bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic Reprogramming of Cis-Regulatory Sites By R882-Mutated DNMT3A Potentiates Aberrant Stem Cell Gene Program and Acute Leukemia Development
Authors: Lu Rui , Wang Ping , Parton Trevor , Zheng Deyou , Wang Gang Greg
Published: October 2019

DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we establish ex vivo and in vivo leukemogenic models recapitulating synergy between a DNMT3A 'hotspot' mutation (i.e., DNMT3AR882H) and kinase activation, which include a leukemia-initiating stem cell (LSC) model. We show that DNMT3AR882H cooperates with constitutively activated RAS (NRAS G12D) in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. Genome-wide transcriptome profiling analysis of these models reveals that DNMT3AR882H potentiates aberrant transactivation of 'stemness' gene expression programs, notably a set of transcription factors Meis1, Hox-A, Mn1 and Mycn. Further examination by ChIP-Seq and enhanced Reduced Representation Bisulfite Sequencing (eRRBS) demonstrate that R882-mutated DNMT3A enzymes directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3AR882H-induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Lastly, with these established leukemogenic model systems, we also demonstrate profound differences between R882-mutated and wild-type DNMT3A in mediating oncogenic transformation, epigenetic dysregulation and gene mis-regulation. Collectively, these findings not only mechanistically explain clonal and malignant hematopoiesis found associated with DNMT3A mutation but also establish targeting transcription elongation machineries as novel therapeutic avenues for DNMT3A-mutated hematological malignancies.

Disclosures

No relevant conflicts of interest to declare.
Pharmacokinetics and Biodistribution of Locked Nucleic Acid (LNA)-Mir-221 Inhibitor: A New Promising Anti-Myeloma Agent
Authors: Gallo Cantafio Maria Eugenia , Gulla Annamaria , Amodio Nicola , Leone Emanuela , Morelli Eugenio , Biamonte Lavinia , Neri Antonino , Tagliaferri Pierosandro , Tassone Pierfrancesco , Di Martino Maria Teresa
Published: October 2019

miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression.

We proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys.

We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice.

Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion.

In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials.

Disclosures

No relevant conflicts of interest to declare.
TLR5 Agonist Recombinant Flagellin, CBLB502 Increases Anti-CMV Immunity By Increasing T-Bet and Eomes Expressing T Cells after MCMV Infection
Authors: Hossain Mohammad Sohrab , Al-Kadhimi Zaid , Waller Edmund K
Published: October 2019

We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p<0.001) and both CD4+ and CD8+ T cells increased significantly on day 3 (CD4, p<0.001; CD8, p<0.01) after MCMV infection. CBLB502 treatment had no effect on the ROR gammaT expression on T cells. Interestingly, although EOMES expressing CD4+ and CD8+ T cells increased significantly on day 0 (48 hrs after CBLB502 treatment) (CD4, p<0.01; CD8, p<0.01) and day 3 (CD4, p<0.001; CD8, p<0.001) in the spleens of CBLB502-treated mice compared with the PBS-treated mice, EOMES expression on CMV-specific tetramer+ CD8+ T cells rapidly decreased by day 10 after infection in both CBLB502 and PBS treated mice. However, >90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT.

Disclosures

No relevant conflicts of interest to declare.
Cytokine Gene Polymorphisms and Epstein-Barr Virus Infection after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Lin Ren , Fan Zhiping , Zhao Ke , Jiang Qianli , Sun Jing , Liu Qifa
Published: October 2019

Backgroud:

Epstein-Barr virus (EBV) infection/reactivation and associated diseases remain common complications in recipients of HSCT, leading to severe end-organ diseases and malignance. Single nucleotide polymorphism (SNP) in cytokine genes is considered to be related with EBV-associated post-transplant lymphoproliferative disorders (PTLD) in solid-organ transplantation recipients. In this study, we analyzed the association between cytokine gene polymorphisms and EBV infection/reactivation or diseases in recipients undergoing allo-HSCT.

Methods:

A total of 233 patients who received allo-HSCT between March 2012 to December 2014 were enrolled in this study. Ten SNPs, including IL-1β -511 rs16944, IL-1RN +11100 rs315952, IL-2 -330 rs2069762, IL-4 -590 rs2243250, IL-10 -592 rs1800872, IL-12 +1188 rs3212227, TNF-α -308 rs1800629, TGF-β1-509 rs1800469, TGF-β1 +869 rs1800470, IFN-γ +874 rs2430561, were tested. The SNPs genotypes in patients with or without EBV infection/reactivation and associated diseases were compared. Besides, the risk factors for EBV infection/reactivation were studied.

Results:

Seventy-four patients developed EBV infection/reactivation. The patients with EBV infection/reactivation had higher frequencies of donor IL-1β -511 TT genotype, donor IL-4 -590 TT genotype and recipient TNF-α -308 GG genotype than those without (p=0.021, p=0.004, p=0.020, respectively) while the frequencies of donor IL-1β -511 CC genotype, donor IL-1RN +11100 TT genotype, donor IL-2 -330 TT genotype, donor IL-4 -590 CC genotype and recipient TNF-α-308 GA genotype in patients with EBV infection/reactivation were lower than those without (p=0.041, p=0.029, p=0.005, p=0.011, p=0.042, respectively). Multivariate analysis showed donor IL-4 -590TT genotype (p=0.016, HR=1.907, 95% CI =1.130-3.218) and recipient TNF-α -308GG genotype (p=0.002, HR=3.550, 95% CI=1.613-7.812) were risk factors for post-transplant EBV infection/reactivation while donor IL-1RN +11100TT genotype (p=0.001, HR=0.382, 95% CI=0.218-0.670) was protective factors for post-transplant EBV infection/reactivation. Twenty-one patients developed EBV-associated diseases. The patients who developed EBV-associated diseases had higher frequency of donor IFN-γ +874 AT genotype than those did not (p=0.027). On the contrary, the frequencies of donor IL-1β-511 CC genotype, donor IL-10 -592 AA genotype, donor IL-12 +1188 AA genotype and donor IFN-γ +874 AA genotype in patients with EBV-associated diseases were lower than those without (p=0.019, p=0.018, p=0.018, p=0.010, respectively).

Conclusion:

Several SNPs in cytokine genes might be associated with EBV infection/reactivation and the development of EBV-associated diseases in recipients of allo-HSCT. However, these association should be studied further.

Disclosures

No relevant conflicts of interest to declare.
ASN002: A Potent Dual SYK/JAK Inhibitor Currently in a Phase I/II Study Shows Strong Antitumor Activity in Preclinical Studies
Authors: Rao Niranjan S. , Reddy Sanjeeva , Sandeep Gupta , Damle Nitin K , Aranapakam Venkatesan M , Thompson Scott , Smith Roger , Plasse Terry , Bristow Penelope , Tolcher Anthony , Papadopoulos Kyriakos P , Patnaik Amita , Rasco Drew W
Published: October 2019

ASN002 is an orally bioavailable potent dual inhibitor of spleen tyrosine kinase (SYK) and Janus kinases (JAK) with low nM IC50 values in biochemical assays. It is currently under clinical evaluation in a phase I/II study for safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy in diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL) and Follicular lymphoma (FCL). SYK and JAK are tyrosine kinases that play important roles in various types of lymphomas, solid tumors, myeloproliferative and inflammation disorders. Inhibition of these targets has been shown to suppress tumor growth in various preclinical and clinical studies. ASN002 showed anti-proliferative activity in a broad panel of cell lines representing both solid and leukemia/lymphoma tumor types. ASN002 potently induced apoptosis in a DLBCL cell-line and showed a strong dose dependent tumor growth inhibition in multiple DLBCL xenograft models. ASN002 also showed strong anti-proliferative activity in an ibrutinib-resistant cell line. In mechanistic cell-based studies, ASN002 strongly suppressed IgE and IL-6 stimulated responses that are thought to be mediated by SYK and JAK, respectively. ASN002 exhibited good oral bioavailability and metabolic stability in animal models. ASN002 is being evaluated in patients with solid tumors and hematological malignancies (excluding those with extensive marrow involvement) in the dose escalation phase which employs a 3+3 open label design. Sequential doses of 10, 20, 30, 40 and 50 mg BID are planned. Patient accrual is continuing and no dose limiting toxicities were identified in the first cohort. Data on safety, tolerability, pharmacokinetics and pharmacodynamics will be presented.

Disclosures

Rao: Asana BioSciences: Employment, Equity Ownership. Reddy:Asana BioSciences, LLC: Employment, Equity Ownership. Sandeep:Asana BioSciences, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Thompson:Asana BioSciences, LLC: Employment, Equity Ownership. Smith:Asana BioSciences, LLC: Employment, Equity Ownership. Plasse:Asana BioSciences, LLC: Consultancy. Bristow:Asana BioSciences, LLC: Consultancy. Tolcher:AbbVie: Consultancy; ArQule: Consultancy; Bayer: Consultancy; BioMed Valley Discoveries: Research Funding; Janssen R&D: Consultancy; Asana BioSciences, LLC: Consultancy, Research Funding. Patnaik:Asana BioSciences, LLC: Research Funding. Rasco:Asana BioSciences, LLC: Research Funding; Celgene: Research Funding.
Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia: Single US Center Experience
Authors: Khoury H Jean , Heffner Leonard T , Arellano Martha , Jillella Anand P , Kota Vamsi K , Winton Elliott F.
Published: October 2019

Imatinib (IM) can be safely discontinued in patients with chronic myeloid leukemia (CML) with sustained complete molecular remission. Previous publications from France and Australia have shown that overall 40% maintain MMR or MR5 for up to 5 years after IM is stopped. We report single US center experience with tyrosine kinase inhibitor (TKI) discontinuation. Between 06/2010 and 7/2015, 22 patients with CML in chronic (CP, n=19), accelerated (AP, n=2) and lymphoid blast phase (LBP, n=1) discontinued IM (n=17), dasatinib (DAS, n=3) or bosutinib (BOS, n=2), and were monitored by qPCR for BCR-ABL1 monthly for the first 3 months, quarterly for the following 2 years and then bi-annually. TKI was restarted in case of confirmed loss of MMR on a repeat qPCR. Reason for TKI discontinuation was predominantly driven by patients' request and TKI intolerance. Median age was 66 (range, 21-84). The 3 who discontinued DAS had IM-resistant (loss of CCyR; n=1), IM-intolerant CP (n=1), or received DAS as first-line agent (n=1). BOS was discontinued for IM-intolerant CP (n=1), or while in CR2 in a patient with LBP that transformed from IM resistant CP and relapsed following chemotherapy (HCVAD). Median duration of TKI therapy pre-discontinuation for the entire cohort was 89 months (range, 26-106). Three patients are not evaluable due to short follow-up (TKI stopped between 5/2015 and 7/2015).

With a median follow-up of 40 months (range, 8-60), 7 (41%, 6 CP and 1 AP), all previously on IM lost MMR a median of 3 months (range, 3-24) after TKI was stopped and restarted IM. Loss of CHR occurred 13 months after loss of MMR in 1 patient who elected not to restart IM at the time MMR was lost, due to complications from cardiac transplant rejection. All 7 achieved MMR following restart of IM. Median duration of TKI therapy pre-discontinuation for these 7 patients was 60 months (range, 48-98). 12 patients (59%, 10 CP, 1 AP, 1 LBP) remain off TKI and have not lost MMR, 8 with continuously undetectable BCR-ABL1; and 4 had 1-2 transient detectable BCR-ABL1 at MR4 levels. Median duration of TKI therapy pre-discontinuation for these 12 patients was 87 months (range, 26-106). Loss of MMR-free survival is depicted in the Figure. We conclude that, similar to previous reports, TKI can be safely discontinued in patients with CML without reappearance of BCR-ABL1 in 50-60%.

Figure 1. Figure 1.

Disclosures

Jillella: Seattle Genetics, Inc.: Research Funding. Kota:Leukemia Lymphoma Society: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.
Pharmacologic Modulation of STAT1 and Wnt Signaling in Chronic Lymphocytic Leukemia
Authors: Wu Christina , Lao Fitzgerald S , Choi Michael Y. , Carson Dennis A.
Published: October 2019

BACKGROUND: The STAT and Wnt signaling pathways play critical roles in early lymphocyte development and function, with considerable cross-talk between them. STAT1 has been reported to be constitutively phosphorylated at SER727, and often at TYR701, in CLL patients. STAT1 activation can regulate the function of the IRF8 transcription factor. Polymorphisms of the IRF8 gene have been associated with both familial and sporadic CLL, and can influence Wnt signaling. Hence, both STAT1 and Wnt are potential pharmacologic targets for CLL therapy, particularly in patients that are resistant to, or intolerant of BTK and PI3K inhibitors. We previously reported that the electrophilic drug dimethylfumarate (DMF, Tecfidera, Fumaderm) could inhibit Wnt signaling in CLL. Here we have investigated its effects on constitutive and inducible STAT1 signaling, and on IRF8 expression, in primary CLL cells.

METHODS: Primary leukemia cells from patients with CLL were cultured with DMF at pharmacologically relevant doses (3 uM to 30 uM) for 4 hours prior to analysis of STAT1 phosphorylation and IRF8 expression. We assessed the effect of DMF with and without pre-stimulation by lipopolysaccharide (LPS), Wnt3a, or a TLR7 agonist. To further evaluate the effect on CLL cells in a microenvironment that stimulates these pathways, we utilized a murine CLL xenograft model. We implanted primary leukemic cells from patients with CLL into the peritoneum of Rag2 deficient immune-compromised mice. Groups of mice were then treated with DMF at a dose of 10 mg/kg by oral gavage. CLL cells were retrieved 4 to 5 hours later by peritoneal lavage and analyzed.

RESULTS: We confirmed that both SER727 and TYR701 are phosphorylated in the majority of primary CLL cells from both VH mutated (high risk) and unmutated (low risk) clones, but not in normal peripheral blood mononuclear cells (PBMC). A 4 hours exposure of CLL cells to DMF inhibited TYR701 phosphorylation, but had no effect on SER727 phosphorylation nor on total STAT1 levels. We also confirmed that IRF8 levels are 6-8 fold elevated in CLL cells, compared to normal PBMC. IRF8 expression was inhibited by DMF treatment in 7 out of 11 CLL patients. Similar effects were observed in vivo. Wnt or LPS stimulation increased IRF8 levels in normal PBMC, but did not alter the already elevated IRF8 levels in CLL cells.

CONCLUSIONS: Activation of the STAT1/IRF8 pathway, like the Wnt pathway, is highly characteristic of CLL. DMF treatment of CLL cells can interrupt these signaling cascades, interfering with leukemia cell survival.

Disclosures

Choi: AbbVie: Consultancy, Other: Advisory Board, Research Funding; Gilead: Consultancy, Other: Advisory Board, Speakers Bureau.
Switching to Warfarin after 6-Month Completion of Anticoagulant Treatment for Cancer-Associated Thrombosis
Authors: Chai-Adisaksopha Chatree , Iorio Alfonso , Crowther Mark A. , de Miguel Javier , Salgado Estuardo , Zdraveska Marija , Monreal Manuel
Published: October 2019

Background: Low-molecular-weight heparin (LMWH) is considered to be an anticoagulation therapy for the treatment of cancer-associated thrombosis. The duration of treatment is recommended to maintain at least 3-6 months after the diagnosis. However, the data on continuing LMWH treatment beyond six months remains unclear.

Methods: Consecutive cancer-associated thrombosis patients who were enrolled in RIETE Registry were evaluated. We systematically selected the patients who completed treatment with LMWH for 6 months. The patients were divided into two groups whether they continued to receive LMWH or switched to warfarin. The main outcomes were recurrent venous thromboembolism (VTE), major bleeding and total bleeding. Survival curves were generated using Kaplan-Meier method and the curves were compared using the log-rank test. Hazard ratio (HR) with corresponding 95% confidence interval (CI) were calculated using Cox-proportional hazard (PH) model.

Outcomes: of the 1,502 eligible patients who completed 6-month anticoagulant therapy, 763 patients continued to received LMWH and 739 switched to warfarin. There was no significant difference in terms of recurrent VTE between two study groups (hazard ratio [HR] 0.67, 95% confidence interval [CI]; 0.44-1.02, p=0.06), Figure 1. The cumulative incidence of major bleeding was 2.6% in LMWH group and 2.7% in warfarin group (HR 1.05, 95%CI; 0.79-1.55, p=0.79), Figure 2. The cumulative incidence of total bleeding was 6.7% in LMWH group and 7.0% in warfarin group (HR 0.92, 95%CI; 0.62-1.37, p=0.70).

Conclusions: In patients with cancer-associated thrombosis who completed 6-month of anticoagulation therapy, switching to warfarin is not associated with increase in recurrent VTE, major bleeding or total bleeding when compared to continuing LMWH. Warfarin is an acceptable alternative anticoagulant in cancer-associated thrombosis patients who do not tolerate long-term treatment with LMWH.

Disclosures

Monreal: Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; boehringer: Consultancy, Membership on an entity's Board of Directors or advisory committees; daichii: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Pronostic Value of Pretransplant Echocardiography in Allogeneic Hematopoietic Cell Transplantation
Authors: Mauricio Sarmiento , Espigado Ildefonso , Parody Rocio , Marquez Francisco , Falantes Jose , Caballero Teresa , Calderon Cristina , Carmona Magdalena , Lopez Jose , Perez-Simon Jose A.
Published: October 2019

Several scores have been developed, such as the Sorror index, which allow to identify before transplant subgroups of patients with different risks of death after allogeneic transplantation (AloTH). One of the parameters included in all these scores and routinely used in the pretransplant evaluation include cardiac function studies. However, it has been heterogeneously defined mainly based according to the ejection fraction using different approaches such as MUGA and echocardiography. The aim of our study was to evaluate the prognosis impact of pretransplant echocardiographic findings.

We retrospectively analyzed a total of 276 medical records of patients undergoing AloTH between 2006 and 2014. Additional data regarding demographic parameters, underlying disease, donor type, conditioning, engraftment, acute and chronic graft versus host disease, overall survival and cause of death were also included into the analysis.

The mean ejection fraction was 61%, with 20 patients displaying an ejection fraction <50%. 19 patients (7%) had diastolic dysfunction, 5% valvular disease and 4% pericardial effusion. Of the 19 patients with diastolic dysfunction, 57% had been treated with anthracyclines: 7 patients (36%) were diagnosed with acute leukemia and 4 (21%)patients with lymphoma. In multivariate analysis we found that diastolic dysfunction was a predictor for survival (HR 5.43, 95% CI: 0,08-20,2, p = 0.028) and non-relapse mortality (HR = 5.7, 95% CI: 0,04-0,7, p = 0.002). Neither ejection fraction nor any other echocardiographic parameter predicted the risk of death.

In the present study pretransplant ejection fraction did not influence the prognosis of patients undergoing allogeneic transplantation; however, impaired diastolic function seems significantly influences post-transplant mortality. Therefore, in the pretransplant evaluation, diastolic function must be assessed in order to better define patient risks..

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

Falantes: Celgene: Honoraria.
Haploidentical Peripheral Blood Stem Cell Transplantation with Post-Transplantation Cyclophosphamide in Primary Refractory Acute Myeloid Leukemia
Authors: Jaiswal Sarita Rani , Chatterjee Sumita , Chakrabarti Aditi , Bhargava Sneh , Kunal Ray , Chakrabarti Suparno , O'Donnell Paul V.
Published: October 2019

In a pilot study, 75 patients with Primary Refractory (PRef) AML without matched family donors were offered post-transplantation cyclophosphamide (PTCY) based haploidentical peripheral blood stem cell (PBSC) transplantation. Twenty-seven patients (36%) opted for haploidentical transplantation with or without further chemotherapy. There was no difference in the patient or disease characteristics amongst patients undergoing transplantation or not. The conditioning regimen comprised of FluCyMel (n=5), FluBuMel (n=17) and FluTreoTBI (n=5). MMF was tapered between days 14 and 21 posttransplant in the absence of GVHD and cyclosporine A was tapered between days 60 and 90. The progression free survival at a median follow-up of 25 months was 36.6% and 0% in the transplant and the non-transplant group (p=0.0001). Prompt engraftment was noted at a median of 14 days irrespective of disease status or conditioning regimens. Cumulative incidences of acute graft-versus-host disease (GVHD) and chronic GVHD were 26.6% and 8% respectively. The overall incidence of infections remained low, with CMV reactivation and invasive aspergillosis occurring in 9 and 2 patients respectively. CMV disease was diagnosed in 2 patients. Non-relapse mortality at 1 year was 16.7%. The incidence of disease progression was 54%. Factors positively impacting progression free survival were < 15% marrow blasts at transplant and a Natural Killer Cell Ligand Mismatch (NKLMM) donor. NKLMM, Haplotype or B scores had no impact on CMV infection or GVHD. However, Bx Haplotype was associated with lower NRM (5%, 95%CI-1-9) compared to 48.6% (95%CI 28.2-69.0) in AA Haplotype (p=0.01). Disease status did not impact the overall survival (p=0.11) in the HSCT cohort. In fact, NKLMM donors with B haplotype had the greatest impact on overall survival in both the HSCT cohort (71.4%, 95%CI 54.3-88.5%) compared to 20% (95%CI 8.8-31.2, p=0.01) in those without the same. Our data suggests PTCY based Haploidentical PBSC transplantation is feasible in patients with PRef AML and donor NKLMM might improve progression free survival, provided the conditioning protocol and the post-grafting therapy offer the optimum platform for alloreactivity of NK cells.

Disclosures

No relevant conflicts of interest to declare.
A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Erythrocytes Sickling in Human Erythroid Culture Model
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai, Wulin , Li Hongzhen , Kumkhaek Chutima , Hsieh Matthew , Rodgers Griffin P
Published: October 2019

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34+ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34+ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34+ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation.

Disclosures

No relevant conflicts of interest to declare.
Higher BTK-Dependent Cell Proliferation in Unmutated Chronic Lymphocytic Leukemia Confers Increased Sensitivity to Ibrutinib
Authors: Guo Ailin , Lu Pin , Galanina Natalie , Nabhan Chadi , Smith Sonali M. , Coleman Morton , Wang Yue Lynn
Published: October 2019

In chronic lymphocytic leukemia (CLL), mutation status of the immunoglobulin heavy chain variable region gene (IGHV) has prognostic significance, with the unmutated phenotype (UM-CLL) having worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK. is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was associated with greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, higher BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling.

Disclosures

Nabhan: Celgene Corporation: Honoraria, Research Funding. Smith:Celgene: Consultancy; Pharmacyclics: Consultancy.
Validation of the Nextseq 500 and Development of a High-Throughput NGS Pipeline for Identifying Clinically-Relevant Gene Variants in Multiple Myeloma Specimens
Authors: Zielinska Agnieszka K. , Leigh Kenton , Gomez Horacio , Van Laar Ryan K
Published: October 2019

As molecular profiling technologies have evolved, our understanding of multiple myeloma heterogeneity and the relative effectiveness of treatment options have increased dramatically. Most recently, next generation sequencing (NGS) studies have provided a new degree of molecular resolution into this disease, however it remains a challenge to translate these methodologies and insights from research tools to widely-available clinical assays which can be performed as part of routine patient care.

MyPRS® is a clinically and scientifically validated high-throughput gene-expression (Affymetrix) based assay available in all 50 US states. The CLIA and CAP-accredited laboratory workflow is able to isolate sufficient RNA from small amounts of fresh bone marrow aspirate, up to 72 hours post collection[1]. In order to expand the content of the MyPRS assay to include DNA-sequencing based variant, copy number and mutation detection, we have validated highly-scalable methods for isolating RNA and DNA separately and in parallel from individual patient specimens of varying quality and quantity.

Yet another challenge of translational NGS profiling is performing complex laboratory procedures, developed primarily for research use only, with the requisite reproducibility and accuracy required for submission to regulatory agencies and ultimately for clinical use. Coupled with the protocols we developed for isolation of both DNA and RNA from small amounts of patient bone marrow aspirate, we performed an investigation of the Illumina NextSeq 500 and "on-site" BaseSpace data processing server. With the multiplexing and parallel processing capability of this platform we estimate being able to sequence, align and variant-call up to 150 myeloma-relevant genes at 1000x coverage per run.

Results from our MM cell-line-, normal human- and NIST reference-DNA whole-exome-profiling studies show extremely high levels of technical reproducibility and agreement with results generated from 3rd party laboratories. In addition, we describe associations between the (RNA-expression based) prognostic, molecular subtyping and virtual karyotyping currently included in the MyPRS assay, with results from DNA-based exome profiling, performed on matched specimens. We believe findings underscore the need for comprehensive DNA and RNA based molecular profiling in order to make the most informed patient management decisions.

1. van Laar R, Flinchum R, Brown N, Ramsey J, Riccitelli S, Heuck C, Barlogie B, Shaughnessy Jr J: Translating a gene expression signature for multiple myeloma prognosis into a robust high-throughput assay for clinical use. BMC Medical Genomics 2014, 7 (1):25.

Disclosures

Zielinska: Signal Genetics: Employment. Leigh:Signal Genetics: Employment. Gomez:Signal Genetics: Employment. Van Laar:Signal Genetics: Employment.
Plasmocytosis and Immunoparesis but Not Neutrophil to Lymphocyte Ratio Can Predict Time to Treatment in Smoldering Myeloma
Authors: Romano Alessandra , Consoli Marzia L , Parisi Marina , Cavalli Maide , Parrinello Nunziatina L , Conticello Concetta , Di Raimondo Francesco
Published: October 2019

Objectives: We recently identified the ratio between absolute neutrophils count and absolute lymphocyte count, NLR ≥2, as a predictor of progression free survival (PFS) and overall survival (OS) in patients younger than 65 years with symptomatic Multiple Myeloma (MM). We retrospectively examined the NLR in a cohort of 140 smoldering Myeloma (sMM) defined accordingly to the updated IMWG 2014 guidelines accessed our Center between June 2006 and June 2014.

Methods: NLR was calculated using data obtained from the complete blood count (CBC) at diagnosis and subsequently correlated with time to treatment (TTT) for symptomatic MM. All patients underwent bone marrow evaluation to estimate plasma cells infiltration (BMPC), Magnetic Resonance Imaging (MRI) to detect bone lesions, serum free-lite chain evaluation (sFLC) starting from January 2012 when they became evaluable in our center.

Results: The mean NLR was 2.0 ± 0.1, lower than the value previously found for MM (2.7 ± 0.2, p= 0.005). Higher NLR was independent of BMPC amount, cytogenetics and sFLC. Using NLR ≥2 as predictive biomarker we could not predict TTT.

In univariate analysis only BMPC ≥ 30% (p<0.0001), immunoparesis (suppression of one or more uninvolved immunoglobulins, p=0.017) and presence of at least one bone lesion at MRI (p<0.0001) could predict TTT. In multivariate analysis, these three parameters were independent (p<0.0001). Since new guidelines consider BMPC≥ 60% and positive MRI as myeloma-defining events, we proposed a simplified score model based on BMPC ≥ 30% (1 point) and immunoparesis (1 point).

We identified patients with score 0 (20%) as low-risk (TTT at 60 months 0%), with score 1 (61%) as intermediate risk (TTT at 60 months 61%) and with score 2 as high-risk (TTT at 60 months 22%, p<0.0001).

Conclusion: We confirmed BMPC and immunoparesis as strong predictors of outcome in sMM.

Disclosures

No relevant conflicts of interest to declare.
Different Levels of Alkaline Phospatase (ALP) in Multiple Myeloma (MM) and SOLID Tumors with BONE Lesions
Authors: Annibali Ombretta , Petrucci Maria Teresa , Santini Daniele , Lisi Elisabetta , Mariani MArco , Pisani Francesco , Bongarzoni Velia , Venditti Olga , Rago Angela , Cerchiara Elisabetta , Fiorini Alessia , Franceschini Luca , Felici Stefano , Russano MArco , PAntano Francesco , Avvisati Giuseppe
Published: October 2019

Background: alkaline phosphatase (ALP) is an enzyme localized in different tissues and its levels may increase in diseases with skeletal involvement.

Aim: To compare ALP levels in patients with Multiple Myeloma and osteolytic lesions, and in patients with solid tumors (breast, prostate, lung, stomach, kidney and colon) and osteolytic, osteoblastic and mixed (osteolytic and osteoblastic) bone metastatic lesions.

Patients and Methods: From 1991 we collected 400 patients with MM and 308 patients with solid cancer with bone involvement. We grouped patients according to the metastases type (osteolytic, osteoblastic mixed), number of metastasis (1, 2-3, more than 3), ISS and D&S Stage. Among patients with solid tumors 45% had lytic lesions, while 31,5% had osteoblastic lesions and 23,5% had mixed lesions. Comparing MM vs. bone metastasis from solid cancer respectively 64.5% vs. 59% had normal ALP values, while 29% vs. 28% had <2xUNL, and 6.5% vs. 13% had >2UNL values of ALP. Patients with MM had significantly lower ALP values when compared to patients with osteoblastic bone lesions (P<0.05). In particular, comparing ALP levels of MM patients with the levels observed in cancer patients with >3 osteoblastic lesions difference is even more significant (P<0.01). Moreover, patients with Breast and Prostate cancer had ALP values significantly higher than MM pts (P<0.05 and P<0.05 respectively).In the group of patients with solid tumors, ALP levels were significantly higher in those with >3 osteoblastic lesions than in those with only osteolytic lesions (P<0.05).

Conclusion: These preliminary results indicate that ALP should be part of the initial work up in pts with bone lesions. This simple and cheap test, if normal or reduced in presence of osteolytic bone lesions, suggests an initial complete protein study including serum and urine protein electrophoresis associated to Bone Marrow aspirate, in case of the presence of a paraprotein.

Disclosures

Petrucci: Celgene, Janssen-Cilag, Amgen, Mundipharma, BMS: Honoraria.
Hyper-Active Non-Homologous End Joining Selects for Synthetic Lethality Resistant and Pathological Hematopoietic Stem Cells
Authors: Du Wei , Amarachintha Surya , Andrew Wilson , Pang Qishen
Published: October 2019

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca-/- or Fancc-/- HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1-/-Parp1-/- but less effective in Fanca-/-Parp1-/- cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with "synthetic lethality" and reduces human engraftment. Our results suggest that hyper-active NHEJ may select for "synthetic lethality" resistant and pathological HSPCs.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell Mobilization Chemotherapy with Gemcitabine Is Effective and Safe in Myeloma Patients with Bortezomib Induced Neurotoxicity
Authors: Mueller Beatrice U. , Keller Sandra , Seipel Katja , Mansouri Taleghani Behrouz , Pabst Thomas
Published: October 2019

Background: Vinorelbine chemotherapy with G-CSF stimulation is a widely applied non-myelosuppressive mobilization regimen in Switzerland for myeloma patients, but its neurotoxic potential limits its use in patients with bortezomib induced polyneuropathy. Methods: In this single-center study, we alternatively evaluated safety and effectiveness of gemcitabine chemotherapy with G-CSF for mobilization of autologous stem cells. Results: Between 03/2012 and 02/2013, all bortezomib pretreated myeloma patients planned to undergo first-line high-dose melphalan chemotherapy received a single dose of 1250mg/m2 gemcitabine, with G-CSF started on day four. The 24 patients in this study have received a median of four cycles of bortezomib-dexamethason based induction. Bortezomib-caused polyneuropathy was identified in 21 patients (88%) by clinical evaluation and a standardized questionnaire. Administration of gemcitabine mobilization did not induce novel or aggravate preexisting neuropathy. Stem cell mobilization was successful in all 24 patients, with a single day of apheresis being sufficient in 19 patients (78%). The median yield was 9.51 x 106 CD34+ cells/kg. Stem collection could be accomplished at day 8 in 67%. Conclusion: Our data suggest that single-dose gemcitabine together with G-CSF is an effective mobilization regimen in myeloma patients and a safe alternative non-myelosuppressive mobilization chemotherapy for myeloma patients with bortezomib induced polyneuropathy.

Disclosures

No relevant conflicts of interest to declare.
Seroprevalence for Toxoplasma Gondii in Allogeneic Hematopoietic Stem Cell Transplantation Recipients
Authors: Atilla Erden , Ataca Pinar , Balli Sevinc , Isikoglu Baris , Topcuoglu Pervin , Ozcan Muhit , Akan Hamdi
Published: October 2019

Introduction:

Toxoplasmosis is a rare but serious and life-threatening infection following Allogeneic Stem Cell Transplantation (Allo-HSCT). The main cause of toxoplasmosis is the reactivation of latent infection in pre-transplant seropositive patients. In our study, our aim is to present toxoplasma gondii seroprevalence in our center.

Patients and Methods:

A total of 251 allogeneic stem cell transplant recipient were evaluated retrospectively from 1998 to 2015. During Allo-HSCT preparation procedures all recepients were serologically tested. Donor serology records were not adequate. Toxoplasma gondii-specific IgG and IgM antibodies were determined by Chemiluminescence by ARCHITECT lab analyzers using commercially available kits (Abbott, USA). All patients were followed for reactivation.

Results:

Toxoplasma IgG positivity was detected in 51 of patients (20.3%). There was no statistically difference detected between related or unrelated transplants. The mean age of the group was 36 (range 14-71). 144 recipients were male (57%). 208 (83%) patients were transplanted for malign hematological diseases. The majority of patients were grafted from full-matched related donors (173, 69%). The most common source of stem cell was peripheral blood in 192 patients (77%) followed by bone-marrow in 51 patients (51%), bone-marrow plus peripheral blood in 7 patients (2.8%) and cord in 1 patient (0.4%). 192 (77%) patients received myeloablative conditioning regimens. All patients received prophylactic twice weekly trimethoprim/sulfamethoxazole (TMP/SMX) (160/800mg PO daily) after engraftment. Two patients had clinical symptoms of toxoplasmosis however they were seronegative in serology. None of the patients developed an active toxoplasmosis after Allo-HSCT.

Conclusion:

Since less than 30% seropositivity defined as low rate, our institution had low rate seropositivity for Toxoplasma Gondii in Allo-HSCT recipients. Routine use of TMP/SMX prophylaxis and difficulties in diagnosis of toxoplasmosis may lead to low frequency active toxoplasmosis.

Disclosures

No relevant conflicts of interest to declare.
Outpatient Hematopoietic Grafting in Patients with Multiple Sclerosis Employing Autologous Non-Cryopreserved Peripheral Blood Stem Cells: A Feasibility Study
Authors: Ruiz-Arguelles Guillermo J. , León-Peña Andrés , Medina-Ceballos Emilio , Ruiz-Arguelles Alejandro , Ruiz-Delgado Manuel A , Ruiz-Delgado Rodrigo J , Ruiz-Reyes Guillermo , Priesca-Marin Manuel , Ruiz-Delgado Guillermo J
Published: October 2019

Background:

Multiple sclerosis (MS) is a chronic, inflammatory, debilitating disease that causes destruction of central nervous system (CNS) myelin, with varying degrees of axonal damage. With the goalofd re-setting the immune system, autologous hematopoietic stem cell transplantations (HSCT) have been done in patients with MS since 1996 and more than 700 HSCTs have been performed around the world. The risk of transplant related mortality in HSCT for MS has declined over the past years.

Material and methods:

Consecutive patients with MS were autografted in a single center using: Hematopoietic stem cells (HSC) were mobilized with cyclophosphamide (Cy), 3 gr/m2 and G-CSF, the procedure was conducted on outpatient basis employing peripheral blood non-frozen HSC and conditioning with high-dose Cy (100 mg/Kg) and post-transplant G-CSF and rituximab. Antibiotics, antimycotics and antivirals were given orally.

Results:

Thirteen patients with MS were prospectively accrued in the study. There were 7 females and 6 males. Median age was 48 years, range 24 to 65. The expanded disability status scale (EDSS) score of these patients had a median of 5 points (range 1 to 6). All the autografts were started on an outpatient basis and two persons were admitted to the hospital during the procedure (persistent nausea/vomiting and neutropenic fever); they stayed in the hospital for 48 hours. In order to obtain a minimum of 1 x106 viable CD34+ cells/Kg, one to four apheresis were done (median 1). The total number of viable CD34+ cells infused to the patients ranged between 1 and 9.6x106 (median 3.1). Patients recovered above 0.5 x109/L absolute granulocytes on median day 9 (range 6 to 12). No individuals needed transfusions of red blood cells nor platelets transfusions. There were no transplant-related deaths and the 23-month overall survival of the autografted patients is 100%. Median cost of the procedure was 30 000 USD. In 8 persons the EDSS was assessed three months after the graft; it diminished from a median of 4.5 to a median of 2.5. In 5 patients, the three months re-assessment of the EDSS has not been possible as a result of the time elapsed after the autograft.

Discussion:

These data indicate that it is possible to conduct autotrasplants for patients with MS employing a simplification of the conventional procedures by means of non-frozen peripheral blood stem cells and outpatient conduction. Additional information is needed to asses the efficacy of these procedures in the treatment of patients with MS.

Disclosures

No relevant conflicts of interest to declare.