bluuebunny/crossref_metadata_2025 huggingface dataset:
Belimumab, a Neutralizing BAFF (B Cell Activating Factor) Antibody, Enhances the Susceptibility of Chronic Lymphoid Leukemia (CLL) Cells to Abt-199 Treatment
Authors: Schmidt Jonas , Wild Julia , Strunz Benedikt , Kanz Lothar , Salih Helmut R
Published: October 2019

Abt-199 is a small molecule BCL-2 inhibitor which has recently been approved for treatment of CLL and mediates its effects by induction of apoptosis in the leukemic cells. The TNF family member B cell activating factor (BAFF) contributes to disease pathophysiology in mature B cell malignancies including CLL by sustaining survival and preventing apoptosis of the malignant B cells. BAFF also plays a prominent role in autoimmune diseases, and accordingly a BAFF-neutralizing antibody termed Belimumab (Benlysta®) has been developed and is approved for treatment of systemic lupus erythematosus. Notably, elevated levels of BAFF have also been detected in sera of B cell lymphoma patients. We demonstrated recently that BAFF protects CLL cells from NK cell cytotoxicity including Rituximab-induced antibody-dependent cellular cytotoxicity (ADCC), the major mechanism by which this CD20-antibody mediates its therapeutic effects. In turn, susceptibility of CLL cells to both direct and Rituximab-induced NK cell killing could be restored by BAFF neutralization with Belimumab (Wild et al, Leukemia 2015). Here we studied whether Belimumab could also serve to increase the susceptibility of CLL cells to Abt-199 treatment. As no information on Abt-199 plasma peak levels in CLL patients is available, we first exposed primary CLL cells of leukemia patients to varying concentrations of Abt-199 and identified 5 nMas in vitro LC50 concentration in analyses of leukemia cell metabolic activity. Next we studied how BAFF influenced the therapeutic efficacy of Abt-199. Analyses of CLL cell metabolic activity and apoptosis/cell death revealed that BAFF concentration-dependently protected primary CLL cells from the therapeutic effects of Abt-199. Notably, CLL cell susceptibility to Abt-199 could be restored by neutralization of BAFF by Belimumab. Together, our findings demonstrate that BAFF contributes to CLL cell resistance to Abt-199 treatment. BAFF neutralization by Belimumab may in turn constitute a promising strategy to increase/restore the therapeutic efficacy of Abt-199, which warrants future clinical studies of combinatorial approaches to improve CLL treatment.

Disclosures

No relevant conflicts of interest to declare.
CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization
Authors: Fooksman David R , Mazumder Amitabha , McCarron Mark
Published: October 2019

Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations.

Disclosures

No relevant conflicts of interest to declare.
Influence of CD34+ and CD3+ Graft Content on Gvhd Development after Haploidentical Allogeneic Transplantation with Post-Transplant Cyclophosphamide
Authors: Bastos-Oreiro Mariana , Balsalobre Pascual , Champ Diana , Churruca Juan , Pascual Maria Jesus , Herrera Pilar , Bento Leyre , Piñana Jose Luis , Jimenez Santiago , Humala Karem , Montesinos Pau , Heras Inmaculada , Iglesias Almudena , Perez-Corral Ana , Pascual Cristina , Martinez-Laperche Carolina , Kwon Mi , Gayoso Jorge , Diez-Martin Jose L.
Published: October 2019

Introduction:

CD34+ and CD3+ cell dose in peripheral blood stem cell (PBSC) graft have been related with increased incidence of graft versus host disease (GVHD) in some transplant settings. Our aim in this study was to evaluate the impact of CD34+ and CD3+ cells graft composition on GVHD incidence in the setting of haploidentical transplant (haplo-HSCT) with post-transplant cyclophosphamide (PT-Cy), in a multicenter analysis.

Materials (or patients) and methods:

We retrospectively evaluated 175 patients with hematologic malignancies who were treated with a haplo-HSCT from 2011 to 2014 with PBSC in GETH centers. All patients received Busulfan-Fludarabine as conditioning regimen and GVHD prophylaxis was performed with PT-Cy (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5. We analyzed the impact of CD34+ and CD3+ cell doses (low vs. high with cut-off at the median value, and the 25-75 percentiles) on the development of acute and chronic graft versus receptor disease. Death before GVHD development was considered as a competitive event. The analysis was adjusted for conditioning intensity. STATA software was used for data analysis

Results:

We analyzed 175 patients. Patient characteristics are resumed in table 1. Median CD34+ and CD3+ cell dose was 5.31x10e6/kg (p 25-75: 4.31-6.1) and 2.07x10e8/kg (p25-75: 1.19-2.94), respectively. Cumulative incidences of grade II-IV acute GVHD and all grades chronic GVHD was 32.6% and 30% respectively. The median follow-up was 12.3 months (r:6-46). No difference in terms of GVHD (acute and chronic), was found between low and high CD34+ cell doses. However, if we focus on T cells, high doses of CD3+, for values above the median, have been has been associated with increased incidence of acute GVHD II-IV (day 150: 47% vs 26% p=0.01, figure 2), as well as cGVHD (30 month: 39% vs 16%, p= 0.01 figure 1). If we focus the analyses regarding the intensity of conditioning regimen, the influence of CD3+ doses remains for the group of reduced-intensity conditioning (RIC) (aGVHD, day 150: 54% vs 24%, p=0.003; cGVHD, 30 month: 47% vs 16%, p=0.006 figure 2), but lost for patients who receive myeloablative intensity (aGVHD, day 150: 33% vs 33% p=0-8; cGVHD, 30 month: 21% vs 16%, p=0.81)

Conclusion:

In our series, in the setting of haplo-HSCT with Pt-Cy, the highest dose of lymphocytes was associated with a higher incidence of aGHVD II-IV and cGV HD, especially for the RIC procedures.

Disclosures

No relevant conflicts of interest to declare.
Natural Toll-like Receptor Agonists for in Situ Vaccination Lymphoma Immunotherapy
Authors: Peng Paul , Brody Joshua , Hammerich Linda
Published: October 2019

Introduction: There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination-an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist-has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells. While the latest clinical trial NCT01976585 employs Poly-ICLC-a synthetic TLR3 agonist-to activate DCs, we hypothesize that "natural" TLR agonists (nTLRa) contained within prophylactic vaccines (e.g. live/attenuated bacteria or viruses) could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver.

Methods: Twenty-four prophylactic vaccines from our hospital's pharmacy were screened using in vitro assays for TLR ligand activity. DC subsets CD11c+ MHC-II+, CD11c+ CD11b+, and CD11c+ CD103+ were gated for flow-cytometric readouts of: (i.) activation markers (e.g. CD40, CD80, CD86, MHC class I and II), (ii.) ability to co-stimulate T cells in the context of TCR activation, as assessed by T cell activation markers (e.g. CD69, intracellular IFNγ), and (iii.) ability of nTLRa-activated DCs to induce T cell proliferation. TLR knock-out cell lines were used for dissecting the mechanistically-distinct properties of each prophylactic vaccine. Best single and combination nTLRa candidates were evaluated with the in situ vaccination maneuver in an in vivo A20 murine lymphoma model.

Results: We identify a combination of vaccines Typhim, BCG-TICE, and MMR that demonstrates the ability to induce high expression of costimulatory molecules on DCs (Figure A) in vitro, as well as possessing the ability to co-stimulate in vitro T cell activation and proliferation. DC costimulation of T cell activation and proliferation is highly dependent on the "live" status of several vaccines, including BCG, MMR, Zostavax, which suggests the involvement of other pattern-recognition receptors in addition to TLRs. Cohorts of mice implanted with A20 lymphoma tumors that received i.t. injections of Typhim, BCG, MMR demonstrated slower A20 growth status post in situ vaccination maneuver, as compared to the control cohort receiving only Flt3L and irradiation (XRT).

Conclusion: Prophylactic vaccines contain natural ligands to TLRs and are immediately translatable as sources of clinical-grade stimulators of dendritic cells. Combinations of the best nTLRa candidates display synergistic activation of DCs and demonstrate in vivo anti-A20 murine lymphoma immunity.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
EUTOS Score Predicts Long-Term Outcome in Chronic Myeloid Leukemia Patients Transitioning from Pre-Imatinib Era to Imatinib upon Availability
Authors: Deb Pratik , Nath Uttam , Aich Rajarshi , Ray Siddhartha Sankar , Chakrabarti Prantar
Published: October 2019

Background: The introduction of Tyrosine Kinase Inhibitors (TKI) in the management of Chronic Myeloid Leukemia (CML) has been a paradigm shifting phenomenon, yet the prognosis of CML still relies on pre-TKI era methods. Recently, European Treatment and Outcome Study (EUTOS) for CML has proposed a novel scoring system to prognosticate the outcome of treatment with TKIs that only relies on spleen size and basophil count. Although the validity of the newly proposed score has been tested in various populations worldwide, such a study is yet to be performed in the Indian population. We have looked at all three prognostic scores, namely Sokal score, Hasford/Euro score and EUTOS score, in 377 patients initiated with Hydroxyurea but later transitioned to Imatinib when it was available in India.

Methods: A cohort of 377 patients who were diagnosed as Philadelphia chromosome positive CML over the period of 5 years (Jan, 2003 - Dec, 2008) in Institute of Hematology and Transfusion Medicine, Medical College, Kolkata had been included in the study. All the patients were primarily treated with Hydroxyurea but received Imatinib Mesylate at a later time point. All patients received Hydroxyurea for 6 months or more and received Imatinib for at least 1 year at the time of final follow-up. No patient was treated with second generation tyrosine kinase inhibitors. We divided the patients using all three scores: Sokal, Hasford and EUTOS. The prognostic power of each of the scores were tested based on Overall Survival (OS) and Event Free Survival (EFS). Survival probabilities were estimated by the Kaplan-Meier method and compared by pair-wise log rank, Breslow (Generalized Wilcoxon) test and Tarone-Ware test. The pair-wise method was applied in order to detect distinguishing capacity of each test among different groups.

Results: Only EUTOS score stood out as informative as it was able to distinctly separate high and low risk group patients significantly (p < 0.01). The other two scores, namely Sokal and Hasford could not discriminate between the low and intermediate risk group of patients in terms of OS. When we considered EFS instead of OS for the same analysis, the superiority of EUTOS score over its counterparts again became obvious (p < 0.01).

Conclusion: EUTOS score was able to significantly discriminate between high and low risk patients both in terms of 5 year OS and EFS, while Sokal and Hasford/Euro score failed to discriminate between low and intermediate risk patients. We found EUTOS score to be valid in terms of predicting the outcome of CML patients who transitioned from the pre-imatinib era to Imatinib therapy in our population.

Disclosures

No relevant conflicts of interest to declare.
The JAK1/2 Inhibitor Momelotinib Does Not Inhibit Thiamine Uptake in Vitro
Authors: Giacomini Marilyn , Hao Jia , Liang Xiaomin , Chandrasekhar Jayaraman , Twelves Jolyn , Whitney Andy , Lepist Irene , Ray Adrian
Published: October 2019

The clinical development of a selective JAK2 inhibitor, fedratinib, was terminated due to cases of Wernicke's encephalopathy, a disease related to thiamine insufficiency. Furthermore, a recent publication reported potent inhibition of the thiamine transporter 2 (THTR2; SLC19A3) by fedratinib1. This study aimed to determine the potential for other JAK inhibitors to inhibit thiamine transport and to better elucidate the structural basis for inhibition. The effect of the aminopyrimidine containing JAK inhibitors fedratinib, AZD1480, momelotinib, and XL-019 on thiamine transport in Caco-2 cells and HEK293 cells transfected with human THTR1 (SLC19A2) and 2 was assessed. Consistent with the prior publication, fedratinib inhibited thiamine uptake in Caco-2 cells (IC50=0.94 ± 0.08 μM) and THTR2 transfected cells (IC50=1.6 ± 0.52 μM). We also observed inhibition of THTR1 by fedratinib (IC50=7.9 ± 0.97 μM). Another JAK inhibitor, AZD1480, also inhibited thiamine transport, but to a lesser degree (Caco-2, IC50=8.1 ± 0.1 μM). In contrast, momelotinib and XL-019 did not inhibit thiamine transport in any of the systems tested. A pharmacophore model derived from the minimized structure of thiamine suggests that the 2,4-diaminopyrimidine containing JAK inhibitors fedratinib and AZD1480 can adopt a conformation matching several key features of thiamine, while inhibitors lacking the 4-amino group and/or an additional hydrogen bond donor poorly align with thiamine. In summary, these results suggest that not all JAK inhibitors have the potential to inhibit thiamine transport. In particular, momelotinib, currently in Phase 3 clinical trials for the treatment of pancreatic cancer and myelofibrosis, showed no potential to affect thiamine levels.

Disclosures

Giacomini: Gilead Sciences: Employment. Hao:Gilead Sciences: Employment. Chandrasekhar:Gilead Sciences: Employment. Twelves:Gilead Sciences: Employment. Whitney:Gilead Sciences: Employment. Lepist:Gilead Sciences: Employment. Ray:Gilead Sciences: Employment.
A New Prognostic Model for Multiple Myeloma Patients in the Era of Novel Agents
Authors: Cho Hyungwoo , Yoon Dok Hyun , Kim Kihyun , Yoon Sung-Soo , Min Chang-Ki , Eom HyeonSeok , Kim Jin Seok , Lee Je-Jung , Lee Jae Hoon , Suh Cheolwon
Published: October 2019

Introduction

The prognosis of multiple myeloma (MM) is heterogeneous and survival varies from a few months to more than 10 years. Numerous prognostic factors have been identified and several prognostic models were developed to stratify patients into various risk groups. Currently, the international staging system (ISS) is the most widely accepted prognostic model. However, since ISS was established using data collected from 1981 through 2002, only a small portion of patients treated with novel agents (e.g., thalidomide, bortezomib, and lenalidomide) were included in the analysis. Therefore its validity is being challenged in the era of novel agents. We aimed to develop an alternative prognostic model based on easily obtained clinical and laboratory data in the era of novel agents.

Patients and methods

Clinical and laboratory data were collected between January 2000 and April 2013 from 8 tertiary hospitals in Korea. Among 2893 newly diagnosed patients with MM, 1338 were treated with novel agents including thalidomide, bortezomib and lenalidomide at least once. Half of these 1338 patients were randomly selected as a training sample and the other half as a validation sample. A new prognostic model was developed in the training sample, based on significant adverse prognostic factors in univariate and multivariate analysis. This model was then validated in the validation sample.

Results

With a median follow-up duration of 50.8 months (range, 47.1- 54.5) in surviving patients, the median overall survival (OS) in the training sample (669 patients) was 67.8 months. The median OS of patients according to ISS 1, 2, and 3 were 118.0, 67.8, and 50.5 months, respectively. There were no significant difference in the median OS between ISS stage 2 and 3 patients (Figure 1). We identified four independent adverse prognostic factors in the training sample; Serum beta2-microglobulin (Sβ2M) ≥ 3.5 mg/L, elevated serum lactate dehydrogenase (LDH), Eastern Cooperative Oncology Group performance status (ECOG PS) ≥ 2 and bone marrow plasma cell percentage (BMPC) ≥ 60%. We used these four adverse prognostic factors to develop a new prognostic model and stratified the patients into three groups; Group 1, no adverse factor; Group 2, 1 adverse factor; Group 3, ≥ 2 adverse factors. We found that there were significant differences in median OS among these three groups (Not reached, 73.0, 40.6 months for group 1,2, and 3, respectively) (Figure 2). This new prognostic model was further validated in the validation sample (Median OS was 128.7, 68.4, and 38.5 months for group 1, 2, and 3, respectively) (Figure 3)

Conclusion

Our study suggests that this new prognostic model based on easily obtainable clinical and laboratory data (Sβ2M, LDH, ECOG PS, BMPC) might be a simple and useful tool to predict prognosis of patients with MM in the era of novel agents.

Disclosures

Kim: CELLTRION, Inc,: Research Funding.
Deletion of Pleiotrophin (PTN), a Vascular Niche Paracrine Factor, Abrogates Chronic Myeloid Leukemia Pathogenesis In Vivo
Authors: Himburg Heather A , Zhao Liman , Chute John P
Published: October 2019

Tyrosine kinase inhibitor (TKI) therapy revolutionized the treatment of patients with chronic myeloid leukemia (CML), but TKI therapy alone is not curative. Leukemic stem cells (LSCs) are considered to be resistant to TKI therapy in CML, although the mechanisms which confer LSC resistance to TKI-mediated eradication remain poorly understood. Our laboratory has previously described the role of PTN, a paracrine factor which is secreted by bone marrow endothelial cells (BM ECs) and is necessary for the self-renewal and regeneration of normal hematopoietic stem cells (HSCs) in vivo. We hypothesized that PTN signaling in the BM vascular niche might also be important in CML pathogenesis and perpetuation of LSCs. In order to test our hypothesis, we crossed SCL-tTA/BCR-ABL mice, which develop CML over time under the control of doxycycline-treatment, with either PTN-/- or PTN+/+ mice, and compared CML pathogenesis in vivo. SCLtTA/BCR-ABL;PTN+/+ mice displayed evolution of chronic phase CML (splenomegaly, increased WBCs, left-shifted myelopoiesis, decreased survival) within 90-120 days. In contrast, SCLtTA/BCR-ABL;PTN-/- mice displayed normal range WBCs, decreased splenomegaly and significantly increased survival over the same time period. At 300 days post-induction of BCR-ABL, 33% percent of SCLtTA/BCR-ABL;PTN-/- mice remain alive compared to only 7% of SCLtTA/BCR-ABL;PTN+/+ mice (p<0.0001). Importantly, treatment of human CML cells with a specific anti-PTN antibody (BD Biosciences) significantly decreased CML colony formation in methylcellulose (P=0.02), whereas normal human CD34+ cells were unaffected. This result suggested the novel possibility that human CML cells may produce PTN in an autocrine manner as a means to propagate disease. These studies suggest that genetic or pharmacologic antagonism of PTN signaling is a potent strategy to inhibit CML pathogenesis. Ongoing studies will determine if PTN-targeted therapy, with or without TKIs, can mediate the eradication of LSCs which are responsible for perpetuation of this disease.

Disclosures

Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.
Outcomes of Low-Molecular-Weight Heparintreatment for Venous Thromboembolism in Patients with Primary and Metastatic Brain Tumors
Authors: Chai-Adisaksopha Chatree , ALKindi Said Y. , Cheah Matthew , Iorio Alfonso , Crowther Mark A. , Linkins Lori Ann
Published: October 2019

Background: Venous thromboembolism (VTE) is one of the most common complications of patients with brain tumors. There is limited data available in the literature on VTE treatment in these patients compared to other cancer types. We evaluated the efficacy and safety of low-molecular weight heparin treatment for newly diagnosed VTE in patients with primary and metastatic brain tumours at a tertiary care centre.

Methods: We conducted a matched retrospective cohort study of patients with primary or metastatic brain cancer who were diagnosed with cancer-associated VTE. Cases were selected after completion of a retrospective chart review of consecutive patients who were diagnosed with cancer-associated VTE between January 2010 and January 2014 at the Juravinski Thrombosis Clinic, Hamilton, Ontario, Canada. Controls were age- and gender-matched patients with cancer-associated VTE from the same cohort, but without brain tumours. The primary outcome was first recurrent VTE and secondary outcomes were major bleeding and clinically relevant bleeding.

Results: A total of 364 patients with cancer-associated thrombosis were included (182 with primary or metastatic brain tumors and 182 controls). The median follow-up duration was 6.7 (inter quartile range 2.5-15.8) months. The incidence rate of recurrent VTE was 11.0 per 100 patient-year (95% confidence interval [CI]; 6.7-17.9) in patients with brain tumors and 13.5 per 100 patient-year (95% CI; 9.3-19.7) in controls, incidence rate ratio [IRR]; 0.8 (95% CI; 0.4-1.5, p-value=0.43). There was no significant difference in the rate of recurrent VTE in the two groups (log-rank p-value=0.26, Figure 1). The incidence of major bleeding was 8.9 per 100 (95% CI; 5.2-15.4) patient-year in patients with brain tumors versus 6.0 per 100 patient-year (95% CI; 3.4-10.9) in controls, IRR; 0.8 (95% CI; 0.4-1.5, p-value=0.51). There were no significant differences in the risk of major bleeding (Figure 2) and clinical relevant bleeding between the two groups, log-rank p-value 0.9 and 0.8, respectively. When compared to controls, the rate of major gastrointestinal bleeding was lower in patients with brain tumours (0.6% versus 6.0%, p-value=0.003) whereas the rate of intracranial bleeding was higher (4.4% versus 0%, p-value=0.004). Subgroup analysis revealed that the incidence of intracranial bleeding in patients with primary brain tumors was higher than those with metastatic brain tumors, but did not reach statistical significant (6.0% vs 3.5%, p=0.008).

Conclusions: Recurrent VTE, major bleeding and clinical relevant bleeding were not significantly different in patients with cancer-associated VTE in the setting of primary or metastatic brain tumours compared with controls. However, intracranial bleedings occurred more frequently in patients with brain tumours.

Disclosures

Linkins: Pfizer: Honoraria; Bayer: Honoraria, Research Funding.
Time until Transfusion Independence in Haplo-Cord Transplant Is Comparable to Matched Unrelated Stem Cell Transplant, a Single Institution Experience
Authors: Tsang Hamilton C. , Bachegowda Lohith S. , Goel Ruchika , Koshy Nebu V. , Gergis Usama , Hsu Yen-Michael S. , Mayer Sebastian A. , Shore Tsiporah B. , van Besien Koen , Cushing Melissa M. , Vasovic Ljiljana V.
Published: October 2019

Introduction: Stem cell transplant (SCT) is a curative therapy for several advanced hematologic malignancies. In the peri-transplant period, red blood cell (RBC) and platelet (PLT) transfusions are often necessary until engraftment. Time until transfusion independence (TI) is influenced mainly by time to engraftment. Haplo-cord (HC) transplant combines an umbilical cord blood unit graft with a CD34 selected adult haplo-identical graft for the purpose of establishing long term cord engraftment. No prior studies have compared TI between HLA matched related donors (MRD), unrelated donors (MUD) and HC allotransplant recipients.

Methods: Patient demographics, ASBMT disease risk classification, relapse and mortality data for Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) patients undergoing MRD, MUD, and HC SCT between 1/2012-1/2015 were included. Time until TI for RBC and PLT was defined as the day of the last transfusion with no transfusions in the following 30 days (d). The cohorts were followed for 180d post-transplant. Kaplan-Meier survival method was used to compare time until RBC and PLT transfusion independence between different transplant types and ASBMT risk categories. Data were censored on disease relapse or death. Log-rank test was used to compare the survival probabilities. Statistical analyses were performed using SAS 9.4.0 (SAS Institute, Cary, NC).

Results: A total of 194 AML/MDS patients received MRD (n=63, 32%), MUD (n=76, 39%), and HC (n=55, 28%) SCT. TI was achieved for RBC transfusion in 84%, 74%, and 67% of patients, and for PLT transfusion in 84%, 91%, and 85% of patients for MRD, MUD, and HC respectively. The time to attain RBC and PLT TI was not statistically different between HC and MUD transplants (log-rank p=0.16 and 0.09 respectively) (Fig. 1: A, C). Within the ASBMT high risk strata, no statistical differences in the time to attain RBC (log-rank p=0.15) and PLT (log-rank p=0.55) TI between any transplant types could be detected (Fig. 1: B, D).

Conclusion: Recipients of SCT often have substantial transfusion requirements. No statistically significant difference in time to TI was seen between HC and MUD recipients. No statistically significant differences in time to TI were seen between HC, MUD and MRD recipients in the ASBMT high risk strata. Time until transfusion independence in haplo-cord transplant is comparable to matched unrelated stem cell transplant.

Figure 1. Figure 1.

Disclosures

van Besien: Miltenyi Biotec: Research Funding.
Modular Domains within a Super Enhancer Determine Drug Resistance in Leukemia
Authors: Ohtani Yumi , Xu Shuquan , Petrovic Jelena , Xu Lanwei , Aster Jon C , Pear Warren S.
Published: October 2019

Notch plays roles in T cell development and is a common oncogenic driver in T cell acute lymphoblastic leukemia. Myc is a critical target of Notch in normal and malignant pre-T cells. We and others identified a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter (1, 2). Deletion of a 1 kb region surrounding this enhancer resulted in a block in T cell development and inhibited Notch induced T-ALL induction and maintenance (2). Nevertheless, this 1 kb region contains binding sites for multiple transcription factors; thus, the requirement for specific transcription factors has not been resolved. To determine the precise sequences required for function of the distal enhancer, we used CRISPR/CAS9 to specifically mutate transcription factor binding sites in the Notch-dependent Myc enhancer (NDME). Specific mutation of the RBPj binding site blocked Notch/RBPj binding and inhibited Myc expression and T-ALL cell growth/survival. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Specifically mutating the single RBPj binding site abrogated H3K27Ac across this entire 600 kb region. Thus, Notch binding at a single site in the NDME is required for enhancer function and H3K27Ac across a very broad region. Furthermore, the Notch-regulated distal enhancer is comprised of individual enhancers that are uniquely sensitive to Notch or BET domain inhibitors. By selecting tumor cells resistant to each inhibitor, we show that the change in drug sensitivity results from selective use of these individual enhancers that are located within the broad Notch regulated region. These data not only provide insights into enhancer function but suggest strategies to treat T-ALL, including drug resistance, by therapeutically targeting these enhancers.

References:

1. Ohtani et al., Proc Natl Acad Sci USA 111:E4946, 2014, PMID: 25369933

2. Herranz et al., Nature Med, 20:1130, 2014, PMID: 25194570

Disclosures

Pear: Rockefeller University: Patents & Royalties; Pfizer: Equity Ownership; Amgen: Equity Ownership.
Identification of Activity Enhancement Residue of Human FVIII and Novel Factor VIII Variant for Human Gene Therapy
Authors: Firrman Jenni A. , Wang Qizhao , Xiao Weidong
Published: October 2019

Gene therapy for Hemophilia A using the recombinant Adeno-associated virus (rAAV) offers an alternative to FVIII protein infusions; However, due to limitations associated with rAAV and the FVIII protein itself, the end result is transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances human FVIII coagulation activity. Through the process of engineering, evaluation, positive and negative selection of kLC, key amino acids in canine factor VIII were identified. These amino acids were incorporated into human factor VIII and a new version of human factor VIII, hFVIII.JF12, was engineered.

The hFVIII.JF12 is a human FVIII B-domain deleted construct containing 12 amino acid changes in the light chain that work together to enhance coagulation activity. In vitro, hFVIII.JF12 resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. CD4KO/HA mice injected with rAAV vector carrying the hFVIII.JF12 gene produced an average 4.6 fold increase in clotting activity compared to those injected with hFVIIIBDD. An ELISA revealed no significant difference in protein production between these two groups of injected mice. In order to determine the mechanism of enhancement, the hFVIIIBDD and hFVIII.JF12 proteins were purified and functional properties analyzed. Results demonstrated that the hFVIII.JF12 protein produced a specific activity of 39,153.69 Units/m. This is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hFVIII.JF12 protein generated a higher amount of FXa at a quicker rate.

The hFVIII.JF12 construct is novel because it enhances FVIII activity both in vitro and in vivo through modifications to the light chain based on the kLC. This will be beneficial in the context of both gene and protein therapy because the protein is more specifically active. This research is also innovative because it demonstrates a novel method of enhancing transgene expression of FVIII delivered by an AAV vector through modifications to the gene itself.

Disclosures

No relevant conflicts of interest to declare.
Heat Shock Factor 1 Promotes NF-Kb and B-Cell Signaling in a Preclinical Model of Chronic Lymphocytic Leukemia
Authors: Rao Rekha , Home Trisha , Yacoub Abdulraheem , Shi Huidong , Saluja Ashok , McGuirk Joseph , Ganguly Siddhartha
Published: October 2019

Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. In this study we determined the mechanisms by which HSF1 promotes HSP90 function and CLL pathogenesis using CLL as model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. Mechanistically, we demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). RNA-sequencing of control and HSF1-knockdown CLL cells revealed that HSF1 regulates the transcription of upstream modulators of the NF-kB pathway including MYD88 and TLR1. Consequently, treatment with triptolide or knockdown of HSF1 inhibits NF-kB signaling in CLL cells. In an in vivo model of CLL, tail vein injection of luciferase-expressing Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by daily intraperitoneal injection of minnelide (a pro-drug of triptolide) for 28 days reduced in vivo disease burden and conferred significant survival advantage (p<0.0003) to treated mice compared to vehicle controls. Minnelide treatment also attenuated NF-kB and BTK signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 in CLL and test the activity of minnelide against human CLL.

Disclosures

Saluja: Minneamrita Therapeutics: Equity Ownership.
The Clinical Significance of Argonaute 2 Expression and Angiogenesis in Multiple Myeloma
Authors: Zhang Qiguo , Wu Hongyan , Ouyang Jian , Chen Bing , Dong Haibo , Zhou Rong-Fu , Zeng Hui , Zhou Min
Published: October 2019

Objective To assess the argonaute 2 (Ago2) expression in the bone marrow of multiple myeloma (MM) and determine its association with angiogenesis.

Methods Fifty-nine MM patients and sixteen normal controls were included. The bone marrow sections were made from plastic (methyl-methacrylate)- embedded bone marrow biopsies. The expression of Ago2 and CD34 in the bone marrow sections was detected by EnVison immunohistochemistry and the microvessel density (MVD) was then assessed. The bone marrow Ago2 expression level was also compared by western blot. The clinical significance of Ago2 expression and MVD in MM was analyzed.

Results Western blot analysis indicated that the Ago2 expression was significantly increased in the bone marrow of MM patients as compared with normal controls. As assessed by immunohistochemistry, fifty MM patients were positive for Ago2 and nine patients were negative. However, sixteen normal controls were all negative for Ago2 (χ2=42.586,P<0.001). The β2-microglobulin levels were significantly higher in the Ago2 positive group as compared with the Ago2 negative group (Z=-2.014 , P=0.042). The MVD was significantly higher in bone marrow samples of MM patients compared to the normal controls (7.89±4.88 vs. 2.16±1.32, Z=-3.283 , P<0.001). Analysis of the correlation coefficients showed that Ago2 expression was positively associated with MVD in MM patients (r=0.461, P=0.023). MVD was also significantly higher in the Ago2 positive group than in the Ago2 negative group (Z=-2.449,P=0.014).

Conclusion Ago2 expression was found to be significantly increased in the bone marrow of MM patients. Ago2 may take part in the pathogenesis of MM and mediate the angiogenesis.

Disclosures

No relevant conflicts of interest to declare.
The Impact of Genetic Abnormalities (GA) and Other Factors on Survival in Patients after 65 Years with Multiple Myeloma (MM)
Authors: Garifullin Andrei , Martynkevich Irina , Voloshin Sergei , Kuvshinov Alexei , Martynenko Ludmila , Kleina Elizaveta , Salogub Galina , Karyagina Elena , Schmidt Alexander , Abdulkadyrov Kudrat
Published: October 2019

Background. The increase of life expectancy in patients after 65 years with MM is the main aim of treatment. Lack of carrying out aggressive anti-multiple myeloma therapy increase influence of different factors on OS.

Aims. To compare influence GA and other different factors on overall survival in 65 and more years old patients with MM.

Methods. We retrospectively analyzed 40 patients 65 and more years (median age 71 years, range 65-86; male/female - 1:1.35). The incidences of genetic abnormalities were determined in all cases. Cytogenetic analysis was performed on bone marrow samples using standard GTG-method. Metaphase FISH analysis was performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1). Stratification of patients was carried in groups of risk according to the modified molecular classification mSMARTmod 1.0 and mSMARTmod 2.0. Patients with 2 and more chromosomal aberrations were additionally entered in high-risk group of both systems.

Results. GA in multiple myeloma after 65 years old patients were detected in 22.8% (9/40). The occurrence frequency of t(11;14) was 26.0% (6/23), del(13q) - 20.8% (5/24), t(4;14) - 4.3% (n=1/23), del(17p) - 0% (n=0/11). 33/40 (82.5%) patients entered into standard risk group, 4/40 (10%) - into intermediate risk, 3/40 (7,5%) - into high-risk. Median OS (MOS) according to mSMARTmod 1.0 in standard risk group (33/40) was 78 months, in high-risk (7/40) - 54 months. Median OS according to mSMARTmod 2.0 in standard risk group (33/40) was 78 months, in intermediate-risk (4/40) - 56 months, in high-risk (3/40) - 49 months. In patients groups without renal failure (35/40) MOS was 78 vs. 46 months with renal failure (5/40). MOS isn't reached in patients with ISS I (4/27), but MOS in patients with ISS II (13/27) and ISS III (10/27) were 50 and 54 months, respectively. MOS in patients group (10/40), who have both (bortezomib and lenolidomide) anti-myeloma agents was 110 months vs. 57 months in group (30/40) only with bortezomib-based regimen of treatment.

Conclusions. Many factors influence on OS in 65 and more years old patients with MM. However, patients, who had treatment with bortezomib and lenelidomide had the best results of OS.

Disclosures

No relevant conflicts of interest to declare.
Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease
Authors: Flodr Patrik , Latalova Pavla , Pusciznova Petra , Pika Tomas , Bacovsky Jaroslav , Scudla Vlastimil , Minarik Jiri
Published: October 2019

Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens.

Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2.

Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%).

Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A).

Supported by the grant NT 14393.

Disclosures

No relevant conflicts of interest to declare.
Distinct Clinico-Pathological Profile of Plasma Cell Leukemia Patients from North India
Authors: Sharma Praveen , Bommannan Karthik B.K , Sachdeva Man Updesh Singh , Varma Neelam , Srinivasan Radhika , Malhotra Pankaj , Varma Subhash
Published: October 2019

Introduction: Plasma cell leukemia (PCL) is a rare and aggressive plasma cell neoplasm where clonal plasma cells comprise ≥ 20% of peripheral blood (PB) leukocytes and/or absolute clonal PB plasma cell count is ≥ 2×109/L. Primary plasma cell leukemia (PPCL) originates de novo, whereas, secondary plasma cell leukemia (SPCL) evolves from pre-existing multiple myeloma (MM). Literature does reveal a few large case series on PCL, however, data on this rare neoplasm is very sparse from Indian sub-continent. We present clinico-pathological profile 14 cases of PCL from single tertiary care centre of north-India.

Materials and methods: Archival files were screened for period of 8 years (Jan 2007 to Dec 2014) and clinico-pathological profile, including overall survival, of patients of PCL was systematically analysed, retrospectively.

Results: Ten PPCL and four SPCL patients were traced in duration of 8 years, all belonging to north-Indian states of Punjab and Haryana. Eight PPCL and three SPCL patients had complete clinical data. Our patients had less frequent renal failure (12.5%), more frequent hepatomegaly (75%) and non-secretory type (33%) disease. Flow cytometric immunophenotyping revealed expression of CD138 (67%), CD56 (33%) and CD20 (0%), which was different from reported western data. With novel therapeutic agents, these patients fared a higher median survival of 15 months.

Conclusion: Our patients of PCL from neighbouring states of north-India have a distinct clinico-pathological profile, with better survival as compared to existing literature. The significance of our findings must be tested in a larger patient cohort and must be supported with molecular and cytogenetic investigations to unmask any significant pathogenesis.

Disclosures

No relevant conflicts of interest to declare.
SNP rs34172460 (S258A)Â in Gene CIP2A Correlates with Bortezomib Resistance in Multiple Myeloma Patients
Authors: Tang George , Li Mingjie , Sanchez Eric , Wang Cathy , Vardanyan Suzie , Lee Tiffany , Nosrati Jason , Cao Jasmin , Zahab Michael , Mehta Puja , Garzio Gigi , Chen Haiming , Berenson James
Published: October 2019

Introduction: The proteasome inhibitor (PI) bortezomib is a novel anticancer drug that shows activity especially for treating patients with multiple myeloma (MM). However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the genetic and molecular basis of which remains elusive. We propose that the resistance to bortezomib treatment may be related to a polymorphism in a gene known to be a target for bortezomib's activity which has been identified as CIP2A which is a tumor suppressor PP2A inhibitor. We have used a high resolution melting curve (HRM) approach to screen several CIP2A SNPs containing significant genetic changes that may be different between bortezomib sensitive and resistant MM patients. In this study, we evaluated SNPs in this gene in healthy subjects and MM patients who were sensitive or resistant to bortezomib-based therapy.

Methods: A panel of DNA samples was purified from MM patients (N=73) and healthy subjects (N=95). The samples were divided into three groups: bortezomib sensitive MM patients (N=61), bortezomib resistant MM patients (N=12), and healthy subjects. A single nucleotide polymorphism (SNP) of the bortezomib target genes CIP2A was selected. The PCR prime pairs for each SNP marker were designed with the software "Primer Expression 3" (Thermo Fisher Scientific). The genotype of each DNA sample was determined with the high resolution melt curve (HRM) technology on a real-time PCR instrument, StepOne Plus (Thermo Fisher Scientific).

Results: The frequency of heterozygosity and homozygosity of CIP2A, SNP rs34172460(S258A) was 0.049 (3 out of 61 cases) in the bortezomib sensitive group, while it was 0.25 (3 out of 12 cases) in the bortezomib resistant group, a 5-fold increase in the frequency of this SNP. The frequency of heterozygosity and homozygosity of this same SNP in the healthy subjects was only 0.03 (3 out of 95 samples).The minor allele frequency (MAF) in the normal population is 0.046, according to the SNP data base, compatible with what we observed in both our healthy subjects and the bortezomib sensitive MM patients.

Conclusion: This preliminary data suggests that heterozygotic and homozygotic mutants of rs34172460(S258A) may have an impact on resistance to bortezomib among patients with multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.