bluuebunny/crossref_metadata_2025 huggingface dataset:
The Atypical Gtpase Rhou Lies Downstream IL6/STAT3 and Regulates Myeloma Plasma Cells Adhesion/Motility
Authors: Nunes Canovas Sara , Carrino Marilena , Manni Sabrina , Quotti Tubi Laura , Manzoni Martina , Barilà Gregorio , Trentin Livio , Zambello Renato , Neri Antonino , Semenzato Gianpietro , Piazza Francesco
Published: November 2019

RhoU is an atypical G protein of the Rho GTPase family endowed with a very high intrinsic guanine nucleotide exchange activity ensuring a prevalent GTP-loaded conformation. It is present at very low levels in most cells, however some reports have shown that the Wnt-1 and STAT3 pathways might regulate its expression which resulted highly increased in malignant tumors. Since the IL6R/STAT3 signaling is of critical in Multiple Myeloma (MM) growth, homing and adhesion to marrow stroma and RhoU GTPase may mediate the induction of filopodia formation and stress fiber dissolution, we aimed at studying the role of this protein in MM malignancy.

mRNA expression studies unraveled that MM patients have heterogeneous levels of RhoU ranging from -0.7 fold change to 18.5 fold change when normalized over controls. By analyzing available gene expression data sets, we found that RhoU expression changes were associated with distinctive expression of 557 genes. We also found a correlation between RhoU levels and genetic abnormalities that associate with a poor prognosis in MM patients, namely the t(4;14), del(13) and 1q gain. RhoU was positively modulated by IL-6 and by soluble factors from bone marrow stromal cells' culture. Its expression was down-modulated after STAT3 inhibition with the chemical inhibitor Stattic, even in the presence of IL-6. The treatment with Stattic also decreased MM cells' migration capability. Remarkably, RhoU knock down by siRNA caused changes in MM cells' phenotype and deranged adhesion/migration functional properties. Lastly we have looked into changes in RhoU expression after the treatment of MM cells with Lenalidomide, known to act on MM microenvironment and alter MM cells' adhesion to protective bone marrow niches. Surprisingly, treatment of MM cell lines or from patients with Lenalidomide led to an upregulation of RhoU expression in the cells before onset of apoptosis after 72 hours.

Our data point to a functional role in cell adhesion, migration and shape for RhoU GTPase in a subset of MM, in particular in cases with genetic abnormalities correlating with a poor prognosis. RhoU is downstream from STAT3/IL-6 and likely from growth factor delivered by bone marrow stromal cells. The upregulation of RhoU expression after treatment with Lenalidomide was associated to a stall of the cellular cytoskeleton and subsequent apoptosis. Thus RhoU might be important in MM pathogenesis and could become a suitable target to disrupt the MM plasma cell interaction with protective bone marrow niches.

Disclosures

No relevant conflicts of interest to declare.
Rigosertib, a Pan RAS Inhibitor, Decreases Mouse and Human Myeloma Cell Growth in Preclinical Models
Authors: Zangari Maurizio , Shin Ikjae , Branca Antonio , Yoo Hanna , Fallo Sophia , Davies Faith E , Hoffman Benjamin , Fruchtman Steven M. , Yoon Donghoon
Published: November 2019

Introduction

Multiple myeloma (MM) is a B cell cancer mainly characterized by proliferation of malignant plasma cells in the bone marrow, presence of a monoclonal serum immunoglobulin, and occurrence of osteolytic lesions. RAS genes encode a family of small GTPase proteins ubiquitously expressed in all cell lineages and organs that transmit signals for cell growth, differentiation, and survival. Mutations in RAS genes often lead to constitutively activated RAS signaling, resulting in cancer. Various RAS mutations have been described in myeloma patients. RAS mutations are rarely found in pre-malignant stages as MGUS (monoclonal gammopathy of undetermined significance). These findings suggest that RAS mutations may play an important role in malignant transformation of MM.

Rigosertib (ON 01910.Na) is an inhibitor of RAS-related signaling pathways associated with cancer cell division, growth, and survival. Rigosertib binds directly to the RAS-binding domain in RAS effector proteins resulting in disruption of RAS/effector interactions and inhibition of multiple RAS-driven signaling pathways. Clinical trials with rigosertib have been conducted in MDS and solid tumors, but the effect of rigosertib in MM has not yet been fully characterized.

Methods

Various mouse/human MM cell lines (5TGM1, KMS28, RPMI8226, JJN3, OPM2) were tested for their growth with treatment of rigosertib at different concentrations (1, 10, 50, 100, 500 nM) for 4 days.

For a mouse MM model study, 0.5e6 Luciferase infected 5TGM1 cells were injected via tail vein into C57BL6/KaLwRij mouse. Rigosertib (100 mg/kg QD) was administrated via intraperitoneal injection from 14 days post-transplantation. The MM growths were weekly monitored by in vivo IVIS imaging after luciferin (Luciferase substrate) injection.

Results

All cells (5TGM1, KMS28, RPMI8226, JJN3, OPM2) tested showed growth inhibition with rigosertib in a dose-dependent manner. MM progression assessed by in vivo imaging was found to be significantly suppressed by rigosertib treatment as shown in Figure. Furthermore, a survival analysis demonstrated a positive trend towards increased life expectancy with rigosertib treatment (Median survival; control (35 days) vs treated (44.5 days)).

Conclusion

Our results demonstrate that rigosertib suppresses MM growth in both human/mouse cell lines and a mouse MM model and suggest additional exploration of rigosertib alone or in combination with other MM treatments.

Figure 1 Figure 1.

Disclosures

Davies: Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Hoffman:Onconova: Employment. Fruchtman:Onconova: Employment.
Repurposing Leflunomide for Multiple Myeloma Treatment
Authors: Buettner Ralf , Morales Corey , Perret Natalie , Palmer Joycelynne , Krishnan Amrita , Rosenzweig Michael , Rosen Steven T.
Published: November 2019

Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients' (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function.

Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM.

Disclosures

No relevant conflicts of interest to declare.
CD38 Deregulation As Strategy to Make Multiple Myeloma Cells More Sensitive to NAD+ Depleting Agents
Authors: Cea Michele , Cagnetta Antonia , Soncini Debora , Minetto Paola , Bergamaschi Micaela , Rivoli Giulia , Canepa Letizia , Gobbi Marco , Lemoli Roberto M
Published: November 2019

Background: The upregulated NAD+ biosynthesis, which is needed to face increased proliferation and metabolic processes, represents an important feature distinguishing cancer cells from their normal counterparts. As a result, the NAD+ biosynthetic apparatus emerges as highly promising therapeutic target for tumors, as suggested by the use of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors in a number of malignancies, including Multiple Myeloma (MM) and leukemia (Cea et al. Blood 2012; Cagnetta et al. Blood 2013; Cagnetta et al. CCR 2015). The ecto-enzyme CD38 is emerging as a novel therapeutic target for patients with hematological malignancies, including MM, with several monoclonal antibodies already tested in clinical trials with promising results. Importantly, CD38 by regulating intracellular NAD+ stores acts as member of the intricate network supporting metabolic reprogramming associated with cancer. Based on these assumptions, here we explored CD38 activity as innovative strategy to enhance the anti-tumor activity of NAMPT inhibition in Multiple Myeloma cells.

Methods: A panel of different MM cell lines and primary cells, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The effects of Nampt inhibition was evaluated in presence of CD38-gene editing (loss/gain of approaches) by using CTG assay and Annexin-V/propidium iodide staining. Next, the anti-MM effects of chemicals affecting CD38 activity were also evaluated in combination with low doses of Nampt inhibitors. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and enzymatic assays.

Results and Conclusions: By using different approaches, we found that CD38 deregulation makes MM cells more vulnerable to NAD+ depleting agents. Ongoing mechanistic studies suggest the central role played by energetic metabolism in the observed synergism. Overall our data provide the mechanistic preclinical rationale to enhance anti-MM activity of Nampt targeting agents, in order to both overcome drug resistance and improve patients outcome.

Disclosures

Gobbi: Mundipharma: Consultancy, Research Funding; Takeda: Consultancy; Roche: Honoraria; Celgene: Consultancy; Gilead: Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria.
Safety and Eficacy of Low Dose- Bortezomib Plus Melphalan/Prednisone (velcadito protocol) in Elderly Patintes Not Eligible to Transplant in Newly Dignosed Multiple Myeloma. Restrospective Study of 26 Patients
Authors: Gusmão Breno Moreno , Blumenschein Danielle Isadora , Persona Ernesto Perez , Guinea De Castro Jose Maria , Lopes Iracema Esteves , Hamerschlak Nelson
Published: November 2019

Introduction: Protocols based on bortezomib have become the standard therapy in the elderly and not eligible for transplantation. The VISTA study showed OR 71%, CR 30%, regardless of adverse factors such as age, renal failure or cytogenetic changes, but the patient had a high rate neuropathy (44%). The Spanish group and the Italian group modified the dose of bortezomib achieving rates of CR 22% and 20%, reducing neurotoxicity 8% and 5% respectively.

We present the experience of 26 newly diagnosed patients treated with low-dose bortezomib associdados the melphalan / prednisone (Velcadito protocol).

Objective: The aim of this study is to assess the effectiveness and safety of low-dose bortezomib in elderly patients not eligible to transplant. We evaluated according to the criteria IMWG.

Results: We evaluated 26 patients treated at the Hospital Universitario de Alava (Spain) and Hospital Israelita Albert Einstein (Brazil) among 2004 to 2016. The patients received 1 cycle with bortezomib 1.3mg/m2 (days 1,4,8,11) associated with melphalan 9 mg/m2 (1- 4 days) and prednisone 60mg/m2 (1- 4 days) 28 days cycle. From the 2nd cycle the patient received low doses of bortezomib (1 mg / m2, days 1 and 4) no change in other drugs and treatment regimen.

The mean age was 78 years. 50% men/ 50% women. 2 patients had poor cytogenetics Pronóstico, 1 intermediate and 23 standard. According to the IMWG: ISS1 = 7 (26%) ISS 2 = 10 (38.5%), ISS3 = 6 (23%). 38,4% (n=10) of patientes had neuropathy but only 11,5%(n=3) grade 3-4. 61,5% (n=16) presented as neutropenia toxicity, 19,2% (n=5) with grade 3 (neutrophils <1000). 15,3% (n=4) had thrombocytopenia with grade greater than 3. Response rate was 100%, and if we consider at least PR was 92% (CR 38.5% VGPR 31%). The global survival had median of 80 months (95% confidence interval 57,8 - 102,1%) and the survival free of progression had median of 37 months (95% confidence interval 15 - 58,9%) with follow-up of 91 months.

Conclusion: The use of velcadito protocol is safe, very effective and seems to be a good option for elderly patients with newly diagnose MM. A pharmacoeconomic analysis is recommended to assess the impact of the cost of treatment. Being a prospective clinical trial where an analysis of this impact of all patients treated with subcutaneous bortezomib beyond the characteristics of this study.

Disclosures

No relevant conflicts of interest to declare.
Lenalidomide and Low Dose Dexamethasone in Previously Treated Multiple Myeloma Patients with Impaired Renal Function: Efficacy Analysis of PrE1003, a Precog Study
Authors: Mikhael Joseph , Manola Judi , Lonial Sagar , Weiss Brendan M , Oettel Kurt , Hayman Suzanne R. , Kanate Abraham S. , Rajkumar S. Vincent
Published: November 2019

Introduction: Lenalidomide (Len) and dexamethasone (dex) has proven to be a highly effective treatment for multiple myeloma (MM) and serves as the basis of other combinations in relapsed disease. However, nearly 1/3 of myeloma patients have renal insufficiency, and the optimal use of Len in this population is not well known. We undertook this study in 3 cohorts of pts: Group A had creatinine clearance (CrCl) of 30-60 ml/min, Group B with CrCl < 30 ml/min, and Group C, with CrCl < 30 ml/min and requiring dialysis. The Phase I component of the trial has been previously reported (ASCO 2014) demonstrating that full dose Len (25mg daily 21/28 days) can be given to all patients despite renal insufficiency, even when on dialysis. We now present data on response to treatment.

Methods: In the Phase 2 component accrual was slow and the trial was terminated. However, prior to termination a modified exploratory analysis plan was established, including all 34 patients treated at the recommended phase 2 dose. This plan would declare the treatment worthy of further study if 18 or more of the 34 patients responded to treatment. This design had 84% power to distinguish a response rate of 60% from a null rate of 40%, using a 1-sided test with 10% type I error.

Results: The Phase 2 efficacy cohort accrued 34 patients, consisting of 20 patients from Group A, 9 patients from Group B, and 5 patients from Group C. Response of PR or greater was seen in 17 patients, resulting in a 50% overall response rate (CI 37-63%). Treatment was well tolerated with expected adverse events; the most common adverse events were anemia, diarrhea, and fatigue. Eleven episodes of Grade 3 and one Grade 4 pneumonia were reported for 10 patients across all cohorts and dose levels; 3 were considered possibly related to treatment. Seventeen patients have remained on treatment for more than 12 cycles. In the exploratory analyses, median PFS was 7.5 months (95% CI, 3.6 - 19.7 months) and median OS was 19.7 months (95% CI, 10.4 - 42.5 months).

Conclusions: Len Dex can be safely administered to patients with impaired renal function at full dose of 25mg daily 21/28 days, and is associated with a 50% response rate. Further investigation into other combinations with Len Dex in patients with renal impairment should be considered.

Table Table.

Disclosures

Mikhael: Onyx: Research Funding; Sanofi: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Lonial:Merck: Consultancy; Novartis: Consultancy; Onyx: Consultancy; BMS: Consultancy; Onyx: Consultancy; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Janssen: Consultancy. Weiss:Novartis: Consultancy.
Evaluation of Various Strategies to Generate NPM1mut-Specific T Cell Clones
Authors: Ruecker-Braun Elke , Heidenreich Falk , Link Cornelia S , Schmiedgen Maria , Wehner Rebekka , Kühn Denise , Heidrich Katharina , Alakel Nael , Schmitz Marc , Thiede Christian , Ehninger Gerhard , Bornhäuser Martin , Schetelig Johannes
Published: November 2019

Mutated nucleophosmin (NPM1) was identified as a promising leukemia-specific antigen for cytotoxic T lymphocytes (CTL). NPM1 is a multifunctional nucleocytoplasmic shuttling phosphoprotein. In AML patients with normal cytogenetics NPM1 mutations are the most frequent molecular genetic abnormalities, accounting for up to 60% of the patients. The peptide (AIQDLCLAV) derived from the mutated NPM1 (NPM1mut) has been described to elicit a CTL response restricted to HLA-A*02:01. We observed that NPM1mut multimer+ T cells were very rare in peripheral blood. The limitation of the multimer technology is the absence of a positive control; nevertheless it is an attractive tool to generate antigen positive T cell clones.

The goal was to compare strategies for the generation of NPM1mut multimer+ T cell clones systematically. For this purpose we analyzed blood samples from two patients with AML after transplantation and six different healthy donors.

We explored different strategies to isolate HLA-A*02:01 restricted NPM1mut multimer+ T single cells. The first strategy was to isolate multimer+ T cells directly from the blood without any supplements by single cell sorting. The second strategy was to sort multimer+ T cells which were previously CD8+ enriched supplementing the media either with or without IL-21. Published by Yongqing et al.IL-21 enhances the generation of human antigen-specific CD8+ T cells. A further strategy was to previously enrich CD14+ cells for the generation of autologous monocyte-derived dendritic cells (MoDCs). The co-cultivation of MoDCs loaded with the NPM1mut peptide and CD8+ cells were performed either with or without IL-21, as well. We expanded the last strategy by a second round of NPM1mut-specific stimulation.

So far it was not possible to generate NPM1mut-specific T cell clones based on the advanced strategies and consistently there is no data published on NPM1mut multimer+ T cell clones. This fact raises the question why NPM1mut specific clones display such low frequencies. We want to point out that although we varied the strategies and we used eight different donors the isolation of NPM1mut-specific T cells restricted to HLA-A*02:01 apparently is challenging. Greater efforts, e.g. a larger number of donors or the use of immunological checkpoint inhibitors during cell culture are needed.

Disclosures

Thiede: AgenDix: Employment, Other: Ownership. Schetelig:Sanofi: Honoraria.
Imaging Human Immune Cell Infiltration in a Xenograft Graft-Versus-Host Disease Model
Authors: Elssen Catharina Van , Rashidian Mohammad , Vrbanac Vladimir , Freund Christian , Cragnolini Juanjo , Ingram Jessica , Tager Andrew , Ploegh Hidde
Published: November 2019

Graft versus host disease (GvHD) is one of the major complications of hematopoetic stem cell transplantation (HSCT), and is characterized by massive alloreactive T cell infiltration into target organs and tissues. Currently, non-invasive tools to diagnose and follow up disease are lacking. Here we show non-invasive imaging of infiltrating immunocytes in a human xenograft model for chronic GvHD, using a single domain camelid-derived antibody fragment (VHH) specific for human Class II MHC products present on myeloid cells, B cells and activated T cells. NSG (NOD-scid gamma) mice reconstituted with human fetal thymus, bone marrow and liver (BLT mice) spontaneously develop a GvHD-like condition, characterized by alopecia, blepharitis and target organ infiltration by activated human T cells. In diseased animals, we show an increased PET signal in the liver, attributable to infiltration of activated Class II MHC positive T cells. Non-invasive imaging of specific immune infiltration and activation could thus be of importance for diagnosis and treatment evaluation of GvHD and holds promise for other diseases characterized by inflammation.

Disclosures

No relevant conflicts of interest to declare.
Dendritic Cell Production from Allogeneic Donor Cd34+ Stem Cells and Mononuclear Cells; Cancer Vaccine
Authors: Unal Ali , Birekul Ayse , Unal Mehmet Cagri , Karakus Esen , Köker Yavuz , Ozkul Yusuf , Comu Faruk Metin
Published: November 2019

Aim: Active immunotherapy provides better recognition of tumor-related antigens by immune system of patient, enhanced immune system and elimination of malignant cells. This modality employs therapeutic potential of donor specific and tumor specific immune responses. Active immunotherapy targets immunosuppressive and tolerogenic mechanisms suppressed by tumor cells. T lymphocytes and antigen-presenting cells (dendritic cells) are two cell lineages that play crucial role in the battle of organism against cancer. Close similarity between cancer cells and normal cell structure is the most important reason of escape from defense cells, namely T lymphocytes. Stimulation and enhancement of T lymphocytes against cancer cells comprise principal part of therapy.

Material and Method: To generate allogeneic dendritic cells, leukemic stem cells were isolated from bone marrow samples from patients with acute leukemia. Lysate was prepared from leukemic stem cells identified by flow cytometer. Stem cells and mononuclear cells (1x10>6/kg) obtained from sibling donors by apheresis were separated to produce Dendritic Cells. For Dendritic Cell transformation, GM-CSF and IL-4 were added to media where leukemic stem cell lysate from patient and mononuclear cells from sibling donor were treated. From samples taken from the culture medium, after 48, 72 and 96 hours, dendritic cell surface markers (CD 80, CD 83 and CD 86) was assessed by flow cytometry. CD3, CD14, CD19, CD56, CD66b-negative, and HLA-DR, CD86 positive are indicative of immature DC. HLA-DR, CD 80 and CD83 positive are indicative of mature DC.

Results: Mononuclear cells were detected by 27 % among allogeneic hematopoietic cell groups harvested by apheresis. After culture under GMP conditions, mononuclear cell rate was found to be 24% on hours 96 and 120. It was seen that 88% of mononuclear cells transformed to mature dendritic cells after 96 hours culture.

Conclusion: In cancer patients, minimal residual disease can be eliminated by active tumor vaccine after reducing tumor burden by standard methods. Tumor vaccine obtained from allogeneic sibling donor can be used in lieu of autologous tumor vaccine and it is thought to be more effective. Allogeneic dendritic cells produced at 37¡C in CO2 media under GMP conditions can be used in tumor immunotherapy. More effective method would have been used by employing dendritic cells against cancer stem cells rather than cancer cells itself.

Figure Mature dendritic cells (CD83+) Figure. Mature dendritic cells (CD83+)

Disclosures

No relevant conflicts of interest to declare.
The Role of MLL1 and MLL2 in MLL Fusion Oncoprotein-Initiated Leukemia
Authors: Ernst Patricia , Anastassiadis Konstantinos , Kranz Andrea , Stewart A. Francis , Arndt Kathrin , Waskow Claudia , Yokoyama Akihiko , Jones Kenneth , Neff Tobias , Chen Yufei
Published: November 2019

The MLL1 histone methyltransferase gene at 11q23 undergoes many distinct chromosomal translocations to yield poor prognosis leukemia. In these MLL-rearranged (MLL-r) leukemias, the remaining wild-type allele is typically expressed, and the fusion oncoprotein lacks the C terminal histone methyltransferase domain. The role of the wild-type, endogenous MLL1 allele in MLL-fusion protein mediated leukemogenesis is not fully understood, with some evidence suggesting an important functional role. On the other hand, the wild-type allele is lost in some patients and in the MLL-r ML2 cell line, suggesting that the wild-type allele is not absolutely required. Here we rigorously address the role of endogenous MLL1 in MLL-r leukemia. Using precise and independent animal models, we show that MLL1 is dispensable for MLL-AF9- or MLL-AF6-mediated acute myelogenous leukemia (AML). In parallel experiments, deletion of Menin, encoding a chromatin targeting cofactor for MLL-fusion proteins, has severe effects on the proliferation and survival of MLL-r leukemia, as previously published. Potential redundancy between MLL family methyltransferases was addressed by co-deleting the closest paralog, Mll2. Surprisingly, deletion of Mll2 by itself had a significant impact on survival of MLL-AF9 leukemia cells, and co-deletion of Mll1 and Mll2 more effectively killed these cells, suggesting a redundant or synthetic lethal relationship between the two genes. A comparison of gene expression and histone modification changes in Mll1, Mll2 and Mll1;Mll2 double knockout leukemia cells showed that 1) global defects in histone H3, lysine 4 methyation can be exclusively attributed to MLL2, 2) MLL1- versus MLL2-dependent genes are largely distinct, and 3) Mll1;Mll2 double knockout leukemia cells exhibit deficiencies in NFκB and integrin ß3 pathways, but exhibit little evidence of deregulation of either MLL1- or MLL-fusion protein target genes. These findings reshape our thinking about the role of endogenous MLL family proteins in MLL-fusion protein leukemogenesis. The data presented here, in conjunction with the minimal effect of Mll2 deletion on hematopoietic stem and progenitor cells make MLL2 an attractive candidate for targeted therapy in MLL-r leukemia.

Disclosures

Ernst: Amgen: Other: stocks. Yokoyama:Sumitomo Dainippon Pharma: Research Funding. Neff:Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel; Epizyme: Patents & Royalties: patent filed.
Comparison of the Terumo BCT MNC and Cmnc Protocols for Peripheral Blood Stem Cell Collections
Authors: Westbrook Lindsey , Roig Joseph , Bagamasbad Neil , Dilag Reynold , Nasser Melissa , Sauthoff Sheri , Berg Mary
Published: November 2019

Background: Terumo BCT recently offered a new method of peripheral blood stem cell (PBSC) collection using the Spectra Optia¨, an apheresis instrument. The new protocol includes a continuous mononuclear cell collection (CMNC) as opposed to an older version, the mononuclear cell collection (MNC), which involves an additional step where product pools through a cell separation insert. Our institution has used both methods and the purpose of this study was to compare the CMNC to the MNC protocol, including run times and PBSC product characteristics.

Methods: A retrospective review and comparison of parameters from 120 collection procedures using the MNC insert and 60 collection procedures using the CMNC insert was done using the t-test. Data from patients/donors (including 20 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (CD34)-positive (CD34+) throughput, CD34+ collection efficiency (CE%), platelet loss (plt loss/total blood volume [TBV]), and collection product characteristics were included in the analysis.

Results: The MNC donor group included 12 allogeneic donors, which is comparable to the 8 allogeneic donors in the CMNC group. Donor weight and patient weight was not significantly different between the two groups. Pre-procedure laboratory values (WBC, percentage of lymphocytes [lymph%], percentage of monocytes [MNC%], and platelet count) were also similar between the two groups.

Run time was found to be significantly shorter using the CMNC protocol compared to the MNC protocol. Product volume was also significantly lower in the CMNC group compared to the MNC group. Although the volume was lower, the CMNC product had significantly higher white blood cell count (WBC), MNC%, and lymph% when compared to the MNC product. The CD34+ throughput was significantly higher in the CMNC group than the MNC group. The CD34+ CE% was found to be slightly increased in the CMNC group, though not significantly. The platelet loss was nearly identical in both protocols when normalized for total blood volume. Product hematocrit (HCT%) was significantly higher using the CMNC protocol; however, the red blood cell volume never exceeded 20 mL due to the lower product volume with the CMNC protocol. The numerical results are summarized in the Table.

Conclusion: The CMNC protocol collects a smaller volume of a purer product when compared to the MNC protocol with comparable platelet and red blood cell loss. Staff members who perform apheresis procedures are pleased by the shorter run time.

Table Table.

Disclosures

Roig: Terumo BCT: Employment.
Hematopoietic Recovery after in-Vivo T-Cell Depleted Allogeneic Stem Cell Transplant- Effects of ABO Incompatibility, Rhesus Incompatibility and Acute Gvhd
Authors: Gergis Usama , Al-Mulla Naima Ali , Javier Segovia , Hafez Rania , Phillips Adrienne A. , Mayer Sebastian , Shore Tsiporah B. , Hsu Jing-ming , Slotky Ronit , Vasovic Ljiljana V. , Cushing Melissa M. , van Besien Koen
Published: November 2019

Background:

Transfusion needs after allogeneic transplantation are closely associated with morbidity, cost of transplantation and may also be associated with long-term outcome. In an effort to better predict determinants of recovery after reduced intensity conditioning, we analyzed 214 consecutive patients who received allogeneic hematopoietic stem cell transplantation (HSCT) at our institution from January 2012 to December 2013.

Patients and Methods:

We excluded patients who experienced early (in the first 4 months) relapse (n=28), non-relapse mortality (n=28) or who experienced prolonged hospital stay before day 100 for transplant related complications (n=51). One hundred and seven patients had uneventful recovery until day 100 and met the inclusion criteria. The majority of patients received Fludarabine and melphalan conditioning (87%). Approximately one third each of the patients had HLA-identical related, HLA-matched unrelated donors or underwent haplo-cord transplantation. Recipients of HLA-identical related or unrelated donor transplant received alemtuzumab and post-transplant tacrolimus. Haplo-cord recipients received thymoglobulin and post-transplant tacrolimus and mycophenolate. Patients and transplant characteristics are summarized in table 1. T-tests were used for comparison between groups.

Results: In our analysis of 107 patients who underwent T cell depleted allogeneic transplantation and fared well at day 100, the determinants of prolonged anemia were ABO incompatibility (p=0.006), rhesus incompatibility (p=0.01) and acute graft versus host disease (aGVHD) (p=0.02). Whereas prolonged thrombocytopenia was only associated with the development of any grade aGVHD (p=0.04). At day 100, the hematopoietic recovery of haplo-cord grafts is similar to matched related and unrelated grafts.

Conclusion:

ABO incompatibility, rhesus incompatibility and occurrence of acute graft vs host disease are the major determinants of red blood cell recovery. Occurrence of acute graft vs host disease is a major determinant of platelet recovery. Of interest, the use of umbilical cord blood grafts combined with haplo-identical grafts is associated with recovery of red blood cells and platelets that is similar to that of adult donor grafts.

Disclosures

No relevant conflicts of interest to declare.
Comparison of Reduced Intensity Conditioning (RIC) Regimen and Myeloablative Conditioning (MAC) Regimen in Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) for Adult Patients with Acute Leukemia
Authors: Gurman Gunhan , Aydemir Guloksuz Gozde , Atilla Erden , Ataca Atilla Pinar , Civriz Bozdag Sinem , Kurt Yuksel Meltem , Toprak Selami Kocak , Topcuoglu Pervin , Arslan Onder , Ozcan Muhit , Demirer Taner , Ilhan Osman , Akan Hamdi , Beksac Meral
Published: November 2019

Introduction:The development of RIC regimens enabled HSCT also for elderly patients who had contraindications for intensive conditioning regimens. The aim of this study is to evaluate the effects of conditioning regimens on outcomes of alllo-HSCT for adult patients with acute leukemia.

Method:In this study, we retrospectively analysed 490 acute leukemia patients who underwent allo-HSCT between June 1988 to June 2015 at Ankara University School of Medicine Department of Hematology. We compared the outcomes of MAC vs RIC conditioning by Fisher's test and chi-square test. Kaplan-meier curves were used for survival. The factors that effect on survival were evaluated by univariate analysis-log-rank test and multivariate analysis-cox regression.

Results: In our study, the median age of participants was 32 with M/F ratio of 285 (57.9%) / 207 (42.1%). The patients were diagnosed as acute myeloid leukemia (AML) in 344 patients (69.9%), acute lymphoblastic leukemia (ALL) in 140 patients (28.5%) and others in 8 patients (1.6%). Pre-transplant active disease detected in 105 (22.7%) patients. Patients received grafts from related donors in 397 (80.7%) transplants. The most common stem cell source was peripheral blood in 378 patients (76.8%) followed by bone marrow in 99 patients (20.1%). MAC was used in 224 (90.4%) recipients whereas RIC was used in 47 (9.6%). The age, unrelated transplants, number of AML patients and intermediate-high risk patients were significantly higher in RIC group (44 vs 31, p<0.001; 37% vs 18%, p=0.002; 81.4% vs 43.7%, p<0.001). Both 1-year OS and 1-year RFS rate were lower in the patients treated with RIC (65.6% vs 34.9%; 44.4% vs 16.7%). Among the patients treated with MAC, 1 year-OS was higher in patients who developed chronic GVHD (78.6% vs 51.1%, p<0.05). The higher OS was related with patients who were transplanted in remission, received grafts from related donors as well as having acute and chronic graft vs host disease.

Conclusion: Lower OS and RFS were observed in the RIC conditioned patients compared to MAC in our study. In the treatment of young patients who do not have comorbidities for intense conditioning regimens, show good performance status and are high risk; the initial use of standard myeloablative conditioning regimens is feasible. However, the lower number of RIC transplants and the variations between groups in terms of patient and donor characteristics might effect the results of this study. Therefore, conducting further prospective studies with larger cohorts would be beneficial to enhance our knowledge.

Disclosures

No relevant conflicts of interest to declare.
Does Donor Epstein-Barr Virus (EBV) Seropositivity Affect Recipient's Risk of Graft Versus Host Disease (GVHD) in Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT)?
Authors: Atilla Erden , Kaplan Esmanur , Ataca Atilla Pinar , Toprak Selami Kocak , Topcuoglu Pervin , Akan Hamdi
Published: November 2019

Introduction:

EBV seropositivity in general population is 80%. Reactivation of latent infection in pre-transplant seropositive patients causes post-transplant lenfoproliferative disease (PTLD) following Allo-HSCT. The effect of donor EBV positivity on recipient's risk of graft versus host disease is not clear. Our aim is to present EBV seroprevalence and PTLD incidence as well as demonstrating the relation of EBV seropositivity with GVHD.

Patients and Methods:

A total of 364 allogeneic stem cell transplant recipients and donors were evaluated retrospectively from 2006 to 2015. During Allo-HSCT preparation procedures all recipients and donors were serologically tested. EBV specific IgG (VCA-IgG, EBNAIgG, EA-IgG) and IgM (VCA-IgM) antibodies were determined by Chemiluminescence by ARCHITECT lab analyzers using commercially available kits (Abbott, USA). All patients were followed for reactivation.

Results:

EBV IgG positivity was detected in 338 of recipients (92.8%) and 283 of donors (77.7%). There was no statistically difference detected between related or unrelated transplants. The mean age was 37 (range 16-67). 217 recipients were male (60%). 295 (81%) patients were transplanted for malign hematological diseases. The majority of patients were grafted from full-matched related donors (258, 71%). The most common source of stem cell was peripheral blood in 299 patients (82%) followed by bone-marrow in 56 patients (15%), bone-marrow plus peripheral blood in 9 patients (3%). 273 (75%) patients received myeloablative conditioning regimen. All patients received prophylactic acyclovir (in related transplants 400mg 3 times daily, in un-related transplants 800mg 3 times daily) starting from conditioning and up to three months posttranplant period. One pretransplant seropositive 26 year-old aplastic anemia patient had PTLD with EBV IgM positivity within 3 months posttransplant. He received 4 cycles of rituximab and prednisolone and achieved complete response. Three patients had EBV IgM positivity in posttransplant 4, 9 and 24th months with symptoms of infectious mononucleosis. The seropositivity resolved without treatment. Acute GVHD developed in 223 patients (61%) whereas chronic GVHD was detected in 285 (78%) of patients. The incidence of acute GVHD was similar when donor was EBV seropositive compared to seronegative (78% vs 22%, p=0.72). Chronic GVHD incidence was similar between donor EBV seropositive group compared to seronegative group (80% vs 20%, P=0.199).

Conclusion:

EBV seropositivity is common detected in 92.8% of our allo-HSCT recipient cohort. Donor EBV status did not have an effect on developing acute or chronic GVHD.

Disclosures

No relevant conflicts of interest to declare.
Zoledronic Acid Boosts γδ T-Cell Activity in Children Receiving αβ+ T and CD19+ CELL-Depleted Grafts from a Haplo-Identical DONOR
Authors: Barbarito Giulia , Airoldi Irma , Zorzoli Alessia , Bertaina Alice , Petretto Andrea , Inglese Elvira , Lavarello Chiara , Brescia Letizia Pomponia , De Angelis Biagio , Tripodi Gino , Merli Pietro , Moretta Lorenzo , Locatelli Franco
Published: November 2019

A new method of graft manipulation based on physical removal of αβ+ T cells and CD19+ B cells, leaving mature NK cells and γδ T cells in the graft, has been recently developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity against leukemia cells.

Thirty-three patients were treated with ZOL every 28 days at least twice. γδ T cells before and after ZOL treatments were studied till at least 7 months after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay.

Proteomic analysis of γd T cells purified from patients showed that, starting from the first infusion, ZOL caused up-regulation of proteins involved in activation processes and immune response, paralleled by down-regulation of proteins involved in proliferation. These findings are consistent with an induction of Vδ2 cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts. Furthermore, a proteomic signature was identified for each individual ZOL treatment. Patients given 3 or more ZOL infusions had a better probability of survival in comparison to those given 1 or 2 treatments.

In conclusion,ZOL influences Vδ2 cell activity, determines a specific proteomic signature and enhances anti-leukemia activity, this potentially resulting into an increased anti-tumor effect.

Disclosures

No relevant conflicts of interest to declare.
Busulfan-Cyclophosphamide Worsens Peripheral Gvhd While Fludarabine-Busulfan Results in Wide Bone Marrow Involvement in a Murine Model of Acute Gvhd
Authors: He Xin , Xu Xiaojun , Ye Yongbin , Liu Qifa
Published: November 2019

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and A proper conditioning regimen is vital to stop the development of aGVHD. To provide a platform for the study of aGVHD and evaluate the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (BU-Cy) and Fludarabine-BU (Flu-BU). In our study, BALB/c mice were conditioned with BU-Cy or Flu-BU and transplanted with 2×107 bone marrow cells and 2×107splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with BU-Cy had shorter survival (P<0.05) and more severe hepatic and intestinal clinical manifestations and pathological changes associated with increased INF-γ expression and diminished IL-4 expression in serum compared to allogeneic recipients conditioned with Flu-BU. Meanwhile increased donor-derived T-cell infiltration and impaired bone marrow B-cell development could be seen in the aGVHD mice conditioned with Flu-BU. Our study showed that the conditioning regimen with BU-Cy resulted in more severe peripheral aGVHD, while the Flu-BU regimen was associated with aGVHD with wide bone marrow involvement.

Disclosures

No relevant conflicts of interest to declare.
Late Reversal of Dominance after Dual Cord Blood Transplantation
Authors: Berg Mary , Gutman Jonathan A , Vinson Leslie , Draper Nicole
Published: November 2019

Background: Most adult patients are transplanted with two cord units to enhance engraftment after stem cell transplantation. Previous reports have demonstrated that one cord blood unit becomes dominant in the recipient and the other unit regresses. Complete chimerism with the dominant unit is achieved in >90% of patients by day +100 after transplantation. We report a case of a patient who initially demonstrated engraftment with one cord blood unit, but months later was found to be engrafted with the other unit.

A 30 year old man underwent dual cord blood transplantation for Acute Myeloid Leukemia in June 2015. The cords had identical HLA types and were both 6/10 match to the patient. During his recovery, he experienced engraftment syndrome, mild sinusoidal obstruction syndrome, Enterococcus bacteremia, and prolonged count recovery. A bone marrow biopsy done on day +77 demonstrated no evidence of disease; chimerism studies demonstrated 95% donor cells (CD3: 44% cord 1, 56% cord 2; CD33: 100% cord 2; CD56: 16% cord 1, 84% cord 2, see table). He was noted to have converted his blood type from his original O-Positive to B-Positive. He returned to his home and was not seen at our institution for several months. When he returned for follow-up on day +244, he was found to have a blood type of A-Negative. Testing with a new specimen confirmed this finding. Chimerism studies were performed to evaluate the status of the two donors in his peripheral blood on day +306.

Methods: A PCR-based STR assay was done on peripheral blood or bone marrow using the PromegaPowerPlex 16 HS System. The WBC populations were manipulated to create sub-populations enriched for CD3, CD33, and CD56, respectively.

Results: No significant recipient DNA was found in any of the specimens. On day +29, Cord 1 dominated in the CD56 cell population, but Cord 2 dominated in the CD3 and CD33 populations. On day +77, Cord 2 dominated in all cell populations, but Cord 1 still represented nearly half (44%) of CD3 cells. On day +306, Cord 1 dominated all cell populations.

Conclusion: The change in the patient's blood type is consistent with reversal of the dominance of the cord blood units received by this patient. Only one other case of this sort of reversal has been reported. This is not consistent with previously published data that the cord unit with higher CD3 chimerism as early as day +14 becomes the long-term engrafting unit.

Table Table.

Disclosures

No relevant conflicts of interest to declare.
An Innovative in Vitro Potency Assay Designed to Predict the Fate of Chimeric Antigen Receptors Modified T Cell Therapy Post Infusion in ALL
Authors: Wang Junxia , Dudley Mark , Choquette Therese , Jeschke Margit , Rutjens Erik , Kassim Sadik
Published: November 2019

Background: Chimeric antigen receptors modified T cells (CARTs) are T cells engineered to express tumor CARs that consist of T cell activation domains coupled to anti-tumor antigen single-chain variable fragments. Upon binding of tumor antigen to anti-tumor antigen antibody fragments on CART cells, the intracellular T-cell receptor CD3-zeta chain signaling domain induces T cell activation. The activated T cells undergo proliferation, cytokines release and tumor cells killing in an antigen-dependent manner. A number of CART-cell immunotherapies are now advancing to pivotal trials, however, little is known about how to characterize these cellular products before administration; one such gap relates to cell therapy product potency. Methods: Within this context, we developed a novel potency assay that quantifies the in vitro proliferative capacity of CART cell therapy products. We tested this assay using CART cell therapy products from patients with pediatric acute lymphoblastic leukemia (pedALL) and diffuse large B cell lymphoma (DLBCL). Results: In 40 CART products from pedALL patients evaluated for proliferation, and 37 products demonstrated significant (20% higher than background) proliferation. Overall, in vitro proliferation was correlated the expansion of the cell product in the patient after infusion. 23 CART products from DLBCL patients were analyzed, and 20 demonstrated significant specific in vitro proliferation. A strong correlation between in vitro proliferation and cell growth has been observed for the DLBCL patient samples. Conclusions: These data suggest that in vitro proliferation is a highly informative potency assay that may help predict product efficacy and safety of CAR T cell therapies.

Disclosures

Wang: Novartis: Employment. Dudley:Novartis: Employment. Choquette:Novartis: Employment. Jeschke:Novartis: Employment. Rutjens:Novartis: Employment. Kassim:Novartis: Employment.