bluuebunny/crossref_metadata_2025 huggingface dataset:
Development Of a Serum Biomarker Panel That Predicts Imminent Risk Of Multiple Myeloma (MM) Progression From Premalignancy
Authors: Kate Sasser A , Casneuf Tineke , Amaratunga Dhammika , Tryputsen Volha , Kumar Shaji K , Rajkumar Vincent , Hall Brett
Published: October 2019

Premalignant states are known to precede malignancy in many different cancers, including multiple myeloma (MM). Each year, 1% of Monoclonal Gammopathy of Undetermined Significance (MGUS) patients and 10% of Smoldering Multiple Myeloma (SMM) patients transition to symptomatic MM, which remains an incurable disease. By identifying patients at imminent risk (within 2 years) of progression to MM, treatment could begin early and potentially prevent full blown MM and associated morbidity and mortality. Multiple risk-factor models are currently utilized to identify patients at high-risk of developing MM. Commonly cited risk factors include percentage of bone marrow plasma cells, free light chain ratio, serum levels of M-protein, IgG subtype and levels of circulating plasma cells. These tests have individual and combined merit but there remains no consensus for measuring imminent risk of MM progression. To determine whether serum proteomics could help predict progression or provide information on relevant pathways involved in progression to MM, we evaluated 200 MGUS and SMM (187 non-IgM) patient samples on the Myriad DiscoveryMAP(r) platform (250+ serum proteins), supplemented by demographic and clinical information. Of the 187 non-IgM patients, 100 progressed to MM within 2 years (high-risk), and 87 did not progress to MM within 5 years (low-risk). We performed different subset analyses to screen for proteins that were informative and predictive of disease progression from MGUS/SMM to MM. First, a univariate Wilcoxon rank sum test was performed. The top 10 proteins that were found to be differential between high-risk/low-risk for all MGUS and SMM patients were IgM, Progesterone, MCP-2, Kallikrein-5, FGF4, EPO, MCP-4, MIP-1β, MCP-1, and TF. Interestingly, when analyzing MGUS and SMM patients separately, additional proteins that were important in distinguishing high-risk/low-risk patients included IgM, EN-RAGE, BAFF, and several others previously shown to be relevant in MM, as well as a handful of novel proteins with unidentified roles in MM. Second, to determine whether specific combinations of proteins could be utilized together to define high/low-risk, a classification tree was assembled. The combination of 8 features (IgM, Progesterone, Aldose Reductase, E-Selectin, Fas Ligand, Hemoglobin, Age and Creatine kinase-MB) appropriately classified 86% of the 187 patients into high/low-risk. Future analyses will include examining this high/low risk classifier in contrast to current risk factor models, and application to two additional sets of similar patient samples that serve as an independent validation set.

Disclosures:

No relevant conflicts of interest to declare.
C950T and C1181G Osteoprotegerin Gene Polymorphisms In Myeloma Bone Disease
Authors: Sonmez Mehmet , Kazaz Nazli , Yucel Burcu , Topbas Murat , Ucar Fahri
Published: October 2019

Bone disease is one of the hallmarks of multiple myeloma (MM). The role of osteoprotegerin (OPG) in the RANK/RANKL/OPG signaling system is well defined in the myeloma bone disease. Polymorphisms of the TNFRSF11B gene encoding OPG have been studied in various bone diseases. However, relationship between the levels of OPG and development of bone lesions regardless of RANKL is yet unknown. In this study, the effects of OPG gene polymorphism on the development of bone lesions in MM were investigated.

52 MM patients, admitted to Karadeniz Technical University, Medical Faculty, Department of Internal Medicine, Division of Hematology between March 2011 and March 2012, were included into the study. Pathologic fracture and/or destructive bone lesions were confirmed in 36 MM patients using X-ray, computed tomography (CT), or magnetic resonance imaging (MRI). No bone lesions were noted in 16 patients. The control group consisted of 20 age and gender matched volunteers.

Exon 1 of OPG gene was sequenced using DNA samples obtained from peripheral blood samples of 52 MM patients and 20 healthy control subjects. OPG gene C950T and C1181G polymorphisms were identified in 45 (33 with bone lesions) MM patients and in 16 control subjects. Polymorphic allele 1181G was higher in MM patients with bone lesions compared to MM patients without bone lesions and control group (χ2=8.629, p=0.013). Other polymorphic allele 950T was also overrepresented (73%; p=0.042) in MM patients with bone lesions compared to MM patients without bone lesions and the control group. The most frequent genotype observed in patients with myeloma bone lesions was 950 TT homozygous variant (56%, p=0.08) followed by 1181 CC homozygous wild type with 50% frequency (p=0.09). The frequency of 950 CT heterozygous genotype was higher in MM patients without bone lesions (50%, p=0.604). 1181 GG genotype showed higher frequency in MM patients with bone lesions compared to patients without bone lesions and control group (χ2=3.853, p=0.145). Of the combined haplotypes, 950 TT/1181 GG haplotype frequency is higher in patients with myeloma bone lesions (36%) compared to MM patients without bone lesions (13%) and controls (15%) (χ2=4.77, p=0.09). The frequency of TT/GG combined haplotype carriers in bone disease patients is higher than of those who do not have either polymorphic homozygous alleles (CT/CC and CC/CC haplotypes) (χ2=6.057, p=0.048). Additionally, 950 CC/1181CC wild type haplotype frequency was reasonably low in patients with myeloma bone lesions (8.3%, p=0.240).

This is the first study searching for the relationship between OPG gene variants C950T (promoter), C1181G (exon 1) and myeloma bone disease. It was concluded that the presence of polymorphic 1181 G/950 T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease.

Disclosures:

No relevant conflicts of interest to declare.
Combined Bendamustine, Prednisone and Bortezomib (BPV) In Patients With Relapsed Or Refractory Multiple Myeloma and Light Chain Induced Renal Failure
Authors: Pönisch Wolfram , Moll Barbara , Niederwieser Dietger
Published: October 2019

Introduction

Serious renal failure represents a main complication of Multiple Myeloma (MM). An estimated 25% to 50% of patients are affected during the course of their disease. These patients are at increased risk for infections and have a significantly worse prognosis. Small phase I/II studies suggest that treatment with chemotherapy and/or new substances results in recovery of renal function in more than 25%. The window of opportunity for reversal of renal impairment is rather small making an immediate and highly active treatment strategy mandatory. Bortezomib and bendamustine have turned out to be quickly acting and effective drugs in the treatment of MM.

Methods

Between March 2005 and March 2013, 36 patients (median age 64; range 32-81 years) with relapsed/refractory MM and light chain induced renal failure (creatinine clearance <60ml/min) were treated with bendamustine 60mg/qm on day 1 and 2, bortezomib 1.3mg/qm on day 1, 4, 8 and 11, and prednisone 100mg on day 1, 2, 4, 8 and 11. Cycles were repeated every 21 days. Patients were divided into two groups: group A (n=20) consisted of patients with moderate or severe renal dysfunction (eGFR 15-59ml/min) and group B (n=16) of patients with renal failure/dialysis (eGFR <15ml/min).

Results

The median number of the BPV-treatment was 2 (1-7) cycles. 24 patients (67%) responded after at least one cycle of chemotherapy with 3 CR, 3 nCR, 6 VGPR, and 12 PR. Six patients had MR, 2 patients stable disease and 4 patients had a progress. With a median follow up of 22 months of the surviving patients, median PFS and OS for patients with moderate or severe renal dysfunction (group A) were 10 months and 25 months, respectively. Outcome for these patients was significantly better compared to patients with renal failure/dialysis (group B) with a median PFS and OS of 3 months and 7 months, respectively (p<0.02).

Eleven patients showed a CRrenal, 5 patients a PRrenal and 15 patients a MRrenal. Median time to first renal response and best renal response were 21 days and 42 days, respectively. The most common severe side effect was grade 3-4 thrombocytopenia in 81% of the patients. Grade 3-4 neutropenia was observed in 50% of the patients. Moderate to severe infections were seen in 13 patients.

These results indicate that the combination of bortezomib, bendamustine and prednisone is effective and well tolerated in patients with relapsed/refractory MM and light chain induced renal failure.

Disclosures:

No relevant conflicts of interest to declare.
JAK1/2 Inhibition Suppresses T Cell Proliferation and Inflammatory Cytokine Production In An Allogeneic Setting and Ameliorates Graft-Versus-Host Disease (GvHD) In a Murine GvHD Model
Authors: Spoerl Silvia , Chen Sophia , Bscheider Michael , Mathew Nimitha , Schmickl Martina , Mueller Tony , Schmitt-Graeff Annette , Peschel Christian , Duyster Justus , Poeck Hendrik , Zeiser Robert , von Bubnoff Nikolas
Published: October 2019

Graft-versus-host-disease (GvHD) constitutes a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In GvHD, tissue damage is mediated by pro-inflammatory cytokines. Cytokine responses are mediated by activated Janus kinases (JAKs). We hypothesized that JAK1/2 inhibition might reduce T effector cell responses and inflammatory cytokine production in an allogeneic system, thereby ameliorating GvHD. We established an allogeneic (major mismatch) cell culture system using naive BALB/c CD4+ CD62Lhigh T cells that were co-cultured with C57BL/6 (B6) bone marrow derived dendritic cells (DC). JAK 1/2 signaling was specifically blocked using the small molecule inhibitor Ruxolitinib (INCB018424). By using a 3H Thymidine proliferation assay, we found that Ruxolitinib was able to inhibit the proliferation of allogeneic CD4+ effector T cells. In our intracellular cytokine staining experiments Ruxolitinib was able to impair the differentiation of naïve T cells into IFN-gamma and IL17A-producing T effector cells. Both cytokines – IFN-gamma and IL-17A – have been linked to aggravated courses of GvHD severity.

In vivo administration of Ruxolitinib signficantly reduced lethal GvHD after allo-HSCT in a murine major mismatch model. BALB/c recipient mice were irradiated and received C57BL/6 (B6) T cell depleted bone marrow along with CD4+ and CD8+ T cells. Animals that were treated with an oral gavage of Ruxolitinib twice daily displayed significantly lower serum levels of IFN-gamma, TNF-alpha, IL-10 and IL-12p70, lower histological and clinical GvHD scores and improved overall survival compared to the control group.

By using luciferase-positive (luc+) CD4+ and CD8+ T cells, we were able to visualize and quantify T cell migration after GvHD induction. The recipient mice received luc+ T cells along with conventional T cell depleted bone marrow. We were able to detect the luc+ T cell signal with a bioluminescence imaging system. At day 9 after allogeneic bone marrow transplantation, migration of donor T cells to GvHD target sites was significantly reduced in Ruxolitinib treated mice compared to control mice.

In summary, our data demonstrate that suppression of inflammatory cytokine responses by JAK1/2 inhibitors modulates T effector cell responses in an allogeneic setting and improves survival in a murine major mismatch GvHD model. These observations suggest that JAK1/2 inhibition might be used for the treatment of GvHD following allo-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Influence Of Race and Ethnicity On The Collection Of G-CSF Mobilized Peripheral Blood Stem Cells From Unrelated Donors, a Center For International Blood and Marrow Transplant Research Analysis
Authors: Hsu Jack W. , Wingard John R. , Logan Brent R. , Chitphakdithai Pintip , Shaw Bronwen E. , Goldstein Steven C. , O’Donnell Paul V. , Confer Dennis L.
Published: October 2019

Peripheral blood stem cell (PBSC) collection is increasingly used in allogeneic stem cell transplantation. However, a small percentage of healthy donors have a poor mobilization response to G-CSF. Very little information exists on the effect of donor race or ethnicity on PBSC mobilization. We analyzed 10776 unrelated donors from the National Marrow Donor Program (NMDP) who underwent G-CSF mobilized PBSC collection from 2006–2012. We investigated the effect of self-reported donor race/ethnicity on collection efficiency, defined as number of CD34+ cells/L (of donor blood processed), number of mononuclear cells (MNC)/L and CD34+ cells/MNC collected on the first day of apheresis. Categorical variables were analyzed by the Chi-square test and the Kruskal-Wallis test was used for continuous variables. A linear regression model was used to compare the various race/ethnic groups while controlling for potential confounding factors (such as age, BMI, gender, and year of apheresis). The result of our analysis is shown in Table 1. Univariate analysis revealed statistically significant differences in CD34+ cells/L, MNC/L and CD34+/MNC in all races analyzed. In general, African Americans (AA) had the highest collection efficiency while Caucasians had the lowest. Other races/ethnicities had collection efficiencies between the two groups. On multivariate analysis, statistically significant differences in CD34+ cell/L were seen in Hispanics, AA and Asian/Pacific Islanders (API), primarily in the obese (Hispanic, AA, API) and overweight (AA, API) donors. In the API group the differences in collection efficiency were predominately seen in males. No differences were seen between Caucasians and Native Americans. This study reveals significant racial/ethnic differences in the efficiency of collection of CD34+ cells in unrelated donors. Although these differences do not appear to interfere with the ability to collect adequate numbers of PBSC, it is currently unknown why they exist. This is an area for continued research.

Disclosures:

No relevant conflicts of interest to declare.
Minimal Residual Disease Monitoring By Quantitative RT-PCR In t (8; 21) AML Early After Allogeneic HSCT Can Predict Clinical Outcomes and Allow Further Risk Stratification
Authors: Wang Yu , Liu Yanrong , Qin Yazhen , Huang Xiao-Jun
Published: October 2019

Background

Recent results from our prospective multi-center study identified patients with t (8; 21) AML as high-risk according to their MRD status after the second consolidation chemotherapy and allo-HSCT can benefit this part of patients; however, the relapse rate was reported to be 22% even after allo-HSCT for those high-risk patients. To date, there have been no studies to answer the question of whether the specific fusion gene, RUNX1/RUNX1T1 can be used to further distinguish between patients with low and high risks of relapse in allo-HSCT setting, just like the already established standard for MRD measurement in CML and APL.

Methods

Sixty consecutive AML patients with t (8; 21) and identified as high-risk according to the criteria from our recently published report patients who received allo-HSCT were enrolled between January 2006 and January 2013. Serial MRD monitoring by RQ-PCR post HSCT was done. The impact of MRD monitoring on transplant outcomes was assessed.

Results

A > 3 log reduction at 1 month after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis, is associated with 2-year CIR of 17% in the 88% of patients achieving it, compared with 43% in the remaining (p=.02). A > 3 log reduction at 2 months after HSCT in transcripts from diagnosis, is associated with a CIR of 9% and LFS of 66% in the 86% of patients achieving it, compared with CIR of 100% and LFS of 0% in the remaining (both p<.001). A > 3 log reduction at 3 months after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis, is associated with a CIR of 11% and LFS of 73% in the 78% of patients achieving it, compared with CIR of 44% and LFS of 0% in the remaining (both p<.001). Of the 60 patients analyzed, 28 patients had positive MRD, occurring at a median of 110 days (30-540 days) after transplant. The predictive value of sequential monitoring could be demonstrated in 8 patients culminating in clinical relapse (representing 73% of all relapsing patients). The median time from MRD positivity in BM to morphological relapse was 95 days (range, 33-140 days).

Conclusions

A > 3 log reduction at the first 3 months after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis is highly prognostic. We conclude that MRD monitoring by RQ-PCR at regular early time points post HSCT in t (8; 21) AML allows further identification of patients at high risk of relapse even after allo-HSCT and could now be incorporated in clinical trials to evaluate the role of risk directed prophylactic/preemptive therapy.

This work was partly supported by The Key Program of National Natural Science Foundation of China £¨Grant No. 81230013£©, Beijing Municipal Science & Technology Commission (No.Z121107002812033) and Bejing Municipal Science & Technology Commission£¨No.Z111107067311070).

Disclosures:

No relevant conflicts of interest to declare.
High-Dose Chemotherapy and Autologous Stem Cell Transplantation In Patients With High-Risk Neuroblastoma : Results Of Turkish Pediatric Oncology Group- Neuroblastoma 2003 (TPOG-NBL 2003) Protocol
Authors: Aksoylar Serap , Varan Ali , Vergin Canan , Hazar Volkan , Akici Ferhan , Dagdeviren Ayhan , Unal Emel , Oniz Haldun , Anak Sema , Koseoglu Vedat , Ince Dilek , Olgun Nur
Published: October 2019

Introduction

TPOG-NBL 2003 national protocol was designed to improve treatment results of the high risk patients by adjunction of stem cell rescue with intensive multimodal therapy.

Material and Methods

High risk stratification was made according to COG criteria. Just before the third cycle of chemotherapy, patients without progression were allocated into two treatment groups non-randomly by physicians’ and/or parent’s choices guided to the center’s facility, toxicity and social-economical facility to attain the megatherapy. After an induction of 6 chemotherapy cycles, the protocol was divided into two arms which were designed to continue the intensive conventional chemotherapy (CCT), or initiate myeloablative therapy with autologous stem-cell rescue (ASCR). All patients were also given 13-cis-retinoic acid as maintenance therapy.

Results

Fifty-six percent (272 patients) of all neuroblastoma patients was evaluated as high risk. Response rate to induction chemotherapy was 81% (CR/VGPR: 32%, PR: 49%) in patients at the end of induction chemotherapy. Overall EFS and OS at 3-years were 36% and 45%, respectively. Intention-to-treat analysis documented post-induction (after the six cycles of induction chemotherapy) EFS of 46% in CCT arm (137 patients) and 37% in ASCR group (55 patients) (p= 0.037); whereas, OS was 59% and 43%, respectively (p=0.052). Thirty-one patients (11%) died of treatment-related complications.

Conclusion

Survival rates of high-risk neuroblastoma have improved over the last decade in Turkey. The main problems when managing these patients were an effective local control, early progression and death. Megatherapy has not augmented the therapeutic end point in our country’s circumstances. However; the better the supportive care and the higher the patients’ compliance is attained, the higher the survival rates might be obtained in Turkish neuroblastoma patients.

Disclosures:

No relevant conflicts of interest to declare.
Sideroflexin 4 Deficiency Results In An Erythroid Differentiation Defect
Authors: Nwanaji-Enwerem Jamaji C. , Hildick-Smith Gordon J. , Bauer Daniel E. , Huston Nicholas , Paw Barry H.
Published: October 2019

Megaloblastic anemia characterizes molecularly heterogeneous disorders, most commonly marked by macrocytosis, red blood cell enlargement, and abnormal nuclear maturation. Aside from vitamin B12 and folate deficiencies, genetic defects may be responsible for hereditary forms of megaloblastic anemia, though some remain unknown (Ghersim C, et al. 2013 J. Biol. Chem.). Loss of function of the gene Sideroflexin-4 (SFXN4) has been implicated as a cause for combined macrocytic anemia and mitochondriopathy. We have previously shown a defect in SFXN4 in humans and successfully modeled the erythroid and mitochondrial defects in the zebrafish (Hildick-Smith GJ, et al. 2013 AJHG). Here, we expand on prior studies involving SFXN4 in zebrafish and human fibroblasts by generating a murine cultured cell model deficient in SFXN4.

Clustered regularly interspaced short palindromic repeats (CRISPR)-cas targeting technology (Cong L, et al. 2013 Science) was utilized to generate SFXN4 knockout Friend mouse erythroid leukemia (MEL) clones. We conducted a screen of clones using qRT-PCR to select MEL clones with the lowest expression of Sfxn4 mRNA. The CRISPR knockout clone shows significantly reduced Sfxn4 mRNA relative to wild type (Panel A). Furthermore, the CRISPR knockout's reduced Sfxn4 mRNA is comparable to Sfxn4 mRNA levels in clones silenced by shRNA hairpin constructs (data now shown).

We also investigated the differentiation and maturation potential of the CRISPR-derived SFXN4 knockout clone. Initially, the MEL clone was differentiated for 3-5 days with DMSO treatment. o-dianisidine stains revealed that the CRISPR knockout clone had a reduction in hemoglobinization. Furthermore, Wright-Giemsa staining demonstrated that the same clone possessed megaloblastic features with immature maturation and a high nuclear to cytoplasmic ratio (Panel B). Both sets of results from experiments support the hypothesis for an essential role of SFXN4 in vertebrate red cell development.

In summary, our data: (1) supports the hypothesis of SFXN4's involvement in erythropoiesis and candidacy as a gene for combined macrocytic anemia and mitochondriopathy, and (2) demonstrates the promise of shRNA knockdown and now CRISPR SFXN4 knockout clones as tools for further studying the biochemical relationship between SFXN4 and megaloblastic anemia.

Disclosures:

No relevant conflicts of interest to declare.
Barth Syndrome and Neutropenia
Authors: Dale David C. , Bolyard Audrey Anna , Marrero Tracy M. , Bonilla Mary Ann , Phan Lan , Steward Colin
Published: October 2019

Barth syndrome is an X-linked, hereditary cause for neutropenia, cardiomyopathy, muscle weakness and growth retardation. It is attributable to mutations of TAZ, a gene encoding a highly conserved acyltransferase necessary for the maintenance of the phospholipids of the inner layers of mitochrondrial membranes. There is a wide diversity in the TAZ mutations but as yet no recognized genotype-phenotype correlations.

We collected and analyzed data for 83 patients (all males, Severe Chronic Neutropenia International Registry, Barth Syndrome Foundation and NHS Barth Syndrome Service in the UK). Fifty-four are currently less than 18 years old (median age 13.6 years, range 0.2-31.6 years). Absolute neutrophil counts (ANC) varied remarkably (median 1.1 x 109/L, range 0.14-5.4 x 109/L). A few patients have cyclic variations in the ANCs, but most have not had a sufficient series to determine if the variations show periodicity. Mouth ulcers, bleeding gums, skin infections, and upper respiratory infections were the most common complications, but pneumonia, sepsis, and more severe infections occurred sporadically. Comparisons of reported events for the population suggest that patients with baseline neutrophil counts< 0.5 x 109/L have more infections than patients with higher counts.

Data were available for 42 patients with neutropenia sufficiently severe to be treated with G-CSF by their physicians. On average, G-CSF was begun at 5.8 years (median age 4.3, range 0.05-21.5 years), with the median dose of 1.67 mcg/kg/d, mean 2.78 +/- 0.78 (SEM) mcg/kg/d (range 0.45-12.8 mcg/kg/d), with an average exposure of 7.3 years.

With longitudinal follow-up, 15 patients have had cardiac transplants and no patients have developed AML or received bone marrow transplantation and treatment responses to G-CSF are maintained long term. Bone marrow examinations were generally not performed, either before or on G-CSF treatment. Longitudinal observation suggests that the hematological profiles of individual patients are largely unchanged from childhood into adulthood, without evidence of worsening or amelioration. Additional clinical and laboratory data are need to define the genetic determinants and hematological features of Barth syndrome.

Disclosures:

Dale: Amgen: Consultancy, Research Funding.
Making “Sense” Of Persistent Polyclonal B Cell Lymphocytosis
Authors: Tsui Hubert , Paltser Geoff , Adissu Hibret , Dosch Michael , Keating Armand
Published: October 2019

Neuronal regulation of the hematopoietic stem cell niche includes elements of the sympathetic nervous system however, a role for sensory neurons has not been explored. While investigating sensory nerves in hematopoiesis, we unexpectedly found mice deficient in the neurotransmitter substance P (Tac-1-/-) to exhibit peripheral blood lymphocytosis that is persistent from 8 weeks to at least 35 weeks of age. Flow cytometry showed the increased population to be comprised of polyclonal B-lymphocytes. Hematopathology review demonstrated abnormal peripheral blood bi-nucleated lymphocytes and enlarged splenic marginal zones. Lymph nodes were not affected. To confirm that sensory nerve disruption and not hematopoietically derived substance P was the cause, wildtype C57BL/6 mice were chemically denervated and found to exhibit similar peripheral blood lymphocytosis. By immunohistochemistry, sensory nerves synapsed at splenic marginal zones, consistent with previous reports and providing a neuro-anatomical link for the observed lymphoid hyperplasia. A human correlate to this hematological phenotype featuring long-term peripheral polyclonal B cell lymphocytosis, bi-nucleated lymphocytes and expanded splenic marginal zones is Persistent Polyclonal B-cell Lymphocytosis (PPBL), a rare disorder affecting predominantly female chronic smokers. The pathoetiology of this disorder is unknown but its strong association with smoking supports a novel hypothesis where chronic smoking leads to respiratory tract sensory nerve desensitization with possibly more systemic effects, including on the spleen, thereby mimicking the substance P deficient environment of Tac-1-/- mice. Indeed, pulmonary function tests on chronic smokers have shown a sub-population of patients with hyporeactivity to respiratory sensory nerve stimulation, explained molecularly by increased activation thresholds. It will be interesting to determine if PPBL patients fall into this category. In Tac-1-/- mice, further characterization of the expanded B cell population, immunoglobulin profiles, VDJ utilization and effect of neurokinin-1 receptor modulators is proposed. These results support a neuroimmune regulatory network involving sensory nerves with potential relevance in both lymphoid homeostasis and lymphoproliferative disease.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Induces Specific Signaling Pathways Distinct From Simultaneous Stimulation With PAR1 and PAR4 Agonist Peptides In Human Platelets
Authors: Arachiche Amal , Nieman Marvin T.
Published: October 2019

Platelet activation by thrombin plays an important role in hemostasis and thrombosis. Protease activated receptor 1 (PAR1) and PAR4 are G- protein coupled receptors expressed in human platelets and activated by the proteolytic cleavage of their N-terminus with thrombin. We have previously shown by Bioluminescence Resonance Energy Transfer (BRET) that thrombin induces the formation of PAR1-PAR4 heterodimers in HEK293 cells. In contrast, simultaneous stimulation with the PAR1 and PAR4 agonist peptides, TFLLRN and AYPGKF, respectively, does not induce the formation of heterodimers. Signaling events that are specific to PAR1-PAR4 heterodimers in platelets are not known. In the current study we compared signaling in human platelets activated with thrombin or a mixture of TFLLRN and AYPGKF agonist peptides by examining the phosphoproteome using mass spectrometry. A global analysis of proteins phosphorylated on tyrosine, serine, or threonine residues was performed using nanoflow liquid chromatography-tandem mass spectrometry (MS/MS). Human platelets were pretreated with tirofiban, a GPIIb/IIIa integrin inhibitor, in order to block the outside in signaling and limit the analysis to the initial signaling events prior to integrin activation. Platelets were then activated with 3 nM thrombin or both TFLLRN (50 μM) and AYPGKF (500 μM) for 3 min and compared to unstimulated platelets. The results showed the expected signaling pathways activated in both thrombin-stimulated and agonist peptide-stimulated platelets. Importantly we identified 51 proteins that were specifically phosphorylated in thrombin-stimulated platelets. Next we wanted to know if the specific phosphorylation sites identified play a role in the function of these proteins. We identified a single phosphorylation residue on Ser552 of β-catenin, a signaling component of the Wnt pathway, after stimulation with thrombin but not with TFLLRN+ AYPGKF. This phosphorylation is important for the stabilization of β-catenin. We also identified that Disabled-2 (Dab2) and protein kinase C beta (PKC-β) are phosphorylated after stimulation with TFLLRN+ AYPGKF, but not thrombin. Dab2 is a cargo-specific adaptor protein involved in endocytosis and cell signaling. The phosphorylation on Ser401 regulates function and localization of Dab2 whereas the phosphorylation on Thr500 and Thr642 regulates PKC-β function. Taken together, we have identified specific signaling pathways initiated by thrombin stimulation, since thrombin induces the formation of PAR1-PAR4 heterodimers, the signaling events identified in our study may be specific to PAR1-PAR4 heterodimers. In conclusion, the understanding of the molecular arrangement of PAR1 and PAR4 in platelets will provide insight for the development of specific antiplatelet therapies.

Disclosures:

No relevant conflicts of interest to declare.
Histone 3 Methyltransferase (EZH2) Inhibition Enhances TRAIL-Induced Apoptosis In Mantle Cell Lymphoma Cells By Accelerated cFLIP Degradation
Authors: Braun Frank K , Mathur Rohit , Sehgal Lalit , Berkova Zuzana , Samaniego Felipe
Published: October 2019

Introduction

Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma that is characterized by the t(11:14)(q13:p32) translocation. MCL cells have altered cyclinD1 levels, impaired cell cycle regulation, DNA damage response, and likely defects in apoptosis signaling. Furthermore, up-regulated anti-apoptotic mediators such as the target of NF-κB c-FLIP were correlated with decreased apoptosis signaling. Also many cancer cells and malignant tumors show a prevalent resistance to apoptosis induction by TRAIL. Thus, by understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve TRAIL-induced killing of lymphoma cells.

Methods/Results

MCL cell lines (Mino, JeKo-1, JVM-2 and Z-138) were treated with DZNep (3-Deazaneplanocin A; 0.2-5µM) for 24 h followed by incubation with TRAIL (10-20ng/ml, 6-16h). Cell death, DNA fragmentation, and mitochondrial membrane potential (Δψm) were determined by calcein staining, subG1 analysis, and TMRM staining, respectively. Neither DZnep alone nor in combination with TRAIL showed a significant induction of necrosis as determined by LDH-release levels, but DZNep alone showed strong antiproliferative properties at higher concentrations (Promega CellTiter 96 assay). Activation of the caspase signaling cascade (caspase-8, -9, -3, Bid, and PARP cleavage) was analyzed by Western blotting. TRAIL-induced signaling was significantly increased and caspase-8 processing enhanced in DZNep pretreated cells indicating a regulation at the TRAIL/DISC. Although a reduced expression of DR5 in total cell lysates of DZNep treated cell was observed, the surface receptor levels were not altered. Interestingly, downregulation of the well-known caspase inhibitor, cFLIP, correlated with the DZNep-induced increased TRAIL sensitivity in MCL cell lines. However, it appears that cFLIP levels are not reduced due to blocked NF-kB signaling but rather by an accelerated ubiquitin-mediated degradation.

Conclusions

This study reveals that inhibition of histone methyltransferase (EZH2) activity by DZNep has a profound positive impact on TRAIL signaling; it enhances TRAIL sensitivity by promoting processing of caspase-8 through enhanced cFLIP degradation. The capacity of DZNep to target stability of cFLIP, which represents a center piece in DISC regulation underscores its potential for enhancing efficacy of TRAIL-based cancer therapies.

Disclosures:

No relevant conflicts of interest to declare.
Control Of Allograft Rejection With Mitomycin C Incubated Blood Cells
Authors: Greil Johann , Kleist Christian , Mohr Elisabeth , Sandra-Petrescu Flavius , Jiga Lucian , Dittmar Laura , Satagopam Venkata , Demirdizen Haydar , Hundemer Michael , Becker Luis , Lang Peter , Opelz Gerhard , Terness Peter
Published: October 2019

Immunosuppressive treatment in allogeneic transplantation is an effective way to control allograft rejection. But unfortunately it can cause serious side effects, such as infections or malignancies. Cell therapeutic approaches for induction of specific tolerance are on the rise and may have fewer side effects. We observed that monocytes or peripheral blood mononuclear cells (PBMC) of healthy persons after culture with dendritic cell-inducing cytokines and differentiation blockade with the chemotherapeutic drug mitomycin C (MMC) show the ability to suppress allogeneic T cell responses. Interestingly these cells resemble morphologically as well as phenotypically myeloid-derived suppressor cells (MDSC) which occur in cancer patients impairing the anti-tumor immunity. FACS- and gene expression analyses of these MMC incubated blood cells revealed downregulation of the immunostimulatory cell surface molecules CD80, CD83, CD86 and HLA-DR, and upregulated transcription of immunosuppressive and pro-apoptotic genes such as iNOS, arginase-1, IL-10, COX2, TGF-β, placental growth factor, immunoglobulin-like transcript 3, adrenomedullin, SMAD5, LRDD and of the transcription factor C/EBPβ. For use in preclinical transplantation models in vitro culture of MMC treated donor-derived blood cells with dendritic-cell inducing cytokines was not applicable and was therefore omitted. The tolerance inducing properties of donor-derived MMC incubated blood cells were investigated in two different animal models of solid organ transplantation. When injected into rats, donor-derived PBMC incubated with MMC strongly prolonged heart allograft survival. Suppression of rejection was donor-specific. Recipients showed an increased number of CD4+CD25+FoxP3+ regulatory T-cells and tolerance could be transferred to syngeneic recipients with blood or spleen cells. Donor-derived MMC incubated blood cells also prolonged kidney allograft survival in pigs. Furthermore, in a first clinical use a 6-year-old girl with relapse of acute lymphoblastic leukemia and imminent rejection of a third haploidentical stem cell transplant received donor-derived CD3/CD19-depleted peripheral blood stem cells, which have been treated in vitro with MMC, in order to prohibit graft rejection. We describe a simple method for in vitro generation of tolerance inducing donor-derived blood cells for potential use in clinical organ transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Compact Bone-Derived Mesenchymal Stem Cell Transplantation Attenuates The Severity Of Idiopathic Pneumonia Syndrome In a Murine Bone Marrow Transplantation Model
Authors: Qiao Shukai , Ren Hanyun , Shi Yongjin , Liu Wei , Yuan Jing
Published: October 2019

Introduction

Idiopathic pneumonia syndrome (IPS) is a serious and life-threatening lung complication following allogeneic hematopoietic stem cell transplantation (allo- HSCT) and currently no effective therapies exist. The present study was designed to determine whether transplantation of allogeneic murine compact bone derived- mesenchymal stem cells (CB-MSCs) could prevent the development of IPS.

Methods

We tested the effects of allogeneic CB-MSCs transplantation on IPS using an established murine model of IPS, wherein lethally irradiated male BALB/c (H-2d) recipient mice received bone marrow and spleen cells from female C57BL/6(H-2b) donors. Survival rates, body weight change, clinical GVHD scores, lung histological changes and wet-dry lung ratios were assessed after IPS induction. Mechanistically, concentrations of cytokines (TNF-alpha, IFN-gamma and IL-4) and chemokines (CCL5, CXCL9 and CXCL10) in bronchoalveolar lavage fluid (BALF) from the recipient mice were measured at different time point post-transplantation. CD4+CD25+Foxp3+ regulatory T cell (Treg) percentage, CCR5, CXCR3 and CCR7 expression on CD3+ T cells, and lung CXCR3, CCR5, CCR7, T-bet and GATA-3 mRNA levels were also evaluated at different time point post-transplantation.

Results

In the IPS model, co-transplantation of CB-MSCs significantly attenuated the severity of lung injuries and increased the survival rate of mice compared to non-cotransplanted mice. Furthermore, a higher Treg percentages, reduced TNF-alpha, IFN-gamma, CCL5, CXCL9 and CXCL10 levels, CXCR3 and CCR5 down-regulation, as well as CCR7 up-regulation, were observed in MSCs co-transplantation mice. Additionally, the prophylactic effect of CB-MSCs was also associated with a shift of Th1/Th2 balance toward anti-inflammatory Th2 polarization.

Conclusions

Our present data suggested that allogeneic CB-MSCs attenuated the severity of IPS due to its profound immunomodulatory capacity in our murine model. This may offer a novel prophylactic approach for IPS patients after allo-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Prospective Evaluation Of Risk Factors For Clostridium Difficile Colonization Among Allogenic Hematopoietic Stem Cell Transplant Recipients, 2010-2012
Authors: Jain Tania , Croswell Christopher , Reddy Banavasi Harsha Vardhan , Urday-Cornejo Varinia , Liubakka Alyssa , Cutright Jessica , Salimnia Hossein , Lephart Paul , Revankar Sanjay , Chandrasekar Pranatharthi , Chopra Teena , Deol Abhinav , Alangaden George
Published: October 2019

Background

Clostridium difficile (CD) is the leading cause of nosocomial diarrhea. After acquisition of CD, some patients (pts) remain colonized whereas others develop symptomatic CD infection (CDI). Hematopoietic stem cell transplant (HSCT) recipients are at higher risk of CDI than the general population. Rates of colonization and risk factors for CD colonization in the HSCT population are unknown. The goals of this study were to determine the rates and predictors of colonization with CD in HSCT pts at our institution.

Methods

We conducted a 16-month prospective study starting in December 2010 in the HSCT unit at the Karmanos Cancer Center. Pts who signed informed consent had stool samples collected within 72 hours of admission and weekly thereafter until discharge. Pts were followed up for 90 days after enrollment. Stool samples were cultured for CD and culture positive samples were tested by PCR for confirmation of toxigenic CD. Demographic information, risk factors and outcomes were examined.

Results

154 patients who underwent allogeneic HSCT were enrolled in the study. Mean age for both CD colonized and non-colonized pts was 51 ±12.8 years, 59% were males. Overall, 47/154 (30%) pts were colonized with CD at admission; 23/154 (15%) with toxigenic CD. 8/23 (34%) pts colonized with toxigenic CD developed symptomatic CDI. Of the pts colonized with non-toxigenic CD, 7/24 (29%) acquired CD during the hospital stay. The median time to acquisition was week 2 of hospitalization. On bivariate analysis, underlying comorbid conditions and traditional risk factors for CDI like prior hospitalization, length-of-stay, antibiotic use, nasogastric tube and gastrointestinal tract procedures were found to be equally prevalent in both colonized and non-colonized population. The outcome in terms of graft versus host disease (GVHD) was also similar in both groups. The use of proton pump inhibitors was more prevalent in non-colonizers (56%) vs. colonizers (43%). However, it was not statistically significant (p=0.16). Similarly, lymphopenia seemed common amongst colonizers vs. non-colonizers (67% vs. 58%) but was not statistically significant (p=0.2).

Conclusion

Colonization with toxigenic CD is high in our HSCT recipients at hospital admission. Symptomatic CDI occurs in a third of pts colonized with toxigenic CD. Unlike in the general population, traditional risk factors for CD colonization are not predictive of colonization in our allogeneic HSCT pts. Novel approaches such as active surveillance to detect colonized patients to guide infection control measures and to direct “pre-emptive” therapy should be evaluated in this population.

Disclosures:

No relevant conflicts of interest to declare.
Role Of miRNA Expression As Novel Biomarker Of GvHD After Allogeneic Stem Cell Transplantation
Authors: Koldehoff Michael , Steckel Nina K , Maniera Christina , Elmaagacli Ahmet H. , Beelen Dietrich W.
Published: October 2019

Background

MicroRNAs (miRNAs) are a family of 19-24 nucleotide noncoding RNAs, which affect the regulation of gene expression in eukaryotic cells by binding to a 3´-untranslated region which target messenger RNAs. MiRNAs plays important roles in many cellular processes such as development, stem cell division, apoptosis and cancer. We profiled miRNAs expressed on patients (pts) who underwent allogeneic hematopoietic stem cell transplantation (HSCT).

Methods

Here we analyzed in a retrospective study 258 pts (male 124 pts, female 134 pts) for miRNAs expression (miRNA-21; -128; -146; -155; -181; and –let7) in whole blood at different time-points that underwent allogeneic HSCT and analyzed their outcome. MiRNA expressions were performed by miRNA-specific real time RT-PCR.

Results

In this cohort 84 pts received grafts from HLA-identical siblings (32%), 126 pts from matched (49%) and 48 pts from mismatched (19%) unrelated donors. Transplant consisted of unmanipulated peripheral blood stem cells (n=225, 87%) or bone marrow (n=33, 13%). Of all pts, 64 (25%) had relapsed and 41 (16%) died of June 2013. There was no significant correlation between miRNA expressions from siblings and unrelated donors. For miRNA-181, we found a significant up-regulation in mismatched unrelated donors versus matched donors (489 vs. 330%, p<0.005). Analysis of each miRNAs for relapse showed no statistically differences. Among all pts, 210 (81%) developed acute GVHD (93 pts had an aGvHD of grade ≥2). There was a significant differences between low level of miRNA-146 (529 ± 516% vs. 804 ± 1335%; p<0.04), and high level of miRNA-let7 (642 ± 419% vs. 546 ± 335%, p<0.05) of pts with aGvHD ≥ 2. In pts with severe aGVHD (grade >3) comparing all other aGvHD pts., we found a high significant reductions of miRNA-146 (333% vs. 720%, p<0.001), and miRNA-21 (528% vs. 2356%, p<0.02). In regard to chronic GvHD, we found a down-regulation of miRNA-let7 expressions in pts with severe cGVHD (384% vs. 922%, p<0.002) comparing other cGVHD in the first year. The probability of severe aGvHD with low level of miRNA-146 (< 215%, p<0.001) is 20 ± 7% versus 4 ± 2% with higher levels of miRNA-146 levels. However, an estimate for death in remission or overall survival was not significantly associated with miRNAs.

Conclusions

These results suggest that pts with different expression of miRNA-146, miRNA-21 and miRNA-let7 confirms a relevant association of the development of aGVHD and miRNA profiling could be an early indicator of severe aGvHD.

Disclosures:

Off Label Use: HCG will be discussed as new therapy for chronic GVHD.
Update On Phase 2 Clinical Trial To Induce Tolerance In Mismatched Living Donor Renal Transplant Recipients
Authors: Leventhal Joseph , Abecassis Michael , Miller Joshua , Gallon Lorenzo , Tollerud David J , Ildstad Suzanne T.
Published: October 2019

Nineteen subjects have been enrolled in a phase 2 protocol (FDA IDE 13947) to induce donor-specific tolerance in HLA-mismatched related and unrelated recipients of living donor renal allografts (KTx). The protocol is based upon tolerogenic CD8+/TCR- facilitating cells (FC) and nonmyeloablative conditioning. Recipients are conditioned with fludarabine (30 mg/kg/dose, days -5, -4, -3), cyclophosphamide (50 mg/kg/dose, day -3 and +3), 200 cGy TBI (day -1) followed by KTx (day 0). A G-CSF mobilized peripheral blood mononuclear cell product is apheresed from the donor > 2 weeks prior to the KTx, processed to remove GVHD-producing cells yet retain CD34+ cells and FC (FCRx), and cryopreserved until infusion on day +1 post-KTx. Subjects ranged in age from 18 to 65 years. All were HLA-disparate from their donors, ranging from 5 of 6 matched related to 0 of 6 unrelated. 18 of 19 subjects exhibited engraftment at 1 month; one highly sensitized subject (PRA > 50%) did not engraft. Subjects were discharged on post-operative day 2 and managed as outpatients. Mycophenolate mofetil and tacrolimus-based immunosuppression were administered for 6 months. At 6 months the MMF was discontinued if the protocol biopsy was normal and stable renal function present. Tacrolimus was weaned at month 9 (trough levels 3-6) and discontinued at 1 year if chimerism, normal renal function, and normal kidney biopsy were noted. No subjects have experienced GVHD or engraftment syndrome. Two early subjects who received a suboptimal cell dose and one other highly sensitized subject were only transiently chimeric (< 6 months); all transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low-dose tacrolimus monotherapy with stable renal function. The protocol was subsequently modified to exclude subjects with a PRA > 20%. One chimeric subject developed viral sepsis at month 3, thrombosed his kidney, and was successfully re-transplanted off study. A second subject developed CNI-induced hemolytic uremic syndrome (HUS) which resulted in graft loss. He is currently off all immunosuppression and his HUS has resolved. The remaining subjects are either off all immunosuppression (n = 9; 1 to 36 months) or are in the process of tapering. None of the chimeric subjects have developed rejection on protocol biopsy while 3 of the 5 who were not durably chimeric had Banff 1A rejection on protocol biopsy. Recurrence of autoimmune disease has occurred in 2 of 4 non-chimeric subjects, but not in any of the durable chimeric subjects. In summary, high levels of durable chimerism and tolerance without GVHD have been achieved in mismatched related and unrelated recipients of living KTx.

Disclosures:

Tollerud: Regenerex, LLC: Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.