bluuebunny/crossref_metadata_2025 huggingface dataset:
Expansion of Platelets for Transfusion
Authors: Fuentes Rudy , Wang Yuhuan , Poncz Mortimer
Published: October 2019

Abstract SCI-47

The clinical need for platelets transfusion continues to grow. Platelet units have all the usual concerns with other donor-derived blood products and in addition have concerns over the need to store the platelets at room temperature and of the resulting increased risk of bacterial contamination. Several groups have been able to develop platelets from embryonic stem cells, but achieving significant numbers of pro-platelets and/or platelets from in vitro grown megakaryocytes (Megs) remains elusive. Three sites of thrombopoiesis in vivo have been described: 1) intramedullary, 2) at the medullary/vascular junction, or 3) intrapulmonary from circulating Megs. We investigated whether infusing murine Megs into mice can release a large number of functional platelets both to better understand thrombopoiesis and also to see if such an approach may be an alternative to generating platelets for clinical usage. We show that infused fetal-liver-derived Megs results in 100–200 platelets/Meg. Levels as high as 12% of the total platelets in the recipient mouse were from the infused Megs. These platelets had a delayed presentation, peaking at 90 mins. In spite of these being new platelets, their half-life was only ∼75% of infused platelets. The shortened half-life was not due to the fact that these platelets were fetal in nature in that infused adult-liver-derived Megs had a similar temporal profile. These derived platelets had normal-size distribution, were not activated, and had not been affected by ADAM17 during in vitro culture as determined by surface glycoprotein 1b alpha. Platelet functionality was shown by their incorporation into clots in arterioles and venules in a cremaster laser injury model and in a ferric chloride carotid artery injury model. Using BrdU-labeled Megs we show that most of the Megs are trapped in the lungs with a few also appearing in the splenic red pulp. None were seen in other tissues. Staining with species-specific CD41 antibodies showed that detectable Meg cytoplasm peaked in the pulmonary vasculature at 30 min, and like the nuclei, was mostly gone by 5 hrs. We estimate that only ∼1% of the available pulmonary capillary bed is affected by the infused Megs, and most studied animals survived the infusion of the Megs. These studies support the lungs as a potential site for thrombopoiesis and that in vitro differentiated Megs can be infused directly avoiding the challenge of developing ex vivo technology to release large number of non-activated, but functional platelets.

Disclosures:

No relevant conflicts of interest to declare.
Expansion of Lymphocytes for Cell-Based Therapeutics
Authors: Heslop Helen E.
Published: October 2019

Abstract SCI-48

Adoptive transfer of T cells can lead to targeted and long-lived anti-tumor or anti-infective activity and can also modulate alloreactivity. T cell therapies have been used in both autologous and allogeneic settings. Donor-derived cytotoxic specific T lymphocytes (CTLs) have already proved highly effective in preventing or treating viral infections and Epstein-Barr virus (EBV) lymphomas developing after allogeneic hemopoietic stem cell transplant while T cells expanded ex vivo with CD3/28 beads have enhanced immune reconstitution after autologous stem cell transplantation. Clinical responses have also been observed following T cell therapy in patients with melanoma, lymphoma or nasopharyngeal cancer. Although successes have been obtained, these studies have also provided insights into the requirements for more effective cellular immunotherapy, which may be obtained by genetic modification of T cells. Approaches under evaluation in the clinic include transfer of artificial T cell receptors to target tumor cells, transfer of genes to render T cells resistant to tumor evasion mechanisms and transfer of a suicide gene that can be activated should adverse effects occur to allow the cell to be destroyed on exposure to a specific signal. The suicide gene strategy using the herpes simplex viral thymidine kinase (Tk) gene allows ablation of infused donor T cells if they induce graft-versus-host disease after allogeneic HSCT and has reached phase III clinical trial. In all these adoptive T cell immunotherapy strategies, broader application is limited by suboptimal persistence of transferred T cells, and by the complexity of current manufacturing techniques. Current research focuses on defining the optimum type of cell for transfer and the ex vivo selection and expansion procedures that favor long term persistence, since broader applicability will require ex vivo expanded T cells to show clinical activity and to have robust manufacturing processes and a clinical distribution paradigm. Strategies for simplifying and accelerating manufacture of T cell products have included the use of artificial antigen presenting cells expressing co-stimulatory ligands to provide a rapid source of antigen and optimized costimulation; bioreactors to grow cells in a closed system; and cytokine combinations to optimize cell growth and survival. Integration of these multiple approaches will be required to determine the best cell type and culture conditions for generating T cells for subsequent adoptive transfer into a complex immune network.

Disclosures:

Off Label Use: T cell products in studies conducted under INDs.
Prognostic Import of French-American-British (FAB) System As Embedded in 2008 Revision of World Health Organization Classification of AML: Review of SWOG Data
Authors: Newell Laura F , Othus Megan , Walter Roland B. , Petersdorf Stephen H. , Chauncey Thomas , Anderson Jeanne E. , Godwin John E. , Kopecky Kenneth J. , Appelbaum Frederick R. , Estey Elihu H.
Published: October 2019

Abstract 1446

Background:

After many years of use, the French-American-British (FAB) system for classification of AML has been replaced by that of the World Health Organization (WHO), as revised in 2008 (Blood 2009;114: 937–51). While unlike FAB, WHO uses genetic and clinical information to specify AML subtypes, the FAB system remains embedded in the WHO category of “AML not otherwise specified” (NOS). Here we ask whether the subdivision by FAB of AML NOS is of prognostic significance.

Methods:

FAB data were available on 970 newly-diagnosed patients treated on various SWOG protocols from 1986 to 2009. The protocols generally called for 3+7 type induction therapy. Exclusion of patients with recurrent genetic abnormalities, therapy-related AML (T-AML), or AML with myelodysplasia related changes (MDS-AML), as stipulated in WHO 2008 (NPM1 and CEBPA mutation status was unavailable), left 447 with AML NOS whose FAB type was recorded. We used multivariate analyses adjusting for age, sex, cytogenetic risk (SWOG criteria), pretreatment performance status, blood counts, and marrow blasts, to assess whether FAB was independently associated with outcome in these 447 patients and used areas under the receiver operator characteristic curve (AUC) to quantify the predictive ability of FAB, with AUC of 1.0 indicating perfect prediction and an AUC of 0.5 equivalent to a coin flip.

Results:

The 447 patients had median age of 58 years, CR rate 56% and median survival 17.2 months. Given the association between T-AML or MDS-AML and FAB types M6 or M7 (p=0.02 in this dataset), only 2% of the 447 patients were M6 or M7; 8% were M0, 27% M1, 26% M2, 25% M4, and 12% M5 (we excluded patients with M3). CR rates were 47% M0, 56% M1, 53% M2, 54% M4, 72% M5, and 45% M6 or M7. Survival probability was longest with M6/M7 followed by M5. The multivariate analyses showed the following for the effect of FAB (considered as M0, M1, M2, M4, M6/M7):

Results were similar regardless of whether we considered FAB as above or grouped as M0, M1/M2, M4/M5, M6/M7. In particular, FAB was not associated with any outcome and its removal from more complete adjusted models had little effect on the models' predictive ability.

Conclusion:

These results question the utility of the FAB system as embedded in WHO 2008.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Cell Transplantation – Specific Comorbidity Index As An Instrument to Predict Survival in Patients Admitted to Intensive Care Unit Early After Allogeneic Transplantation
Authors: Bayraktar Ulas D. , Shpall Elizabeth J , Liu Ping , Ciurea Stefan O , De Lima Marcos , Rondon Gabriela , Price Kristen J , Champlin Richard , Nates Joseph L
Published: October 2019

Abstract 147

Background:

Hematopoietic cell transplantation – specific comorbidity index (HCT-CI) based on pre-transplant comorbidities was shown to be predictive of survival and non-relapse mortality in patients undergoing allogeneic stem cell transplantation (ASCT). We assessed HCT-CI as a predictive instrument for survival in patients admitted to intensive care unit (ICU) early after ASCT.

Methods:

Patients older than 18 who were admitted to ICU between the preparative regimen initiation and post-transplant day 100 were included. Demographics, pre-transplant comorbidities, disease characteristics, transplant data, dates of transplantation, ICU admission, and last follow-up were gathered from institutional registries. Reason for ICU transfer, date and survival status at the time of hospital discharge were gathered from individual medical records. HCT-CI scores were calculated as previously described by Sorror et al. Patients who were transferred to a hospice and died within 7 days of the transfer were considered to have died in the hospital for the purpose of current analyses.

Results:

Of 3039 patients who underwent ASCT between June 2001 and December 2011 at MD Anderson Cancer Center, 377 patients (12%) were transferred to ICU within 100 days of the transplant. Disease and transplant characteristics of the patients are demonstrated in the Table. The most common reasons for ICU transfer were respiratory failure (n=230), septic shock (n=44), altered mental status (n=33), hemorrhage (excluding intracranial, n=28), and arrhythmias (n=20). Median age was 53 years with a range of 19–80. The most common pre-transplant comorbidities were pulmonary in 302 (80%) and cardiac in 73 patients (19%). Overall 240 patients (64%) died in the hospital. A comprehensive logistic regression model demonstrated HCT-CI score ≥3 to be associated with a higher risk of death in the hospital (Table). Patients with myeloproliferative diseases and those admitted to ICU during the preparative regimen had less risk of dying in the hospital while those who received ablative regimens had an increased risk. One-year overall survival (OS) rates were 22%, 16%, 16%, and 10% among patients with HCT-CI scores of 0–1, 2, 3, and ≥4, respectively. A Cox proportional hazards model demonstrated HCT-CI score of ≥4 to be a poor prognostic factor on OS (HR: 1.72, 95% CI: 1.21–2.44). The only other significant prognostic factor on OS was the age.

Conclusion:

Overall prognosis in patients who are transferred to ICU within 100 days of ASCT is poor. HCT-CI score could aid clinicians to identify the worst-to-perform patients within this population.

Disclosures:

No relevant conflicts of interest to declare.
The SirT1 Inhibitor Tenovin-6 Induces Monocytic Differentiation in APL Cells
Authors: Sunami Yoshitaka , Araki Marito , Morishita Soji , Hironaka Yumi , Edahiro Yoko , Komatsu Norio , Ohsaka Akimichi
Published: October 2019

Abstract 1542

Tenovin-6, an inhibitor for nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase Sirtuins, shows cytotoxicity to cancerous cells and thus recognized as a potential therapeutic compound for cancer treatment (Lain S., et al. (2008) Cancer Cell. 13,454–63). Since there is limited information for the cytotoxic property of this compound for hematopetic malignancies, we treated an APL (acute promyelocytic leukemia) cell line NB4 with Tenovin-6. As expected, Annexin V assay revealed that Tenovin-6 induced apoptosis in NB4 cells. However, to our surprise, at modest concentration, Tenovin did not induce cell death, rather inhibited NB4 cell proliferation and altered cell morphology. The fluorescence-activated cell sorting (FACS) analysis revealed that tenovin-6-treated NB4 cells are positive for CD11b and CD36 with decreased level of CD13 and CD33. Moreover, tenovin-6-treated NB4 cells presented nitroblue tetrazolium reduction capacity, suggesting that tenovin-6 induced monocytic differentiation in NB4 cells.

To assess how Tenovin-6 induces cellular differentiation in NB4 cells, we investigated downstream target of Sirtuins. Although Tenovin-6 reportedly promotes acetylation of p53 by inhibiting SirT1, a founder of Sirtuin family proteins, the acetylation status of p53 is unchanged at the concentration where we observed differentiation. This suggests that Tenovin-6 induces NB4 differentiation through inhibiting other Sirtuin family proteins. These findings demonstrate a potential of Tenovin-6 as a differentiation-inducing reagent in APL cells.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Comparision of Surgical Resection Followed by R-CHOP and R-CHOP Alone for Primary Localized Intestinal Diffuse Large B Cell Lymphoma
Authors: Lee Ho Sup , Park Lee Chun , Lee Eun Mi , Shin Seong Hoon , Ye Byeong Jin , Oh Sung Yong , Song Moo-kon , Lee Sang Min , Lee Won-Sik , Kang Byung Woog , Chang Myung Hee , Cho Seok-Goo , Yahng Seung-Ah , Yoon Sung-Soo , Kwon Ji-Hyun , Kim Yang Soo
Published: October 2019

Abstract 1627

Abstract

Background:

Gastrointestinal tract is the most commonly involved extranodal site and is represented 10–15% of all non-Hodgkin¡&hibar;s lymphoma (NHL) cases and 30–40% of all extranodal sites. In this retrospective studies, the purpose is finding appropriate treatment strategy according to comparing the efficacy of treatment in patients with primary intestinal diffuse large B cell lymphoma (DLBL) undergoing surgery followed chemotherapy or chemotherapy alone.

Method:

Seventy six patients were newly diagnosed with DLBL and received chemotherapy between March 2004 and June 2011. Primary intestinal lymphoma which had predominant intestinal lesions was diagnosed by specialized hemato-oncologist. All patients were treated with rituximab combined cyclophosphamide, adriamycin, vincristine, and prednisolone (R-CHOP). Patients were divided into two groups. One included patients who were undertaken surgery followed by R-CHOP (surgery group). The other included patients who were undertaken R-CHOP alone (CT group).

Results:

The characteristics of the patients were as follows: the median age was 56.5 years (range 15–85 years) with a female-to-male ratio of 45: 31. Patients characteristics had no significant difference between two groups. The estimated 3 years progrression free survival rates (PFS) and overall survival rates (OS) of surgery and CT group were 92.2% and 74.8%, (p=0.009) and 94.2% and 80.7%, (p=0.049) respectively. In univariate analysis, PFS and OS were estimated in Lugano stage I, II1 and II2, IIE (p=0.006 and p=0.036), Low, Low-intermediate, and high-intermediate risk (p=0.004 and p=0.000), and surgery and CT alone, (p=0.009 and p=0.049), respectively. In multivariate analysis, there was no independent predictive factors for survival.

Conclusion:

Patients treated with surgery followed by R-CHOP were seemed to have higher survival rate than R-CHOP alone although there was no significant differences for survival rate. There was no significant prognostic factors for survival but there were possible prognostic factors such as Lugano stage, IPI risk, and treatment modality for PFS and OS.

Figure. The superior survival rates were shown in surgery group than those in R-CHOP chemotherapy group.

Disclosures:

No relevant conflicts of interest to declare.
Treatment with a Bortezomib-Containing Regimen Is Associated with Better Therapeutic Outcomes in Patients with Waldenstrom's Macroglobulinemia Who Have Familial Disease Predisposition
Authors: Treon Steven P , Hunter Zachary , Hanzis Christina , Tripsas Christina , Ioakimidis Leukothea , Patterson Christopher , Manning Robert , Sheehy Patricia , Turnbull Barry
Published: October 2019

Abstract 1643

We examined the impact of familial predisposition on treatment outcome in 135 Waldenstrom's macroglobulinaemia (WM) patients, 26.7% of whom had first or second degree relative(s) with a B-cell lymphoproliferative disorder. All patients were rituximab-naïve and received a rituximab-containing regimen. There were no significant differences in baseline characteristics between cohorts. Overall (93.9% vs. 75.0%; p=0.029) and CR/VGPR (23.2% vs. 16.7%; p<0.0001) responses, time to progression (45.5 vs. 21 months; p=0.015), and to next therapy (50.0 vs. 33.0 months; p=0.024) favored sporadic patients. By multivariate analysis, familial predisposition was an independent marker for disease progression (Hazard ratio 0.554). We next assessed whether the type of treatment used impacted therapeutic outcome in patients with familial WM. Overall, major and CR/VGPR responses were better among patients with familial but not sporadic WM who received a bortezomib versus non-bortezomib containing regimen (Figure 1). A longer TTP was also observed for familial but not sporadic WM patients who received a bortezomib versus non-bortezomib containing regimen. For patients with familial disease, the median TTP was estimated at >33 versus 20.6 months for bortezomib and non-bortezomib containing therapy, respectively (p=0.08). For sporadic WM patients, the median TTP was estimated at >35 versus 45.5 months for bortezomib and non-bortezomib containing therapy, respectively (p=0.68). The findings of this study convey that familial disease predisposition is an important determinant of response, TTP and TTNT in patients with WM. Treatment with a bortezomib-containing regimen is associated with better therapeutic outcomes in patients with WM who have familial disease predisposition, and may be revealing of signaling pathways that are present in familial patients and amenable to select targeting by proteasome inhibition.Figure 1.Overall, major, and VGPR/CR responses for 135 WM patients with sporadic (A) or familial (B) disease who received either bortezomib or a non-bortezomib containing regimen. *p=0.05; and **p=0.0006 among familial WM patients who received bortezomib versus non-bortezomib containing therapy.Figure 1. Overall, major, and VGPR/CR responses for 135 WM patients with sporadic (A) or familial (B) disease who received either bortezomib or a non-bortezomib containing regimen. *p=0.05; and **p=0.0006 among familial WM patients who received bortezomib versus non-bortezomib containing therapy.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA-130a Targets ATG2B, AGO4 and DICER1, Inhibits Autophagy and Induces Cell Death in Chronic Lymphocytic Leukemia
Authors: Kovaleva Valentina , Mora Rodrigo , Stilgenbauer Stephan , Lichter Peter , Seiffert Martina
Published: October 2019

Abstract 1768

B-cell chronic lymphocytic leukemia (CLL) is characterized by deregulated expression of microRNAs (miRNAs). These post-transcriptional regulators of gene expression play a crucial role in controlling multiple cellular processes. By microarray analysis and quantitative RT-PCR we observed significantly lower levels of miR-126, miR-130a, miR-143, miR-181a and miR-326 in primary CLL cells compared to normal B cells. Transfection of synthetic miR-130a or miR-143 induced a significant reduction in cell viability of both primary CLL cells and the CLL cell line MEC-1. As autophagy is connected to cancer cell survival and resistance to apoptosis, we investigated the effect of these two miRNAs on autophagy by following the specific autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). Therefore, we generated MEC-1 cells stably expressing GFP-tagged LC3 and analyzed autophagosome formation by using an imaging flow cytometer quantifying GFP-positive dots. These experiments revealed that autophagy is induced in these cells upon starvation, and that introduction of miR-130a, but not miR-143, resulted in a reduction of autophagosome formation (see Figure). These findings were verified by LC3 Western blot analysis, and extended to primary CLL cells, showing for the first time that autophagy is an active process in these cells and that miR-130a inhibits autophagy in primary CLL cells as well. To further elucidate the molecular mechanism of miR-130a-mediated CLL cell survival and autophagy, we aimed at identifying putative target genes of this miRNA and identified ATG2B, an autophagy-related gene, as well as DICER1 and AGO4, two components of the miRNA processing machinery, as direct target genes of miR-130a in CLL cells. The relevance and role of these three novel target genes in miR-130a-regulated cell death/cell survival programs is under current investigation.

Figure: Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC). Figure:. Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC).

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Outcome of B-CLL Patients Treated in a Prospective Trial Evaluating Combination of Oral Fludarabine and Cyclophosphamide As Frontline Therapy
Authors: Guièze Romain , Tournilhac Olivier , Maloum Karim , Leprêtre Stéphane , Haioun Corinne , Feugier Pierre , Delmer Alain , Leporrier Michel , Cazin Bruno
Published: October 2019

Abstract 1795

Introduction.

The combination of fludarabine and cyclophosphamide (FC) has become the core combination of modern frontline chemotherapy for fit and young B-CLL patients (pts). Recently the association with rituximab to FC (IV administration) has been shown to increase the response and extend both progression free survival (PFS) and overall survival (OS) in untreated pts (Hallek et al., Lancet 2010). So far few data are available concerning the very long term outcome of pts treated by FC containing regimen. Methods. We previously reported a prospective phase II trial (Cazin et al., BJH 2008) of the oral combination of FC over 5 days in 75 patients with untreated B-CLL and less than 66 years old. The study was conducted between October 1999 and February 2001. Briefly, oral FC then demonstrated high efficacy with overall response rate (ORR) and complete response (CR) rate of 80% and 53% respectively despite the absence of rituximab in this regimen. We here propose to examine 10-year end points of progression-free survival (PFS), overall survival (OS), impact of genomic features, and risk of therapy-related myeloid neoplasm (t-MN) by updating survival data of all the pts incuded in this trial. Results. With a median follow-up of 10.7 years, the median 10-year OS was 51.7%. Responders presented better 10-year OS than non-responders (57% vs. 38%, p=0.031) but quality of response (CR vs. PR) did not significantly impact 10-year OS. A major prognostic impact of IGVH mutational status could be observed since 10-year OS was 81% for mutated patients vs. 44% for unmutated pts (p=0.012). The median 10-year PFS was 30% and clearly influenced by the mutational status (50% if mutated profile vs. 12% if unmutated profile, p=0.02). However there is no trend of a plateau and even long term responding patients still relapse with time. Finally, only one patient developed t-MN but 9 others presented solid neoplasms. Conclusion. Long-term follow-up of B-CLL patients prospectively treated in a phase II clinical trial demonstrates extended OS and PFS with oral FC without high risk of t-MN.

Disclosures:

No relevant conflicts of interest to declare.
Validation of Potential Chemosensitivity Genes in Multiple Myeloma
Authors: Yang Lin , Zhang Xingding , Wang Hua , Orlowski Robert Z. , Huang Xingxu
Published: October 2019

Abstract 1818

Chemotherapy agents are extremely important in the treatment of liquid malignancies, such as multiple myeloma (MM). Unfortunately, chemotherapy resistance in MM therapy is the most significant cause of treatment failure. The ability to predict, treat, or circumvent resistance is extremely likely to improve clinical outcomes. Thus, identification of novel genes that play a crucial role in MM progression and chemosensitivity is necessary to understand this disease better at the molecular level. Moreover, these genes and their products may serve as new therapeutic targets for MM, whose expression could improve patient outcomes or served as a predictor for chemotherapy outcome. To identify potential chemosensitivity genes, establishing a high-throughput method for validation of targets becomes urgently needed. Toward this purpose, we have successfully developed a high-throughput siRNA based functional target validation approach and identified 34 potential chemosensitivity genes.

Our preliminary studies focusing on one of the candidate gene, TJP1 (tight junction protein 1), suggested that targeting TJP1 led to tumor cell resistant to several chemotherapy agents, including doxorubicin (Dox), cisplatin (Cis), methotrexate (MTX), and bortezomib. Further analysis with 264 bortezomib treated MM patients indicated that expression level of TJP1 correlated with patient response to bortezomib. Two clones and pooled RPMI 8226 MM cell line, which were developed against bortezomib treatment in our lab, showed loss of TJP1 expression, suggesting a role of TJP1 may play in bortezomib resistance development. More importantly, TJP1 targeting in myeloma cells resulted in cell resistance to bortezomib treatment.

Protein tyrosine phosphatase, receptor type, O1 (PTRPO) was identified in our screening as well. We first examined the expression of PTPRO in a panel of myeloma cell lines, and found that PTPRO expression was dramatically decreased in several drug resistant lines compared to their parental sensitive cells. Moreover, using a Lentiviral cDNA expression system to overexpress PTPRO, we observed that PTPRO significantly inhibited cell growth and sensitized drug sensitivity of several drug lines by triggering apoptosis. Finally, evaluation of the publicly available gene expression profiling data gathered from primary plasma cells on the Multiple Myeloma Genomics Portal, and the linked clinical annotation describing patient outcomes, has shown that over-expression of PTPRO is associated with a superior overall survival compared to patients whose myeloma show low levels of PTRPO expression, suggesting this may be a prognostic marker as well.

Together, we have successfully identified a group of potential chenmosensitivity genes, in which two of the genes (TJP1 and PTPRO) have been further validated to sensitize the drug treatment in multiple myeloma cells.

Disclosures:

No relevant conflicts of interest to declare.
The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma
Authors: Roccaro Aldo M , Sacco Antonio , Kuhne Michelle , Azab AbdelKareem , Maiso Patricia , Zhang Yong , Liu Yang , Azab Feda , Issa Ghayas C , Quang Phong , Ngo Hai T , Pan Chin , Choen Lew , Cardarelli Pina M , Ghobrial Irene M.
Published: October 2019

Abstract 1844

Background.

We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents.

Methods.

Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models.

Results.

MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models.

Conclusion.

These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease.

Disclosures:

Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.
Bortezomib Induction Followed by ASCT in Patients with Multiple Myeloma: Achievement of Response After Induction and Achieving CR Post-ASCT Are Both Important Prognostic Markers
Authors: Kim Min Kyoung , Min Chang-Ki , Hyun Myung Soo , Kim Kihyun , Yoon Sung-Soo , Kim Jin Seok , Eom Hyeon Seok , Suh Cheolwon , Kang Hye Jin , Kim Jeong-A , Mun Yeung-Chul , Shin Ho-Jin , Jo Deog-Yeon , Choi Chul Won , Moon Joon Ho , Kim Yang Soo , Yang Deok-Hwan , Bae Sung Hwa , Lee Gyeong-Won , Kwon Jung Hye , Kim Kyoung Ha , Yoon Hwi-Joong , Kim Chul Soo , Park Joon Seong , Lee Jae Hoon
Published: October 2019

Abstract 1866

Background:

In multiple myeloma (MM), the association between the response to induction before autologous stem cell transplantation (ASCT) and long-term outcome is less clear but the situation may change with the introduction of novel agents. We therefore assessed the clinical relevance of response of bortezomib induction treatment or post-ASCT response for patients who received bortezomib-combined induction chemotherapy followed by ASCT.

Methods:

We retrospectively assessed 183 MM patients who received bortezomib-containing induction therapy (BTZ-IT) followed by ASCT in 24 institutions throughout Korea between 2003 and 2010. Records of these patients were reviewed using the Korean Myeloma Registry database (www.myeloma.or.kr). Each institution was requested to reconfirm the data using additional case report forms. Patients who had overt MM based on International Myeloma Working Group diagnostic criteria were selected.

Results:

One-hundred seventy eight patients were eligible. Their median age was 56 years (range, 28–69 years) and 96 (53.9%) were male. Forty nine (27.5%) received bortezomib as front-line therapy and 129 (72.5%) as second-line treatment. All patients underwent ASCT and 22 (12.4%) were treated with tandem ASCT. Ninety-seven (54.5%) patients were treated with maintenance therapy after ASCT. After BTZ-IT, the response rates in this selected series of patients were 37.6% CR, 12.4% VGPR, 41.0% PR, 7.3% SD and 1.7% PD (Figure 1A, 1B, 1C); the corresponding post-ASCT rates were 69.2% CR, 14.0% VGPR, 11.0% PR, 2.9% SD and 2.9% PD. At a median follow-up of 46.6 months, the 3-year overall survival (OS) and event-free survival (EFS) rates were 70.0% and 31.9%, respectively. Multivariate analysis showed that factors independently predictive of OS and EFS included achievement of BTZ-IT response °Ã PR (P=0.025 and P=0.014, respectively) and the treatment with maintenance therapy (P=0.048 and P=0.001, respectively). Post-ASCT CR vs. °Â VGPR was also an independent prognostic factor for OS and EFS (P=0.0001 and P=0.002, respectively).

Conclusion:

At least PR to BTZ-IT and CR after ASCT were predictive of survival. These findings suggest that patients who responded to BTZ-IT may benefit from ASCT due to an enhanced quality of response. Maintenance therapy can also affect patient outcomes.

Disclosures:

No relevant conflicts of interest to declare.
Upregulation of the Chemokine Receptor CCR5 on CD16+ Myeloid Blood Dendritic Cells in Allogeneic Hemopoietic Cell Transplant Patients Predicts for Acute Graft Versus Host Disease
Authors: Shahin Kifah , Sartor Mary M , Hart Derek NJ , Bradstock Kenneth F
Published: October 2019

Abstract 1982

Introduction:

Dendritic cells (DC) are centrally involved in the development of acute graft-versus-host disease (GvHD) following allogeneic hemopoietic cell transplantation (alloHCT). We previously showed that the activation status, as assessed by CMRF-44 antigen expression, of CD11c+ myeloid blood DC is highly associated with the onset and severity of acute GvHD (Transplantation. 2007;83:839–846). We also reported a positive correlation between acute GvHD and the expression of the chemokine receptor CCR5 on CD11c+ myeloid DC (Blood. 2009;114 Suppl.:2251). Given the phenotypic and functional heterogeneity of the CD11c+ DC population, we investigated CCR5 expression on the CD11c+ DC subsets and then monitored the informative CD11c+ CD16+ DC subset expression of CCR5 in the peripheral blood of 42 patients post alloHCT, and correlated the findings with GvHD.

Methods:

Peripheral blood was collected twice weekly up to Day 100 post transplant from 42 alloHCT patients. The expression of CCR5 receptor on CD11c+ and CD11c- DC subsets was evaluated using multiparameter flow cytometry.

Results:

Only the CD11c+ CD16+ DC subset lacked CCR5 and induced it upon alloactivation. Seventeen of 42 patients developed acute GvHD (5 grade I, 12 grades II-IV). The percentage of CD11c+ CD16+ DC expressing CCR5 correlated with the development of acute GvHD grades II-IV. The maximum CCR5 expression detected on CD11c+ CD16+ DC in patients prior to developing grades II-IV GvHD (mean 22.7 ± 4.3%, n=12) was higher than in those with grades 0-I GvHD (11.4 ± 1.7%, n=30) (p=0.0285). CCR5 levels >20% on CD16+ myeloid DC predicted grades II-IV GvHD with a sensitivity of 66.7% and specificity of 86.7%. Levels of expression of CCR5 on the CD11c+ CD16- DC and CD123+ plasmacytoid DC were not predictive of GvHD.

Conclusion:

Expression of CCR5 on circulating CD11c+ CD16+ myeloid DC post alloHCT correlated with the development of moderate to severe GvHD. This observation may reflect DC activation or altered DC homing during the alloimmune response. Detection of increased CCR5+ CD11c+ CD16+ DC may allow pre-emptive therapeutic intervention prior to the clinical diagnosis of GvHD.

Disclosures:

No relevant conflicts of interest to declare.
Second Stem Cell Transplantation (SCT) for Relapsed Leukemia Provides Only Modest Prolongation of Survival
Authors: McIver Zachariah A. , Hughes Thomas , Ito Sawa , Battiwalla Minoo , Koklanaris Eleftheria K. , Haggerty Janice , Hensel Nancy F. , Barrett A. John
Published: October 2019

Abstract 2001

Patients with leukemia relapsing after SCT have a dismal prognosis. Treatments ranging in intensity from palliation to second SCT are offered to such patients but the overall survival remains in the region of 3–4 months. Second SCT is typically restricted to selected patients deemed more likely to survive and experience further leukemia control. However, since a second SCT is associated with an increase risk of transplant related morbidity and mortality, it is important to determine whether this option has a survival benefit. We studied 60 consecutive relapsed patients who had received a myeloablative matched-sibling T-cell depleted SCT for a variety of hematological malignancies including AML, ALL, CML blastic phase, and MDS. Median patient age at time of first SCT was 36 years (range, 10 – 68), median T cell dose varied between 1–5 x104/kg. Thirty-five patients relapsed early (< 6 months post SCT) with a median survival after relapse of 170 days (range 45 – 402), and 25 patients relapsed late (> 6 months post SCT) with a median survival after relapse of 1005 days (range, 240 – 4459). Salvage therapies included tyrosine kinase inhibitors (for CML), intrathecal chemotherapy (for CNS leukemia), and systemic chemotherapy combinations predominantly involving fludarabine and cytarabine. Twenty-four patients (13 early relapse, and 11 >6 months), with a good performance score and showing some preliminary response to non-transplant therapy, received a 2nd SCT from their original donor. Our rationale for selecting these patients for a T replete 2nd SCT was based on the potential enhancement of graft-versus leukemia effects following the failed T-cell depleted SCT. Sixteen received myeloablative conditioning with either Busulphan or Melphalan, and 8 patients received a nonmyeloablative regimen specific to their disease. Of the 36 patients not transplanted, 14 received a donor lymphocyte infusion, and 22 received palliative chemotherapy or hospice care only. Compared with non-transplant treatment patients, only those relapsing early after their first SCT experienced a significant but small survival benefit from 2nd SCT, with most patients dying from relapsed disease (median 55 vs 154 days, untransplanted vs transplanted; HR = 0.4, p = 0.01). In untransplanted patients survival was not different between any treatment option. Although patients selected for 2nd SCT represented a more favorable group, it was disappointing that intensive treatment only resulted in a 3 month prolongation of median survival. These results do not indicate an important benefit for second transplant in any patient relapsing with leukemia after SCT.

Disclosures:

No relevant conflicts of interest to declare.
The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21
Authors: Curtis David J. , Nguyen Nhu-Y , Salmon Jessica
Published: October 2019

Abstract 214

The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Potential Benefits of Extended Genotype Matching to Chronically Transfused Patients with Sickle Cell Disease
Authors: Helman Ricardo , Cancado Rodolfo , Mota Mariza A , Dezan Marcia R , Kutner Jose Mauro , Chiattone Carlos S , Castilho Lilian
Published: October 2019

Abstract 2144

Background:

Management of RBC alloimmunization in Sickle Cell Disease (SCD) patients has been the subject of much debate, and currently there is no standard approach. Many programs transfuse SCD patients with RBCs that are phenotype-matched for D, C, c, E, e and K. Although these approaches reduce the incidence of alloantibody production, patients still become alloimmunized. Based on this we aimed to identify the rates of alloimmunization in chronically transfused SCD patients and compare the phenotyping with genotyping methods to find a better way to match RBC units to those patients. Methods: We selected 45 SCD patients (homozygous for hemoglobin S) with multiple transfusions, previously phenotyped for ABO, Rh (D, C, c, E, e) and K1. Phenotypes were determined by hemagglutination using gel cards (Diamed® ). Genotypes were determined by a DNA array using the Human Erythrocyte Antigen BeadChip (“HEA”) from Bioarray Solutions. All SCD patients included in this study were in chronical transfusion program; receiving multiple transfusions. The median age was 24y; there were 28(62%) females and 17(37.8%) males. The median of transfusions were 53 (5–78) and 40 (88.9%) patients received more than 20 phenotype-matched units for Rh (D, C, c, E, e) and K1. Results: Of the 45 SCD patients selected, 11 (24.4%) had alloantibodies. The antibody specificities found in these patients were anti-D, -C, -CW, -E, -Jka, -Jkb, -Fya, -Dia, -s. Although the patients were receiving Rh and K phenotype-matched units 8 (17%) of them became alloimmunized to Rh antigens and on those patients we found discrepancies between the previous phenotype and genotype-derived phenotype. Our results showed that the risk of immunization increases in patients over 40 years old (p= 0.05) and with the number of transfusion events. Patients with more than 20 RBC transfusions have a tendency for alloimmunization (p=0.65). We also observed that genotyping was more effective than hemagglutination in determining patient's correct phenotype. Conclusion: Our data show that even with the implementation of Rh and K phenotype-matching in chronically transfused patients with SCD, they still become alloimmunized to other antigens with high immunization risk and also to Rh antigens due to the limitations of the hemagglutination. The relevance of genotype determination of blood groups for the management of multiple transfused patients with SCD has been demonstrated by allowing the determination of the true blood group genotype, by assisting in the identification of suspected alloantibodies and in the selection of antigen-negative. As donor genotyping for the most clinically relevant blood group antigens by automated DNA techniques are becoming available, extended genotype matching should be considered in this group of patients.

Disclosures:

No relevant conflicts of interest to declare.
Delayed Embryonic to Adult Globin Switching in HMGB2 Knock Out Mice
Authors: Marini Giuseppina M. , Asunis Isadora , Cabriolu Annalisa , Porcu Loredana , Sau Anna Maria , Manchinu Francesca , Porcu Susanna , Marongiu Franca M. , Poddie Daniela , Ronfani Lorenza , Bianchi Marco , Ronchi Anotonella , Ristaldi Maria Serafina , Cao Antonio , Moi Paolo
Published: October 2019

Abstract 2152

Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal regulation of the beta globin gene cluster. Screening a λgt11 cord blood cDNA library with the tandem NFE2 repeat of HS2 as recognition site probe, we isolated 14 cDNA clones of HMGB2, a chromatin non histone protein. Binding to the HS2 region was confirmed in vivo by ChIP assay. Transactivation analysis in K562 cells showed mild repression of a luciferase reporter driven by HS2 and the γ-promoter. The DNA bending capacity and the increased expression of HMGB2 during erythroid differentiation are properties well suited to facilitate LCR looping toward the β-globin genes, the mechanism thought to mediate globin gene activation. Hence, HMGB2 binding to HS2 may be relevant for the regulation of the β-globin gene cluster. To assess the function of HMGB2 as a possible regulator of the globin genes we analyzed the hematological phenotype of the HMGB2 knock out mice during erythroid differentiation. In peripheral blood from E18 fetus or adult mice no hematological differences were found among normal, heterozygous or homozygous HMGB2 mice. However, at earlier stages of development, at E12, β-minor and major were slightly reduced in HMGB2−/− mice, whereas embryonic εy and βH1 globin genes were overexpressed 4–5 times compared to normal littermate mice. These results support a repressing activity of HMGB2 on embryonic globin genes and a possible role in the switching of hemoglobins during early stages of differentiation. The lack of any hematological phenotype in the latter stages of development may be explained by a temporal restriction in the expression of HMGB2 or by the vicarious activity of other HMGB proteins during definitive erythropoiesis

Disclosures:

No relevant conflicts of interest to declare.
TLR-Signaling Induces Granulocytic MDSCs Generation by Directing Myeloid Lineage Commitment During Polymicrobial Sepsis
Authors: Kong Yaxian , Zhang Weimei , Liu Wei , Miao Peng , Zeng Hui
Published: October 2019

Abstract 2160

Introduction:

Elevated leukocyte frequency at early stage of polymicrobial sepsis is a common phenomenon, which is considered to be a result of rapid migration of neutrophils from the bone marrow (BM). However, a dramatic increase of granulocytic myeloid-derived suppressor cells (MDSCs) of sepsis usually occurs after sustained stimuli of the pathogens, which implies that emergency granulopoiesis is involved in negative regulation of immune response. In the present study, we try to reveal the mechanisms by which TLR signal regulates emergency myelopoiesis during infection.

Methods and models

:B Mice were subjected to cecal ligation and puncture to make polymicrobial sepsis model. Blood, spleens and bone marrow were harvested post operation at 1, 3, 7 and 12 day, and analyzed using flow cytometry. Hematopoietic stem cells and special progenitors were isolated using an FACS Aria sorter, and then total mRNA was extracted and reverse-transcripted. Some lineage commitment-related genes including Ikzf1,Gfi1, Cebp/a, PU.1, Csf1r, Csf2r, Csf3r, Notch1, IL-7r, Flt3 were analyzed by Real-time PCR. The methylcellulose colony assay of single-cell suspensions of bone marrow cells was used to identify myeloid progenitors in sepsis model.

Results:

We found that the elevated frequency of myeloid cells was associated with an expansion of hematopoietic stem cell (HSC) pool accompanied by augmented proportion of cells entering cell cycle. Furthermore, increased HSC numbers resulted in gradually elevated proportions of common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs), but a decrease of the frequency of common lymphoid progenitors (CLPs). In line with the decreased ratio of lymphoid/myeloid progenitors, real-time PCR revealed impaired expression levels of lymphoid differentiation-related genes in HSCs and CLPs, including Flt3, IL7R, Notch1, Ikaros and Rag1. By contrast, the expression levels of myeloid differentiation-related genes (eg., CEBP/a, Gfi1, PU.1, GM-CSFR) were up-regulated or remained unchanged in the stem cell and myeloid progenitor compartments. Moreover, consistent with elevated frequency of granulocytic MDSCs, an increase of Gfi-1 expression which made a granulocytic decision and a decrease of M-CSFR expression which played a crucial role in monopoiesis were noticed in CMPs and GMPs. More importantly, similar results were obtained in the mice challenged with LPS peritoneal injection.

Conlusion:

Our study sheds light on its importance of TLR-signaling in generation and replenishment of granulocytic MDSCs by affecting early myeloid /lymphoid lineage decision and late granulocyte /monocyte lineage decision.

Disclosures:

No relevant conflicts of interest to declare.