect on survival, likely due to the overriding influence of high risk status.

CONCLUSIONS:

In our experience, alloHSCT was feasible in elderly patients of sufficient performance status, with acceptable toxicity and relapse rates, and with insignificant impact from comorbid conditions. Up to the age of 65 years, outcome was not inferior to that of patients below 55 years of age.

Disclosures:

No relevant conflicts of interest to declare.
Targeting the Leukemia Initiating Cells in a Mouse T-ALL Model.
Authors: Tatarek Jessica , Cullion Kathleen , Kelliher Michelle
Published: October 2019

Abstract 3474

Poster Board III-411

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is largely caused by aberrant activation of the TAL1/SCL, LMO1/2 and NOTCH1 oncogenic pathways. Although most patients respond to cytotoxic therapy, 20-30% relapse and currently we cannot distinguish children likely to relapse from those who will respond to therapy. Evidence is emerging that some malignancies are driven by a rare initiating population that is capable of extensive self-renewal and is resistant to conventional chemotherapy. Although the leukemia-initiating cells (L-ICs) have been well documented in AML and CML, it is unclear whether T-ALL is driven by a rare L-IC and whether relapse reflects an inability to eliminate L-ICs. To determine whether T-ALL is driven by a rare L-IC, we utilized a murine model of T-ALL in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development via E47/HEB inhibition and 75% of tumors develop spontaneous gain of function mutations in Notch1. We have shown that treatment with γ-secretase inhibitors (GSIs) to inhibit Notch1 activity induces apoptosis of mouse T-ALL cells in vitro and when administered in vivo extends the survival of leukemic mice. We demonstrate that clonal mouse T-ALL tumors are phenotypically heterogeneous, containing immature CD4- and CD8-negative, double negative (DN) 3 and 4 thymic progenitors and differentiated double positive (DP) and/or single positive (SP) leukemic blasts. Importantly, the DN3 or DN4 progenitors are maintained upon serial transplantation of the tumor into syngeneic recipient mice. Injection of serial dilutions of murine T-ALL cells reveal that the tumors are also functionally heterogeneous; with 1/5000-1/50,000 tumor cells exhibiting leukemia initiating activity. We found the CD44-, CD25+ DN3 progenitors enriched in disease potential, whereas mice injected with DP leukemic blasts failed to develop leukemia. Consistently, our preleukemic studies reveal a 3.5-fold increase in the percentage of Notch1 active, DN3/4 thymic progenitors, raising the possibility that Notch1 drives L-IC growth. Collectively, these studies suggest that activation of the Notch1-c-Myc pathway may confer self-renewal capabilities on committed thymic progenitors. The effects of Notch inhibitors on mouse L-IC survival and activity will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
MYBL2 Is a Candidate Tumor Suppressor Gene In MDS
Authors: Heinrichs Stefan , Conover Lily , Bueso-Ramos Carlos E. , Kilpivaara Outi , Levine Ross , Stevenson Kristen , Neuberg Donna , Bejar Rafael , Ebert Benjamin L. , Garcia-Manero Guillermo , Kantarjian Hagop M. , Look A. Thomas
Published: October 2019

Abstract 1865

Cytogenetic changes, mainly deletions, can be found in about 30–50% of patients with Myelodysplastic Syndromes (MDS). To identify a tumor suppressor candidate within a commonly deleted region on chromosome 20q, we performed gene expression analysis on CD34+ bone marrow cells obtained from 8 patients with a 20q aberration and 18 with a normal karyotype. However, we were unable to identify genes that were significantly differentially expressed in aberrant 20q karyotype as compared to normal karyotype MDS patients. In contrast, a comparison of CD34+ cells from all MDS cases analyzed (n=26) with CD34+ cells obtained from normal bone marrow (n=4) revealed 108 genes that were differentially expressed. Interestingly, one of the top-scoring genes was MYBL2, which is located on chromosome 20q. MYBL2 levels were downregulated more than two-fold in 18 out of 26 cases. RNAi-mediated knockdown of MYBL2 in CD34+ normal bone marrow cells revealed a signature of genes functionally associated with the G2/M cell cycle phase confirming the well-documented role of MYBL2 as key transcription factor governing the onset of cell division. We hypothesize that in a subset of MDS cases the control of cell division may be impaired by low levels of MYBL2 such that altered cell fates established during cell division in early hematopoietic stem and progenitor cells will lead to clonal expansion with imbalanced or impaired differentiation. Indeed, gene set enrichment analysis revealed a strong enrichment of MYBL2 signature genes in MDS CD34+ cells. In support of a potential role as tumor suppressor, resequencing of MYBL2 (144 patients) identified 2 somatic mutations, pinpointing an additional mechanism to reduce expression of normal levels of wild-type MYBL2.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Impact of Chromosomal Abnormalities In Elderly Patients with Multiple Myeloma Treated with High-Dose Melphalan (MEL140) and Autologous Stem Cell Transplantation
Authors: Liebisch Peter , Straka Christian , Einsele Hermann , Hinke Axel , Sandermann Andreas , Dohner Hartmut , Langer Christian
Published: October 2019

Abstract 1914

Background:

Although chromosomal aberrations (CA) have emerged as important outcome predictors in multiple myeloma (MM), the prognostic significance of many recurring genomic changes is still unknown. Moreover, there is only scarce data on the implication of CA in elderly patients (pts.) receiving high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT).

Aims:

To evaluate the prognostic relevance of recurring CA as detected by cIg-FISH for elderly pts. with MM receiving HD-CTX and ASCT.

Patients and Methods:

Between 05/2001 and 08/2006, 549 pts. 60–70 yrs. of age with newly diagnosed symptomatic MM entered the DSMM II trial of the Deutsche Studiengruppe Multiples Myelom to receive two cycles of HD-CTX (melphalan 140 mg/sqm) followed by ASCT after 3–4 cycles of dexamethason-based induction chemotherapy (IC; arm A1) or no IC (arm A2). cIg-FISH and DNA probes mapping to chromosome bands 1p22, 1q21.2, 6q21, 8p11.2, 9q34, 11q25, 13q14, 14q32, 17p13, and 22q11 were applied to all pts. with sufficient bone marrow specimen at diagnosis. 289 pts. with a median age of 64 yrs. were included in the present analysis.

Results:

After a median follow-up of 82 weeks (w) the following CA were associated with a significantly shorter event-free survival (EFS): 17p13 deletion (17p–; 55 vs. 93 w, p<.0001); translocation t(4;14) (t(4;14); 65 vs. 95 w, p<.001), 1q21.2 gains (+1q21.2; 79 vs. 99 w, p=.0002); 13q14 deletion (13q–; 82 vs. 99 w, p=.03); and 8p11.2 deletion (8p–; 80 vs. 95 w, p=.04). Overall survival (OS) was significantly shorter in pts. with +1q21.2 (148 vs. 231 w, p<.0001); 17p– (118 vs. 219 w, p=.001); and t(4;14) (t(4;14); 195 vs. 223 w, p=.017). In a multivariable analysis t(4;14) (p<.001); 17p– (p=.0089); +1q21.2 (p=.031); and beta-2-microglobulin (p=.033) independently predicted outcome.

Conclusion:

In this large cohort of uniformly treated elderly pts. with newly diagnosed MM, CA – in particular 17p–, t(4;14), and +1q21.2 – remain strong predictive markers for outcome.

Supported by a grant from the Else Kröner-Fresenius-Stiftung to P.L. and H.D.

Disclosures:

No relevant conflicts of interest to declare.