bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymus-Derived PTH (TPTH) Is Increased after Thyroparathyroidectomy in C57BL6/Kalwrij Mice and Modulates Mouse Sensitivity to 5TGM1 Myeloma Cell Line
Authors: Zangari Maurizio , Yoo Hanna , Suva Larry , Yoon Donghoon
Published: October 2019

We have previously reported a significant correlation between serum PTH levels and myeloma responses to proteasome inhibition and have recently demonstrated a crucial role for the parathyroid hormone receptor 1 (PTHR1) in vitro and in vivo. In this study the effect of abrogation of parathyroid-derived PTH was tested in 5TGM1 myeloma transplanted C57BL6/KaLwRij mice. To interrogate PTH activity in vivo, thyroparathyroidectomy (TX-PTX) was performed on the C57BL6/KaLwRij mice before or after the transplantation of 5TGM1 cells. Surgical success was confirmed by the rapid loss of measureable circulating PTH. In TX-PTX mice bearing transplanted 5TGM1 tumors, serum PTH levels was noted to recover 3-4 weeks after surgery and to achieve levels consistently higher than baseline levels pre TX-PTX. These data raised the compelling possibility of an alternative PTH source. To determine the potential source of the recovered PTH levels, multiple organs were examined. Whole tissue lysates were analyzed by Western blot and revealed a faint but specific band from thymic tissue, as expected with no obvious bands detected in 13 other tissues investigated including the liver, lung, kidney, ovary and testis. In addition, murine PTH mRNA transcripts from thymus RNA were measured by Real-Time PCR, using a specifically designed primer set that spans exons 1 and 2, since intron 1 is >1kb. These primers specifically discriminate mRNA transcripts from genomic DNA and improve the assay sensitivity for the detection of PTH transcripts. Using these custom-designed primers, control thymus expressed measureable PTH mRNA transcripts that was significantly elevated in TX-PTX mice. Since the thymus is considerably bigger than the parathyroid glands, PTH was immunoprecipitated before performing Western blot analysis. Following immunoprecipitation, a distinct PTH protein band was observed from the thymus in both TX-PTX and control mice.

These data suggest that the thymus is the major source of serum PTH in TX-PTX mice and that the organ can compensate for the loss of serum PTH from the parathyroid gland. We propose that thymus-derived PTH in the TX-PTX mouse may contribute to the control of the 5TGM1 murine myeloma model.

Disclosures

Zangari: University of Arkansas for Medical Sciences: Employment; Novartis: Research Funding; Millennium: Research Funding; Onyx: Research Funding.
Minimal Residual Disease Analysis in Multiple Myeloma Patients By 6-Color Flow Cytometry: An Experience from Tertiary Care Center of North India
Authors: Sharma Praveen , Sachdeva Man Updesh Singh , Varma Neelam , Bose Parveen , Malhotra Pankaj
Published: October 2019

Therapeutic advances in multiple myeloma (MM) incorporating the use of high-dose melphalan, novel therapeutic immunomodulatory agents, proteasome inhibitors and supporting autologous stem-cell transplantation (ASCT) have improved response rates and overall survival. The detection of minimal residual disease (MRD) is recognized as a sensitive and rapid approach to evaluate treatment efficacy as a tool for predicting patient outcomes and guiding therapeutic decisions. MRD analysis is reflected by many different techniques, however, multiparametric flow cytometry is a sensitive, feasible and adequate method for monitoring residual disease. Studies from India related to this context are lacking. In the present study, we compare MRD levels in patients of multiple myeloma after chemotherapy/ASCT assessed by multiparametric flow cytometry, with M band status, immunofixation (IFE) and percentage of plasma cells on bone marrow aspirate.

Seventeen patients of multiple myeloma were included in the study over a duration of one year, (Male=13, Female=4) with mean age of 56.8 years (range 44-80 years). MRD was analyzed using a dual laser 6 color-flow cytometer in 9 patients of ASCT (day 100) and 8 patients on chemotherapy alone (post-induction). Pre-titrated cocktail of CD38, CD138, CD19, CD45, cytoplasmic Kappa light chain, cytoplasmic lambda light chain, CD81, CD27, CD28 CD200 and CD10 were used in 6-color combination of three tubes for MRD analysis. MRD was detectable in 5 patients, mean of 0.61% (range of 0.07 - 6.44%). M band and IFE were positive in 2 patients, each. Bone marrow plasma cells ranged from 0 to 22%. MRD levels did not show significant correlation with percentage of plasma cells in bone marrow aspirate, however it had an statistical agreement with presence or absence of serum M-band and IFE. Patients are on regular follow up for their clinical and hematological response.

Disclosures

No relevant conflicts of interest to declare.
Orthotopic Heart Transplant Facilitated Autologous Hematopoietic Stem Cell Transplantation in Light-Chain Amyloidosis
Authors: Kamble Rammurti T , Obi Gloria , Scholoff Audrey , Carrum George , Rice Lawrence , Bhimrajan Arvind , Estep Jerry
Published: October 2019

Dominant cardiac involvement by primary systemic amyloidosis (AL) precludes effective AL treatment and is associated with short survival. Between January 2009 and June 2015, total of 9 patients who presented with severe cardiac dysfunction as their major manifestation of AL underwent orthotopic heart transplantation (OHT). Four of these 9 patients were able to complete the planned second phase of treatment with autologous hematopoietic stem cell transplantation (ASCT) 13-16 months after OHT. Diagnosis of cardiac AL was established via endomyocardial biopsy, Congo red staining and immunohistochemistry. All patients had end stage heart failure and developed cardiogenic shock requiring intra-aortic balloon pump support (median 20 days, range 10-165) as a bridge to OHT.

The median age at AL presentation was 54 years (42-65) in 4 females and 5 males. At median follow-up of 30 months (1-54) from OHT, 6 (66 %) patients are alive (table-1). Two patients died of post-operative complications at 1 and 7 months post OHT; a 3rd patient died 36 months after OHT (23 months post ASCT) of AL progression. Four patients received ASCT at median of 13 months (13-16) after OHT. In the remaining 3 patients ASCT was not feasible due; to low DLCO; recently treated disseminated Cryptococcus; or prior ASCT.

All 4 patients with ASCT were on tacrolimus and prednisone at the time of stem cell mobilization and hematopoietic transplant; two patients were also receiving mycophenolate mofetil and valganciclovir. We collected 5.7, 6.1 and 6.2 x 106/kg CD-34+ cells in 2 days after filgrastim administration (5 ug/kg, twice, daily) and plerixafor (16 mg/kg based on day- 4 CD-34+ counts) in 3 subjects. The fourth patient initially failed to mobilize but 4.3x106/kg CD-34+ cells were subsequently obtained after stopping mycophenolate mofetil for 4 weeks. The creatinine clearance at the time of the high-dose chemotherapy given prior to ASCT was 36, 30, 41 and 43 ml/minute. All 4 patients received a renal adjusted dose of melphalan at 140mg/m2. Mycophenolate mofetil and valganciclovir were withheld during neutropenia until engraftment. No patients received post-transplant filgrastim. Patients were hospitalized for 17, 18, 18 and 20 days. Renal function remained stable during ASCT and non-hematological toxicity was limited to grade I-II apart from one grade III oral mucositis and colitis. Two patients achieved hematologic complete remission (patient2&4) while 2 patients had a partial response following ASCT. Post OHT and ASCT 3 patients are alive and well at follow-up of 54, 37 and 52 months.

The strategy of OHT followed by ASCT is therefore feasible in select patients with dominant cardiac involvement and advanced heart failure

Disclosures

No relevant conflicts of interest to declare.
Bortezomib-Induced Proinflammatory Macrophages Contribute to Multiple Myeloma Cell Aggressiveness
Authors: Beyar Katz Ofrat , Ben-Tsedek Neta , Avivi Irit , Alishekevitz Dror , Timaner Michael , Miller Valeria , Lindzen Moshit , Yarden Yosef , Shaked Yuval
Published: October 2019

Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. Previous studies revealed that chemotherapy induces pro-tumorigenic host-mediated effects which could explain tumor re-growth and metastasis(Gingis-Velitski, Loven et al. 2011, Katz, Shaked 2014). Here we show that plasma from bortezomib-treated mice significantly increases migration, viability and proliferation of human MM cells in vitro, compared to plasma from control untreated mice. Comparable results were demonstrated with plasma obtained from patients with MM treated with bortezomib. Additionally, bortezomib induces the mobilization of pro-angiogenic bone marrow cells. Mice treated with bortezomib and subsequently intravenously injected with MM cells succumb to MM aggressiveness earlier than mice treated with the vehicle control(Figure 1). We show that pro-inflammatory macrophages contribute to MM cell aggressiveness in response to bortezomib treatment, in part by secreting interleukin-16(IL-16). Blocking IL-16 in conditioned medium obtained from bortezomib-treated macrophages generated reduced viability of MM cells in vitro. Accordingly, co-inoculation of MM cells with pro-inflammatory macrophages from bortezomib-treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti-tumor activity of bortezomib, this drug can induce host-driven pro-tumorigenic effects that may promote MM aggressiveness.

Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system. Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system.

Disclosures

No relevant conflicts of interest to declare.
Thromboxane a2 Exerts Promoting Effects on the Proliferation of Multiple Myeloma Cells By Reducing G2/M Phase Arrest through the Activation of p38/JNK MAP Kinase Pathway
Authors: Xiong Hong , Yu Yu
Published: October 2019

Background: Multiple myeloma is a kind of frequent hematological malignancy and its effective cure is still deficiency. Whether TXA2/TP affects the proliferation of MM through the MAPK signaling pathway are still uncovered.

Results: SQ29548 inhibits the proliferation of MM cells by arresting cells at the G2/M phase through attenuating cyclin B1/CDK1 expression and p38 MAPK/JNK activation, and inducing cell apoptosis.

Conclusion: SQ29548 has potential as a novel agent to target kinase cascades for MM therapy.

Significance: We have defined a novel physiological role for TXA2/TP in MM cells.

Multiple myeloma (MM) is a plasma cells malignant proliferation disease, and a satisfactory cure is unavailable till now. Thromboxane A2 (TXA2)/TXA2 receptor (TP) has involved the modulations of some carcinomas progression, while their effects on the proliferation of MM cells remain unclear. In this study, we detected cyclooxygenase enzymes (COX) downstream prostaglandin (PG) profiles in human myeloma cell lines U266 and RPMI8226, analyzed the effect of COX-1/-2 inhibitors, SC-560 and NS-398 on the MM cells proliferation. The observations showed that COX-2 involved in the modulation of cell growth. Then, MM cells were incubated with 4 antagonists of DP2, EP2, EP4 and TP receptors which were chosen according to their high transcriptions, and only TP antagonist, SQ29548, suppressed MM cells proliferation. Moreover, SQ29548 promoted MM cells G2/M phase arrest accompanied by reducing cyclin B1 and CDK1 expression at mRNA and protein levels. We also found TP agonist, U46619, activated JNK and p38 MAPK by phosphorylation, and JNK inhibitor, SP60015, and p38 MAPK inhibitor, SB203580, depressed U46619-induced proliferation and protein expressions of cyclin B1 and CDK1. In addition, SQ29548 leaded to MM cells apoptotic rate increasing with improving caspase 3 activity. Taken together, our results suggest that TXA2/TP promote the proliferation of MM cells by reducing cells at the G2/M phase through elevating cyclin B1/CDK1 expression and p38 MAPK/JNK activation, and reducing cell apoptosis. It has potential as a novel agent to target kinase cascades for MM therapy.

Disclosures

No relevant conflicts of interest to declare.
Identification of a CLL Cancer Stem Cell
Authors: Kellner Joshua , Yvon Eric , Gordon Veronica , McNiece Ian K.
Published: October 2019

To date cancer research and therapeutic approaches have mainly targeted the cellular mechanisms of aberrant proliferation. These treatments do shrink tumors, but after a remission of variable length, most patients relapse with frequent drug resistance and metastatic dissemination. These features indicate that within a tumor exists a population of cells that is inherently resistant to therapies and are able to reinitiate tumor growth and development. This is the basis of the cancer stem cell (CSC) theory. Targeted elimination of CSCs is actively being explored as a new therapeutic approach in hemato-oncology with a focus on cell surface antigens expressed on CSCs for antibody- or ligand-based therapeutic approaches. Most studies have defined CSCs functionally by their tumor initiating ability, when inoculated into immune-deficient (NSG) mice. These xenograft models have been powerful preclinical tools in a number of malignancies, with the notable exception of chronic lymphocytic leukemia (CLL).

We have recently isolated a primary cell line from the bone marrow BM of a patient with CLL, which we have termed MDA-BM5, and have successfully passaged this line through six serial transplants in NSG mice. The mice die of disease around 1 month post transplant and analysis of organs has demonstrated the presence of CD19+CD5+ cells with Ig light chain restricted to lambda which is consistent with the original patient samples. BM cells from tertiary transplant NSG mice demonstrate a stem cell phenotype; including expression of CD34 and aldehyde dehydrogenase (ALDH). Treatment of MDA-BM5 cells in vitro with inbrutinib is effective in killing 99% of cells, however, continued culture after removal of ibrutinib results in propagation of a "resistant" subpopulation. Based upon these data we propose that the novel cell line (MDA-BM5) contains a CSC subpopulation which replicates human CLL in NSG mice. Further characterization of the CLL-CSC is ongoing to identify potential therapeutic targets.

Disclosures

No relevant conflicts of interest to declare.
The Targeting Repair of UC-MSCs Homing on Immune Inflammatory Microenvironment and Thrombophilia in CIA Rats
Authors: Cai Xinzhen , Ni Jun , Wu Wei , Shi Qingqing , Li Zou , Gu Wei , Gu Jian , Depei Wu
Published: October 2019

Introduction

To preliminary study the repair effect of umbilical cordmesenchymal stem cells (UC-MSCs) homing on local and systemic inflammatory microenvironment and immune inflammatory thrombophilia states of the CIA rata by observing the distribution of the UC-MSCs in the CIA rate and the influence of the UC-MSCs on the expression of the inflammatory cytokines IL-10, TNF-α IL-6, IFN-γ and the thrombosis indicators TF, VWF, DD, FIB's.

Methods

The clean grade, female, 5-week-old SD rats were randomly divided into a control (C) group, model (M) group, UC-MSCs treatment (SU) group, adding AMD3100 to labled UC-MSCs therapy (ASU) group. Except for control group, the other rats were induced as CIA rats model. Treatment group were injected UC-MSCs suspension by tail vein. The rats were sacrificed in the first, the third and the fifth week after transplantation. HE staining was used to observe the pathological changes of joint tissues. The distribution of UC-MSCs in the joint tissue was detected by FISH. ELISA assay was used to observe the expression of inflammation and thrombosis indicators in peripheral blood. The expression of inflammatory factors in the joint tissue were detected by western blot.

Results:

1. One week after injection, the expression of SDF-1 in the injuried joint of the group SU was significantly increased compared with the control group, at the same time, the large number of UC-MSCs occured in injured sites. While, adding AMD3100 to labled UC-MSCs were not expressed in the joint tissue. The expression of SDF-1 in the labled UC-MSCs treating group decreased over time, and the number of UC-MSCs reduced in the inflammatory joints.

2. After given UC-MSCs treatment, the levels of pro-inflammatory cytokines IL-6, TNF-α, IFN-γ in the knee and serum were conspicuously reduced compared with the group M since the first week. While the level of anti-inflammatory cytokine IL-10 was increased (p <0.05). After adding AMD3100, the expression of above indicators in the group ASU showed no significant difference compared to the group C.

3. After given UC-MSCs treatment, the levels of TF in serum and DD, FIB, VWF in plasma were conspicuously reduced compared to the group M since the first week (p <0.05). The expression of the above indicators in the group ASU showed no significant difference compared to the group C.

Conclusion:

1. UC-MSCs homing to the injured joint tissue is influnced by the local inflammation environment, which is an important way to play its role of immune regulation to improve the immune inflammatory thrombophilia state in CIA rsts.

2. SDF-1/CXCR4 axis is important to the UC-MSCs homing, the antagonist AMD3100 can suppress the UC-MSC homing to the injured site.

Funded by Jiangsu Provincial Special Program of Medical Science (BL2012005)

Disclosures

No relevant conflicts of interest to declare.
HO-1-Transduced Mesenchymal Stem Cells Attenuate aGVHD By Inhibiting HIF-1α /ROR-γt and then Down-Regulate Proportion of TH17/Treg
Authors: Wang Jishi , Yu Meisheng , Chen Shuya , Zhao Jiangyuan , Zhang Yaming , Lin Xiaojing , Zhe Nana , Fang Qin
Published: October 2019

Background. In acute graft versus host disease, the balance pattern of Treg/Thl7 cells are destroyed, leading to a series of inflammation and damage to the body, greatly affect the survival of allogeneic hematopoietic stem cell transplantation patients. Mesenchymal stem cells can regulate the body's balance of TH17/Treg cells, clinically used to prevent and treat aGVHD.But between immune adjustment ability of mesenchymal stem cells in clinical trials have not been shown to efficiently prevent and treatment of GVHD. HO - 1 as a significant anti-inflammatory and immunosuppressive molecules in rats and human MSC played a crucial role in immune suppression function.In this article, we discuss slow virus in mice mediated transfection MSC HO-1 key nuclear transcription factor can inhibit the TH17 STAT3 activation to further decline, the proportion of TH17/Treg and whether more effectively adjust the related the secretion of cytokines to alleviate aGVHD caused by inflammation of the target organ damage, in addition to observe the clinical patients undergoing allogeneic transplantation patients occurred aGVHD HO-1 high expression and the relationship between the expression of STAT3 and the secretion of peripheral blood serum of TH17/Treg related factors.

Methods.Lentivirus transfection raised for rat mesenchymal stem cells between the expression of HO-1, trailing edge vein injection of mice treated, fluorescence microscopy MSC colonize the target organs of mice, pathological biopsy to detect target organ damage, immunohistochemical detection of target organs and the spleen and the expression of pSTAT3, HO-1 flow cytometry to detect the spleen, drainage of lymph node ratio of TH17/Treg cells, Realtime PCR and ELISA to detect the related inflammatory factor levels.

Results. The mouse in accepting HO-1 high expression of mesenchymal stem cells after infusion, significantly reduce aGVHD, immunohistochemical results show that the main target organs damage in the expression of HO-1 is inversely proportional to the expression of STAT3 and spleen pSTAT3 protein and total level of STAT3 protein was lower than other groups, the spleen and lymph nodes of TH17/Treg proportion are lower than other groups, IL-17 A, ROR-ϒt mRNA level and serum concentration is lower than other groups, FOXP3, TGF-β mRNA and serum concentration is higher than in the rest of the group.

Conclusions. These results suggest that activation of HO - 1 May improve the anti-inflammatory and immune suppression ability of MSC, and perhaps by inhibiting the phosphorylation of STAT3, cut ratio of TH17/Treg cells, promote reduce aGVHD.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell Mobilization after Various Induction Regimens in Patients with Multiple Myeloma
Authors: Radocha Jakub , Maisnar Vladimir , Lanska Miriam , Hanousek Jiri , Machalkova Katerina , Beranova Katerina , Zavrelova Alzbeta , Motyckova Monika , Zak Pavel
Published: October 2019

Stem cell mobilization after various induction regimens in patients with multiple myeloma

Introduction: Rapid development of novel therapies for multiple myeloma has led to a significant improvement in response to the treatment. Stem cell mobilization before autologous stem cell transplantation is a source of considerable costs of transplant procedure. Whether modern induction regimens affect outcome of stem cell mobilization has not been extensively studied.

Aim: The goal of this study was to compare efficacy of stem cell mobilization after different induction regimens in patients with multiple myeloma. The primary goal was to compare CTD (cyclophosphamide, thalidomide, dexamethasone), CVD (cyclophosphamide, bortezomib, dexamethasone) and VTD (bortezomib, thalidomide, dexamethasone) and regimens in terms of succesful stem cell collection.

Methods: All patients with multiple myeloma who have been planned for stem cell collection and were treated with one of the above mentioned regimens were included in this retrospective analysis. The demographic data, amount of stem cells collected, number of days needed to reach the target collection were recorded. All patients received high dose cyclophosphamide 2,5 g/m2 prior to stem cell collection and were primed with G-CSF twice daily from day 5. The collection was started at day 10. Collection goal was 8x106/kg CD34+ cells.

Results: 15 patients received CTD, 25 patients CVD and 16 patients VTD regimen before stem cell collection. Groups were comparable according to age, gender and myeloma stages. Mean collected cells at the end of collection were 9.2 (SD 2.8) for CTD, 12.3 (SD 5.6) for CVD and 10.1 (SD 2.1) for VTD (p=0.066). Mean daily harvest was 3.4, 8.0 and 7.6 x106/kg respectively (p=0.01). Mean days needed to reach desired harvest were 3, 2.25 and 1.6 days (p=0.001). No collection failure was observed.

Conclusion: The best collection results were seen in patients after induction with CVD or VTD regimen. VTD regimen also required the least days for collection and seems to be most beneficial for cost of collection. CTD regimen shows the least efficacy in stem cell collection before autologous transplantation. All patients managed to harvest for at least one stem cell transplant.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Chemotherapy Combined with Granulocyte Colony Stimulating Factor (G-CSF) for Mobilization of CD34+ Cells in Lymphoma and Multiple Myeloma Patients
Authors: Zheng Gaofeng , Zheng Yanlong , Luo Yi , Shi Jimin , Zheng Weiyan , Wei Guoqing , Sun Jie , Huang He , Cai Zhen , He Jingsong
Published: October 2019

Objective: To investigate and analyze factors which effect autologous stem cell collection in patients with lymphoma and multiple myeloma (MM) during chemotherapy combined with G-CSF mobilization, for improving quality and effectiveness of autologous stem cell transplantation.

Methods: A retrospective analysis was performed from April 1, 2006 to October 31, 2013 in our hospital and 128 lymphoma and MM patients whose autologous peripheral blood stem cells (PBSCs) were collected including 75 patients with malignant lymphoma,7 cases of Hodgkin's lymphoma and 68 non-Hodgkin's lymphoma (NHL) cases as well as 53 MM patients were enrolled. The stem cells of all patients were mobilized by chemotherapy combined with G-CSF and collected via a continuous flow cell separation instrument (COBE Spectra, Lakewood, CO). Mobilize failure was defined when the amount of CD34 + cells was less than 2.0 x 106 / kg, whereas ≥2.0 * 106 / kg was defined as successful mobilization. More than 5.0x 106 cells / kg or more was considrered as ideal mobilization. Univariate and multivariate regression analyses of factors for mobilization failure, successful mobilization and ideal mobilization acquisition were performed.

Results: There were more CD34+ cells in MM patients than in lymphoma patients (P = 0.064). The collection rates of CD34 + cells in MM patients were ≥ 2.0 x106 / kg in 64.8% (83 cases) and ≥ 5.0 x 106 / kg in 35.2% (45 cases). MM patients with a success collection ratio was 73.6 % (39/53) and the ideal collection rate was 43.4% (23/53), which was higher than in the NHL group with a success rate and ideal rate of 58.7% (44/75) and 30.7% (23/75). A total of 35.2 % (45 cases, including 31MM cases and 14 lymphoma cases) a mobilization was not successful.

Conclusion: In different chemotherapy regimens in patients with lymphoma, remission, ever use MTX and/or Ara-c treatment and collecting the outer peripheral hematocrit could significantly affect the success rate of stem cell collection; In MM patients, who received lenalidomide treatment and multiple courses of treatment, still not got CR, which these reasons were the factors of non- successful mobilization.Although Plerixafor and peripheral blood CD34-positive cell counts could help to improve the success collection rate and predict collection rate, but there is still a need for further improvement of the current mobilization protocols, recognizing the ideal stem cell collection dynamics, efficiency and cost in order to select the appropriate mobilization protocols.

Disclosures

No relevant conflicts of interest to declare.
Reconstitution of Memory T -Cells after Allogeneic Stem Cell Transplantation in Patients with Post-Transplantation Cyclophosphamide As Tolerance Induction
Authors: Vasilyeva Vera , Parovichnikova Elena N. , Kuzmina Larisa A. , Drokov Mikhail , Mikhaltsova Ekaterina , Koroleva Olga , Dubnyak Daria , Galtseva Irina , Savchenko Valeri G.
Published: October 2019

Introduction.

Nowadays high-dose post-transplantation cyclophosphamide (CY) replace standard immunosuppression (IST). Thereby, the investigation of T-cells reconstitution after post-transplant-CY doesn't reach appropriate level, and probably it's very different from what we see after standard IST. We studied the reconstitution of memory T-cells on day of engraftment (WBC>1000 cells\us) after allogenic hematopoietic stem cell transplantation (allo-HSCT) with post-transplant-CY and standard immunosuppression therapy.

Patients and methods.

During 2 years, 29 patients with different hematological malignancies were included in this study. Median of age was 36 years (24-60 years). 16 patients were males, 13 - females. 22 received RIC, 7 - myeloablative regime. Match unrelated donor (MUD) was in 17 cases, "Mismatch" MUD - 2, Match related donor (MRD) - 9, "Mismatch"MRD - 1. 4 patients were in relapse or disease progression. CY as alternative IST was administrated to 6 patients. Standard immunosuppression consisted of CSA, MMF or MTX at standard dose. Peripheral blood samples were collected in EDTA-tubes at day of engraftment after allo-HSCT (Me= 20 day (14-35)). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD4- APC-Cy7 antibody (Becton Dickinson, USA) and FSC/SSC were used for determine population of CD4+T-cells. Anti-CD45R0- FITC (Becton Dickinson, USA) antibody were used to determine memory T-cells as subpopulation of CD4+ T-cells. Due to cyto- and lymphopenia 1,000,000 events was analyzed.

Results.

Mann-Whitney U test was used to test for differences between memory T-cells (CD4+ CD45R0+) after post-transplant-CY alone and in a group with standard IST. The percent of memory T-cells in CD4+ cell population at day of engraftment after post-transplant CY alone was statistically higher (74,3% ± 5,1% ,p=0.048*) than in patients with standard immunosuppression (49,4%±6,7%).

Conclusion.

We may conclude that patients with post-transplant CY had a different "T cell reconstitution profile". Reported data show us that probably post-transplant CY spares memory T-cells in contrast with standard IST, and also probably that CY is more selectively immunosuppressor than "gold standard" (such as CSA, MMF and etc.) not only on effector T-cells population. Despite the fact that the analyzed group is small, obtained data is important and needs further investigation.

Disclosures

No relevant conflicts of interest to declare.
Thymoglobulin Does Not Impact Relapse or Survival after Matched Unrelated Transplantation for AML/MDS Compared to Matched Related Donor Transplantation without Thymoglobulin Use
Authors: Khaled Yasser , Patel Rushang D. , Mori Shahram , Ahmed Wesam B. , Solh Melhem
Published: October 2019

Thymoglobulin does not impact relapse or survival after matched unrelated transplantation for AML/MDS compared to matched related donor transplantation without Thymoglobulin use.

Background: Thymoglobulin has been used customarily after matched related (RD) and unrelated (MUD) hematopoietic stem cell transplantation (HCT) to decrease risk of GVHD and graft rejection. However, its use has been implicated in increasing the risk of relapse. As matched sibling related donors considered the best available donor source for patients with AML /MDS, we hypothesized that production of outcomes (Relapse/Survival) after MUD transplant with use of thymoglobulin similar to RD transplantation without the use of thymoglobulin, would justify the use of thymoglobulin after MUD HCT in AML/MDS.

Method: We compared the outcomes of 98 (29 matched RD/ 69 matched MUD) consecutive patients with AML/MDS who underwent HCT with Fludarabine/ Busulfan based conditioning between 7/2009-12/2014. Patients Characteristics are shown in Table-1. Patients received full intensity conditioning (FIC) with Fludarabine 40 mg/m2 for 4 days and Busulfan 3.2 mg/Kg single daily dose for 4 days (AUC 4200-5000). GVHD prophylaxis for FIC was Tacrolimus/mini Methotrexate and Tacrolimus/Mycophenolate for RIC. Thymoglobulin was added at dose of 1.5 mg/Kg for 3 days for MUD patients only regardless the conditioning intensity.

Results: With median follow up 696 days (RD 746 days, MUD 676 days), the overall survival at 1 year was 75% +/- 8% and 74% +/- 5% for RD and MUD respectively, P=0.66. All patient engrafted except two patients in FIC MUD group. The cumulative incidence of relapse at 1 year was 31% +/- 8% and 23% +/- 5% for RD & MUD respectively, while the treatment related mortality (TRM) was 3.5% +/- 3% and 10% +/- 3% for RD and MUD HCT recipients. Cumulative Incidence acute GVHD grade II-1V was 29% in RD and 49% in MUD. Grade III-IV acute GVHD was 14% in Both RD and MUD recipients. Higher cumulative incidence of extensive chronic GVHD was observed in MUD recipients of 28% vs 20% in RD recipients.

Conclusion: The addition of thymoglobulin to MUD HCT did not increase the cumulative incidence of relapse compared to RD HCT patients who did not receive thymoglobulin. RD HCT remained associated with lower TRM than MUD HCT despite the addition of thymoglobulin. MUD HCT with thymoglobulin produced similar overall survival to RD HCT without thymoglobulin. Our data suggest continued use of low dose thymoglobulin with MUD HCT while avoiding its use with matched RD HCT.Table 1.PatientsRelated DonorUnrelated DonorNumber2969AML2261MDS78Graft Failure02Age (Range)53 (35-71)56 (25-73)Reduced Intensity825Full Intensity2144CR11747CR245Relapsed/Refractory817

Disclosures

No relevant conflicts of interest to declare.
Clinical Analysis of Invasive Fungal Infection after Allo-Geneic Stem Cell Transplantation
Authors: Ni Ming , Chen Baoan , Ding Jia Hua , Lin Qiang , Ma Yu
Published: October 2019

Object: The study was aimed to analyze the incidence, risk factors and outcome of invasive fungal infection (IFI) in allogeneic hematopoietic cell transplantation (HSCT) recipients, and to provide evidence for the prevention and treatment of IFI.

Method: 79 patients undergoing formed HSCT at this institute from Jan 2005 to Sep 2014 with intact datum, were analyzed retrospectively according to the diagnostic criteria of IFI. The data of age, sex, primary disease, the source of stem cell, HLA-identical situation, ABO matching, conditioning regimen, use of ATG, season, aGVHD, neutropenia less than 0.5×109/L, CMV infection, etiology culture, blood routine test, imaging, relapse of disease and survival were recorded. Moreover, the data of patients with IFI including usage of glucocorticoid and the neutrophil count were recorded when IFI occurred and the blood cells count after IFI.

Result: 17 patients has been diagnosed with IFI, in which 13 cases were probable and 4 cases were possible. The infection rate was 21.5%. There were 8 female and 9 male. The median age was 26 years (6-50 years).The total non relapse survival (NRS) rate was 55.6%, The NRS rate in non-infection group was 61.2% and that in infection group was 35.2%. The single-factor analysis showed matching(x2=15.061, p=0.010) and aGVHD (x2=6.983 p=0.008) were risk factors. After IFI, 8 patients died, 2 patients got complete response, 5 patients got partial response and 2 patients got stable response. The death rate was 47%, there was significant difference between two groups. The dose of glucocorticoid before IFI within 30 days was risk factor. Multiple-factor analysis further showed Ⅱ-Ⅳ degree of aGVHD (OR=4.810 p=0.012 CI:1.406-16.459 ) and matching (OR=25 p=0.007 CI:2.380-262.653) were risk factors. Moreover, before IFI, use of methylprednisolone more than 360 mg before infection (OR=0.041 P=0.017 CI:0.003-0.56) was risk factor.

Conclusion: Patients with poor matching, unrelated stem cell and Ⅱ-Ⅳ degree of aGVHD are more likely to occur IFI and those who were used more glucocorticoid are likely to die. Therefore, avoiding the risk factors might reduce the incidence rate of IFI and respect using dose of immunosupperssor before infection within 30 days may improve the patients' prognosis.

Disclosures

No relevant conflicts of interest to declare.
Towards Restoring Coagulation in Hemophilia By Antibody-Mediated Inhibition of Antithrombin
Authors: Boross Peter , Yildiz Cafer , Bakhtiari Kamran , Meijers Joost C.M. , Hack C. Erik
Published: October 2019

Currently hemophilia A is mainly treated with factor VIII (FVIII). However, 20-25% of the hemophiliacs develop inhibiting antibodies (inhibitors) against FVIII. Hemophiliacs with inhibitors are treated with high dose FVIII or with therapies that bypass the need for FVIII such as activated FVII (rFVIIa, NovoSeven) or activated prothrombin concentrate. These have some disadvantages including rapid clearance, price and a risk of thrombo-embolism.

In hemophilia, the balance between coagulation and anticoagulation is shifted towards the latter. This balance can be restored by administration of clotting factors but also by reducing anticoagulation mechanisms. Antithrombin (AT) constitutes a main anticoagulant mechanism as it inhibits thrombin, FXa and other activated clotting factors. In silico studies as well as observations that AT deficiency reduces bleeding in hemophilic mice and humans strongly support the concept that lowering functional AT levels bypasses the need for FVIII to generate thrombin. A Phase 1 clinical trial by Alnylam Pharmaceuticals showed that lowering AT activity by RNA-interference can boost thrombin generation in hemophilia. However, RNA-interference is an experimental therapy with unknown long term effects and is not suitable for acute treatment of bleeding as it takes weeks to become effective.

We hypothesize that monoclonal antibodies (mAbs) that block AT activity can be used as FVIII bypass therapy. Such a mAb-based therapy is suitable for on demand and for prophylactic therapy, is relatively cheap and does not require administration of activated clotting factors.

We have generated a panel of mouse anti-AT mAbs exhibiting varying binding characteristics to human AT. To assess the inhibitory activity of anti-AT mAbs, FXa activity assay was performed after FXa was incubated with normal plasma supplemented with the different anti-AT mAbs. Five anti-AT mAbs were identified that inhibit AT activity at around equimolar ratio. Inhibitory anti-AT mAbs were able to dose-dependently enhance thrombin generation in normal and hemophilic plasma. Heparin caused a marked prolongation of aPTT of normal and FVIII-deficient plasma, which was almost completely normalized by addition of the inhibitory mAb anti-AT 5G1.1, but not by anti-AT 6E7.2, indicating that these mAbs exert their inhibitory activity via different mechanisms.

These data provide initial in vitro proof-of-concept that inhibitory anti-AT mAbs can be used to improve coagulation and may be used as FVIII bypass therapy.

Disclosures

Boross: Prothix B.V.: Employment. Hack:Prothix B.V.: Equity Ownership, Patents & Royalties.
HSCT Improve Long-Term Outcome for Patients with T-Cell Lymphoma / Leukemia (TCL)
Authors: Wang Chunyan , Zheng Runhui , Qin Pengfei , Huang Zhenqian , Tan Huo
Published: October 2019

Objective To evaluate the treatment outcomes of hematopoietic stem cell transplantation (HSCT) in patients with T-cell lymphom / leukemia (TCL),observe hematopoietic reconstitution and transplant-related complications.

Methods Seventeen TCL patients received HSCT from 2005 to 2013 in our hospital were retrospectively analyzed.Of the 17 patients,6 were T lymphoblastic lymphoma / leukemia (T-LBL),3 were Peripheral T-cell lymphoma / leukemia (PTL),2 were Anaplastic large cell lymphoma (ALCL),2 were Extranodal NK / T-cell lymphoma (NK / TCL),1 were Angioimmunoblastic T-cell lymphoma (ATCL) and 3 were Acute T lymphoblastic leukemia (T-ALL) with a median age of 35(19~67) years old.Ratio of male to female is 13F4. The distribution of stages was 2(14.2%),0(0%)and 12(85.7%) patients with stages II,III,and IV,respectively. The distribution according to international prognostic index (IPI) was 8(57.1%) patients in 2 score,1(7.1%) in 3 scores and 5 (35.7%) in 4 scores.

Results ‡@17 patients were successfully engrafted, the median time of absolute neutrophil count(ANC) > 0.5×109/L was 11(8~15) days, the median time of platelet > 20×109/L was 13(8~18) days. 3 cases developed I degree acute GVHD, all patients were improved after treatment. ‡AAt a median follow-up of 36 (range,12-96) months,12 patients were alive and 5 relapsed and died from TCL after ASCT. The estimated 5-year probability of DFS and OS was (72.1±15.2)% and (59.7±15.2)% for all the patients.

Conclusion The results showed that HSCT may significantly improve long-term outcome for patients with T-cell lymphoma / leukemia (TCL).

Disclosures

No relevant conflicts of interest to declare.
Long-Term Outcomes after Myeloablative Allogeneic Hematopoietic Cell Transplantation for Adult Acute Lymphoblastic Leukemia
Authors: Mustafa Ali Moaath , Abounader Donna M , Rybicki Lisa A. , Starn Jamie , Ferraro Christina , Winslow Victoria , Hamilton Betty K. , Gerds Aaron T. , Liu Hien , Dean Robert M. , Hanna Rabi , Kalaycio Matt , Bolwell Brian J. , Advani Anjali , Majhail Navneet S. , Sobecks Ronald
Published: October 2019

Allogeneic hematopoietic cell transplantation (alloHCT) is a curative therapy for high-risk acute lymphoblastic leukemia (ALL). However, long-term outcomes after alloHCT for adult ALL have not been well described. We conducted a retrospective cohort study of 72 consecutive adult ALL patients who underwent a first myeloablative alloHCT at our institution from January 2000-December 2013. Median age at HCT was 38 yrs (range, 18-62), 40 (56%) were male, 18 (38%) had high HCT CI score, 14 (19%) had prior CNS leukemia and 35 (49%) had BCR-ABL+ disease. Donor source was HLA-matched related donor for 50% patients and 90% received PBSC as graft source. All patients were transplanted in CR (72% were in 1st or 2nd CR) and 92% received T-cell replete grafts. Median time from diagnosis to alloHCT was 5 months (range, 2-90). The incidences of grade II-IV and III-IV acute GvHD, chronic GvHD and extensive chronic GvHD were 43%, 13%, 51% and 36%, respectively. The median follow-up for our cohort is 76 months. At 6 years after HCT, probability of overall survival (OS) was 33% (95% CI, 21-44%) and relapse-free survival (RFS) was 30% (95% CI, 19-42%), and the cumulative incidence of relapse was 36% (95% CI, 25-48%) and non-relapse mortality (NRM) was 37% (95% CI, 26-49%). The most common causes of death were relapse (43%) and infection (21%); majority of relapses occurred within the first 2-years post-transplantation. There were no second cancer related deaths. In multivariable analyses, factors significantly associated with OS were HCT CI score (HR 2.69 for high vs. low/int., P=0.002) and CMV status (HR 2.62 for donor+ vs. others, P=0.05). HCT CI score was the only predictive factor for RFS (HR 2.26 for high, P=0.007). We also compared outcomes by BCR-ABL status. BCR-ABL+ patients were older (median age 42 vs. 36 yrs, p=0.02), had low HCT CI score (34% vs 22%, p=0.01), were more likely to be in CR1 (74% vs. 32%, p=0.002), and as a result, proceeded to HCT sooner after diagnosis (median 4 vs 7 months, p=<0.001). For BCR-ABL+ and BCR-ABL- patients, 6 year OS was 41% and 25%, RFS was 40% and 21%, relapse was 27% and 45% and NRM was 38% and 36% (P=NS for all comparisons). Myeloablative alloHCT can provide long-term survival for selected high-risk adult ALL patients. Relapses are relatively uncommon after 2 years post-transplant. Long-term NRM is high in this population and we did not observe a plateau in its incidence until 7.5 years post-transplant, suggesting the need for long-term follow up to prevent and manage late complications of alloHCT.

Figure 1. Figure 1.

Disclosures

Majhail: Gamida Cell Ltd.: Consultancy; Anthem Inc.: Consultancy.
Transfection CXCR3 Gene into Lymphocytic Leukemia Cell Lines By Lentiviral Vector
Authors: Ding Wenjiao , Feng Xiaoqin , Li Chunfu
Published: October 2019

Objective: Chemokine receptors and their ligands play important roles in the leukemia-initiating cell activity ,BCP-ALL progression and CNS infiltrating. To study the role of chemokine receptor CXCR3 in the mechanism of leukemia cell infiltration of the CNS, CXCR3 overexpression cell lines should be established. Lentivirus generally does not interfere with proto-oncogene and can express long-term and stable, so it is a good vector for genetic research on hematopoietic cells and T cells.

Methods: Mouse CXCR3 gene fragment was extracted by PCR from T vector which was kept in our lab. After the extraction, CXCR3 fragment and linearized LV5 lentivirus overexpressing vector(with GFP+-puro) were digested with NotI and BamHI.After this,we used T4 DNA ligase to connect them, making the target gene (CXCR3) cloned into LV5 vector, to obtain recombinant shuttle plasmid. Next,we mixed the shuttle plasmid with packaging plasmids (pGag/Pol, pRev, pVSV-G), packaged lentivirus, collected, tested viral titer and sequenced target gene by DNA sequencing. Then the constructed CXCR3 overexpression lentivirus transfected Jurkat and L1210 cell lines, at the same time MOI achieved to 100, twice infection and adding polybrene 5ug/ml to increase lentivirus transfection efficiency. After GFP expression was observed under fluorescent microscope, we used puromycin to select transfected cells and tested the expression of CXCR3 on cell surface by flow cytometry analysis of Anti-Mouse CD183 (CXCR3).

Results: After we sequenced the target mouse CXCR3 gene, viral titer reached to 1×10^9 TU/ml and GFP expression was observed after transfection 96h (<10%) on lymphocytic leukemia cell lines. Through puromycin selection, GFP+ cells surpassed 90% and CXCR3 overexpression group had upregulated 30%-90% compared with control group.

Conclusion: Chemokine receptor CXCR3 had been successful transfected into lymphocytic leukemia cell lines and had stably high expression. The effect of CXCR3 in leukemic invasion and leukemia progression can be further studied using constructed CXCR3 overexpression lymphocytic cell lines.

Disclosures

No relevant conflicts of interest to declare.
Cell Line-Dependent Synergy Between the PARP Inhibitor Veliparib and the Proteasome Inhibitor Bortezomib in the Killing of Myeloma Cells
Authors: Xu Alex , Mazumder Amitabha , Borowiec James A.
Published: October 2019

One hallmark of multiple myeloma is its genetic instability, leading to extensive molecular heterogeneity. This has led to the hypothesis that a high level of DNA repair is needed to counterbalance the continual genotoxic stress seen in these cells. One important DNA repair pathway involves PARP (poly-ADP ribose polymerase 1 and 2). With the goal of developing improved therapies against myeloma, we examined for potential synergy between the PARP inhibitor veliparib and agents currently used to treat myeloma.

In these pre-clinical studies, various myeloma cell lines including OPM2.2 and RPMI 8226 are being tested. Cells in triplicate are exposed to myeloma therapeutics (e.g., bortezomib, doxorubicin, cyclophosphamide) in the presence and absence of a sub-lethal dose of veliparib. Cells are incubated for 48 h and viability then assayed by luminescence using CellTiter-Glo (Promega).

Dependent on the cell line, we found that veliparib can be synthetically lethal with other agents. For example, testing OPM2.2 cells, we found that 50 µM veliparib caused a ~7-fold decrease in the bortezomib LC50 from 7.1 nM (bortezomib alone) to 1.0 nM (bortezomib and veliparib; see Figure). We note that the concentration of veliparib employed was significantly below the LC50 of veliparib (410 µM) for these same OPM2.2 cells. In contrast, RPMI 8226 cells did not show a significant synergy between bortezomib and veliparib, even though the LC50 of each agent alone was similar to that found for OPM2.2 cells.

We are currently examining the synergy between veliparib and other chemotherapeutic agents and additional myeloma cell lines. These studies will determine the generality of a potential synthetic lethality between these agents, and reveal whether use of a PARP inhibitor has the potential to provide improved treatment options against myeloma.

Disclosures

No relevant conflicts of interest to declare.