bluuebunny/crossref_metadata_2025 huggingface dataset:
Allogeneic and Autologous Transplantation In Patients with Aggressive B- and T-Cell Lymphomas
Authors: Schueler Frank , Hoffmann Madelaine , Pfrepper Christian , Daneschnejad Said , Lange Thoralf , Pönisch Wolfram , Al-Ali Haifa K. , Niederwieser Dietger W , Basara Nadezda
Published: October 2019

Abstract 3544

The prognosis of patients with aggressive relapsed or refractory B- or T-cell lymphomas is poor with conventional chemotherapy. Therefore, high-dose chemotherapy followed by autologous or allogeneic transplantation is the treatment of choice in these patients.

We investigated the outcome of patients with aggressive B- or T-cell lymphomas transplanted between 1998 and 2009 in one centre. A total of 104 patients with diffuse large B-cell lymphoma (DLBCL) (n=69) and T-lymphoblastic lymphoma (T-LBL) (n=35) received autologous (auto) (n=66; DLBCL: 68%; T-LBL: 32%) or allogeneic (allo) (n=38; DLBCL: 63%; T-LBL: 37%) HSCT. Patients ineligible for auto transplantation, who did not respond to prior chemotherapy or had bone marrow involvement, were assigned to allo transplantation. Allo HSCT recipients were more likely to have high risk disease (higher disease stage, more prior chemotherapy regimens, resistant disease). Recipients of auto HCT were more likely to have chemosensitive disease at HSCT compared to patients at allo HCT (70% vs. 30%). Median age at transplantation was 52 years for auto and 42 years for allo HSCT. Median follow up was 12.7 (0.2-106) months after auto and 6.4 (0.4-111) months after allo transplantion. All patients with auto HSCT received BEAM and patients with allo HSCT TBI-based preparative regimen (myeloablative: n=23; reduced intensity: n=15).

In the cohort of 104 patients the estimated 5-years overall (OS) and progression free survival (PFS) were 38% and 44%, respectively. There was no difference in OS and PFS between DLBCL and T-LBL (p=0.44). The estimated OS and PFS after allo HCT were significant lower (25% and 37%) than after auto HCT (45% and 47%; p=0.008 and p=0.03) caused by outcome differences in patients with DLBCL (p=0.02). Interestingly, OS and PFS were not different in patients with T-LBL transplanted with auto or allo HSCT (p=0.55 [OS] and p=0.34 [PFS]). The estimated PFS was significantly better in chemosensitive than chemorefractory patients (p=0.0001), however the OS and PFS were similar in chemosensitive group of patients regardless auto or allo HSCT (PFS: 50% vs. 51%; p=0.57; OS: 49% (auto) vs 32% (allo); p=0.19). Treatment related mortality (TRM) at day 100 was 18% after allo HSCT compared to 8% after auto.

In conclusion, allo HSCT in patients with aggressive B- or T-cell lymphoma ineligible for auto transplantation is an attractive treatment option for these patients with high risk of relapse with a considerably low TRM. The response status at the moment of HSCT is the most important parameter affecting either OS or PFS.

Disclosures:

Niederwieser: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Haploidentical Hematopoietic Stem Cell Transplantation In Hematologic Malignancies with G-CSF Mobilized Bone Marrow Plus Peripheral Blood Stem Cells Grafts without T Cell Depletion: a Single Center Report of 29 Cases
Authors: Wang Hengxiang , Yan Hongmin , Wang Zhidong , Xue Mei , Zhu Ling , Liu Jing , Ding Li
Published: October 2019

Abstract 3552

Introduction:

Haploidentical Hematopoietic stem cell transplantation (Haplo-HSCT) has provided an alternative option since virtually all patients have an immediately available donor. In order to reduce the occurrence of severe acute GVHD, initially we used donor bone marrow with G-CSF mobilization as hematopoietic stem cell source and achieved good effect. However, as this work carried out widely, it is difficult to collect enough bone marrow cells from donor due to the much differences body weight between the recipient and donor, and lead to patients with hematopoietic recovery slowly. From February 2003 we started trying to use bone marrow and peripheral blood stem cell as stem cell source.

Patients and Methods:

Twenty-nine patients with hematologic malignancies were enrolled in this study between February 2003 and August 2007 at the general hospital of air force. Conditioning regimen consisting of high-doses of cytarabine and cyclophosphamide with total body irradiation, while 6 cases were preconditioned with busulfan, cytarabine and cyclophosphamide. aGVHD was prevented by a combination of immunosuppressive drugs including a monoclonal antibody against human CD25 (basiliximab), cyclosporine A (CsA), methotrexate (MTX), mycophenolate mofetil (MMF), and a rabbit anti-thymocyte globulin. Donors were given G-CSF at a dose of 300μg daily for 6 consecutive days prior to marrow harvesting. Peripheral blood stem cell was collected on the 7th day.

Results:

All patients attained successful neutrophil and platelet recovery. The median time to neutrophil engraftment was 17.1days, and that of platelet recovery was 20.9 days. The incidence of grade II-‡W GVHD was 31.03% and grade III-‡W GVHD was 13.79%. The GVHD-related death was 3.45%. The incidence of cGVHD was 48.2%. The incidence of extensive cGVHD was 23.3%. The incidence of diseases relapsed was 13.79%. A median follow-up of 54 months noted that 13 patients died, while 16 survived. The total disease-free survival rate longer than 3 years was 55%.

Conclusion:

G-CSF mobilization bone marrow and peripheral blood stem cell as stem cell source for Haplo-HSCT provided rapid and sustained engraftment without increase of severity GVHD. The rate of disease relapse was reduced. This treatment was particularly suitable for patients with heavier weight.

Disclosures:

No relevant conflicts of interest to declare.
Prolongation of Progression Free Survival In Mantle Cell Lymphoma After RHCVAD: ASCT Consolidation or Rituximab Maintenance
Authors: McQuade Jennifer , Ahmadi Tahamtan , Porter David , Frey Noelle , Loren Alison Wakoff , Goldstein Steven C. , Svoboda Jakub , Stadtmauer Edward , Schuster Stephen J. , Nasta Sunita Dwivedy
Published: October 2019

Abstract 3570

Mantle cell lymphoma (MCL) is a small B cell lymphoma, incurable with standard chemo-immunotherapy. The best initial treatment regimen remains unclear. Although it is generally acknowledged that aggressive approaches using combination chemotherapy and/or high dose chemotherapy can prolong survival, consensus on upfront treatment strategies for advanced MCL is currently lacking without randomized controlled data to guide treatment decisions. We conducted a retrospective cohort analysis to describe and compare the survival experiences of MCL patients at the University of Pennsylvania treated in the first-line setting with R-HCVAD (N=43) with or without autologous stem cell transplant (ASCT) or Rituximab maintenance. The primary study endpoints were PFS and OS as assessed by chart review and confirmed by SSDI database. Median follow up for all pts was 3 years. The median age was 53.7, and 76.7 % (n=33) were stage IV at diagnosis. 15 patients underwent consolidative ASCT. 11 pts received Rituximab maintenance. Comparing patients treated with R-HCVAD vs R-HCVAD + R maintenance vs. R-HCVAD + ASCT, there were no statistical differences in terms of age, ECOG PS, LDH, WBC, beta-2microglobulin, BM or GI involvement, bulky disease or blastoid variant at baseline. Median PFS for all patients was 3.9 years: R-HCVAD alone 2.1 years vs. R-HCVAD+R 3.9 years (P=0.02, HR 3.51, 95%CI: 1.2–10.2) vs R-HCVAD + SCT not reached (p=0.017, HR 3.7, 95%CI: 1.26–10.63). PFS survival rates at 2 years were 50%, 88% and 70%; 33%, 71/% and 63% respectively at 3 years, and 0%, 33% and 33 % at 5 years. 3 year OS for all patients was 84% (95% CI: 65–94) with no significant differences among the three approaches. Notably, only 1/8 patients treated with R-HCVAD + SCT relapsed after 2 years, with a median follow up of 4.8 years for these patients. Our data suggest a further improved PFS when R-HCVAD is consolidated with either Rituximab maintenance or ASCT. While neither of the two consolidative approaches appears superior in our limited data set, both show significant PFS prolongation when compared to R-HCVAD alone. Further prospective investigation of consolidative approaches after RHCVAD in a randomized fashion is warranted.

Figure 1: Figure 1:.

Disclosures:

No relevant conflicts of interest to declare.
Berberine Induces Apoptosis In Acute Lymphoblastic Leukemia Cells by Demethylation of RASSF1A and Self-Ubiquitination of MDM2.
Authors: He Jing , Zhang Hailong , Li Jiansha , Gu Lubing , Zhou Muxiang
Published: October 2019

Abstract 3627

Berberine, an isoquinoline alkaloid derived from a plant used in Chinese herbal medicine, is reported to exhibit anti-proliferative and pro-apoptotic effects on human cancer; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by demethylation of the tumor suppressor RASSF1A, which results in self-ubiquitination and degradation of the oncoprotein MDM2. In contrast to the conventional chemotherapeutic drug doxorubicin, which directly induced p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53, which resulted in apoptosis of ALL cells tested, even if they were doxorubicin-resistant. Our investigation discovered that berberine but not doxorubicin can induce demethylation of RASSF1A in RASSF1A-methylated ALL. The result of RASSF1A demethylation was the increased expression of the tumor suppressor, which disrupted the MDM2-DAXX-HAUSP complex, thereby promoting MDM2 self-ubiquitination and subsequent MDM2 degradation. Consistent with this finding, the most potent apoptosis was induced by berberine in wild-type p53 ALL cells with both RASSF1A methylation and MDM2 overexpression, while no pro-apoptotic effect was detected in berberine-treated RASSF1A normal ALL cells that had no or low level of MDM2 expression. Given that ALL cells with RASSF1A methylation and MDM2 overexpression are commonly chemotherapy-resistant our finding that these cells can be potently killed by berberine through effective induction of RASSF1A and inhibition of MDM2 suggests that this naturally-derived agent may have a highly useful role in the treatment of patients with refractory ALL.

Disclosures:

No relevant conflicts of interest to declare.
An Unintended Benefit of Anabolic Steroid Use: Therapy of Hemophilia B Leiden.
Authors: Rimmer Emily K. , Houston Donald Stuart , Seftel Matthew D.
Published: October 2019

Abstract 3661

Introduction:

In Hemophilia B Leiden, administration of testosterone and anabolic steroids in pre-pubertal boys can raise endogenous Factor IX (FIX) to levels sufficient to prevent most hemorrhages. It is unknown whether this androgen responsiveness may occur even in the post-pubertal male. We present a case that unintentionally demonstrated this phenomenon.

Case:

A 29-year-old man with mild Hemophilia B (baseline Factor IX [FIX] level 12%) presented to the emergency department with acute left quadriceps swelling. He received 1,800 units (18 U/kg) of human factor IX concentrate and the following day FIX level was 80%. One day later, he was seen with persistent pain and swelling despite adequate factor replacement. The patient disclosed that he had been using intramuscular injections of the anabolic steroid stanozolol. On suspicion that the thigh swelling may represent abscess rather than hematoma, blood cultures were drawn and grew Staphylococcus aureus. He received appropriate antibacterial therapy and 1.5 liters of purulent material was drained from the thigh. Intriguingly, FIX level one month after discharge was still 61%. One year later, his FIX level had stabilized at 36%.

Given the persistently elevated FIX level compared to baseline, the FIX gene was sequenced. Genotyping revealed a G to A transition at nucleotide -6 within the promoter region of the FIX gene. Mutations within nucleotides -34 to +19 of the 5′ region (Leiden-specific region) of the FIX promoter are associated with the Hemophilia B Leiden phenotype. This rare form of Hemophilia B improves following puberty. There is an androgen-response element (ARE) located at the -26 position of the FIX promoter, within the Leiden-specific region. The mechanism of recovery is not fully understood, but it is postulated that in the presence of androgen binding to the ARE, a normal FIX gene can be transcribed, thus overcoming the mutation causing the hemophilia phenotype prior to puberty. It has been shown that mutations involving the ARE do not resolve after puberty, which suggests an androgen responsive mechanism to the increase in FIX.

Conclusion:

This case raises the possibility of androgen-responsive increases in FIX that persist after puberty. We propose that a further rise in the FIX levels occurred as a result of exogenous exposure to pharmacologic concentrations of androgens in the post-pubertal period. This deliberate use of exogenous androgens carried the unintended benefit of abolishing the Hemophilia B phenotype.

Disclosures:

No relevant conflicts of interest to declare.
Upper Gastrointestinal Bleeding In Patients with Haemophilia In Iran: Prevalence of Helicobacter Pylori Infection.
Authors: Dolatkhah Roya , Khoshbaten Manouchehr , Kermani Iraj Asvadi , Sanaat Zohreh , Bonyadi Mohammad Reza , Ghojazadeh Morteza , Alimoghaddam Kamran , Kermani Touraj Asvadi
Published: October 2019

Abstract 3662

Background:

Upper Gastro Intestinal (UGI) Bleeding is one of the most life-threatening complications, and occurs in up to 25% of persons with Hemophilia (PWH).Recurrent bleeding is common and can be caused by the Helicobacter Pylori (H.Pylori) infection. Our aim was to evaluate the role of H.Pylori infection in UGI bleeding in PWH, using Serology and Stool antigen tests.

Methods:

Ninety patients with hereditary bleeding disorders, 30 patients(group A) with and 60 patients(group B) without a history of UGI bleeding, who were admitted to our Hemophilia Treatment Center, were included in the study. The prevalence of H.Pylori infection was investigated by stool antigen test, as well as serum serologic tests including IgG and anti-CagA.

Results:

Among 90 patients (81 men,16 women, mean age 31 ± 11 years), 66 patients with Hemophilia A,10 patients with Hemophilia B, 6 patients with VWD, and 3 patients with other factor deficiencies were evaluated for H.Pylori infection. About 46.7% of patients in group A, and 23.3% of patients in group B were anti-Cag A – positive in serum (P=0.02), while 76.7% of patients in group A and 51.7% of patients in group B had H.Pylori IgG antibodies in serum (P= 0.02). H.Pylori antigen in stool was positive in 76.7% in group A and 55% in group B (P=0.03). Gastro endoscopy was done to find the bleeding focus and further evaluations, in 19 patients with acute UGI bleeding. No statistically significant difference was found between type and severity of diseases and risk of UGI.

Conclusion:

There was a statistically significant differences in H.Pylori infection between patients with and without UGI bleeding history. So, H.Pylori infection should be considered as an important cause of UGI bleeding in PWH. We would recommend stool antigen test as a new and none-invasive screening test for diagnosis of H.Pylori infection in all patients with hereditary hemorrhagic disorders.

Disclosures:

Dolatkhah: Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Employment, Research Funding. Khoshbaten:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Research Funding. Kermani:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sanaat:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Employment, Research Funding. Bonyadi:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Employment, Research Funding. Ghojazadeh:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Employment, Research Funding. Kermani:Hematology and Oncology Research Center, Tabriz University of Medical Sciences: Research Funding.
A Phase II Study to Assess the Safety, Efficacy and Tolerability of Rituximab (Mabthera) In Combination with Plasma Exchange In Patients with Acute Thrombotic Thrombocytopenic Purpura (TTP).
Authors: Scully Marie , Cavenagh James D , Hunt Beverley , McDonald Vicki , Machin Samuel J
Published: October 2019

Abstract 3699

Idiopathic adult TTP is an acute life threatening disorder, in which antibodies, primarily IgG, are detected against ADAMTS 13. We undertook a a phase II trial in 40 patients between 2006-09 of Rituximab, 375mg/m2, weekly for 4 weeks, within 3 days of admission of acute TTP, in conjunction with standard therapy (PEX and steroids). Results have been compared to 40 historical controls (2000-2006), who had not received Rituximab, but had received other immunosuppressive treatments. The female to male ratio was 2:1, age 42 years (21-76), compared to 42 years (15-78) in the historical group. A third of trial patients required ITU admission and 15% were intubated and ventilated at presentation. One patient was withdrawn from the trial. Pre the 2nd Rituximab infusion, 68% had a platelet count >50 ×109/L and 38% >150 ×109/L. Six cases received more than 4 Rituximab infusions (maximum of 8), primarily non-Caucasian, guided by ADAMTS 13 assays. There was a significant reduction in days admitted in hospital in the Rituximab group (median 16.5 days) compared to historic controls (median 20 days) (p=0.04, Spearman correlation), specifically in Caucasian patients (12.5 V's 16 days-Rituximab V's Historical groups) (p=0.0005 Pearson Correlation). There was no overall significant difference in the number of PEX to remission. ADAMTS 13 activity on admission was median <5% (NR 55–126%). Median Anti ADAMTS 13 IgG was 40% (6-162%, NR:<4.2). Following 4 Rituximab infusions, median ADAMTS 13 activity was 43% (7-67%), median Anti-ADAMTS13 IgG was 12% (2-74%). In the historical group, 48% relapsed, median 18 months (3-60 months). In the Rituximab group, follow up 16–40 months, 10% relapsed, median 27 months (17-31). There were no excess infections in the Rituximab group. There were three deaths in the Rituximab cohort at days 11,15 and 25, due to progressive cardiac/neurological disease. In conclusion, Rituximab appears to be a safe and effective therapy given during acute TTP in conjunction with PEX and steroids. No significant difference was seen in the number of PEX to remission, compared to historical controls. However, there was a significant reduction in the number of days in hospital in the Rituximab group. The risk of relapse up to 40 months is significantly reduced in the Rituximab cohort. In patients with acute TTP, Rituximab should be considered in conjunction with standard therapy.

Disclosures:

Scully: MRC, BHF: Research Funding. Off Label Use: Rituximab, in TTP. McDonald:Baxter (uk): Research Funding. Machin:Baxter (UK), Bayer (UK), MRC, BHF: Research Funding.
Human CD8+/TCR−/CD56dim/− facilitating Cells Enhance Engraftment of HSC In NOD/SCID/IL2rγnull Recipients.
Authors: Huang Yiming , Elliott Mary J , Ramsey Deborah M , Miller Thomas , Bozulic Larry D , Ocker Ashley A , King Bradley J , Ildstad Suzanne T.
Published: October 2019

Abstract 3709

CD8+/TCR− graft facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC). They are distinct from HSC since they do not generate multilineage engraftment when infused alone, nor do they form colonies in vitro. The function of human FC remains to be defined. Here, we phenotypically characterized the human CD8+/TCR− FC subpopulations and evaluated their function in vivo and in vitro. Human CD8+/TCR− FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (PBMC). Approximately 55% of FC express CD56dim/−, and the remaining population is CD56bright. In the CD8+/TCR−/CD56bright cell population, approximately 65% of cells express CD11c+ and 67% of cells express CD11b+. In CD8+/TCR−/CD56dim/− cell population, the majority of cells express CD3ε+ (80%), 17% were CD11c+, 16% were CD19+, 14% were CD11b+, 11% were CD123+, and 30% were HLA-DR+. To evaluate whether human CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo, we transplanted 100,000 HSC alone or plus 300,000 CD8+/TCR−/CD56dim/− FC into NOD/SCID/IL2rγnull (NSG) recipient mice conditioned with 325 cGy of total body irradiation. At 30 days after transplantation, 8 of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of recipients (n = 16) receiving HSC plus CD8+/TCR−/CD56dim/− FC engrafted, and donor lymphocyte and monocyte chimerism in PB was 0.53% ± 0.16% and 3.93% ± 1.28%, respectively. At 6 months after transplantation, NSG recipients of HSC alone lost donor chimerism in PB and no donor cells were detected in spleen and BM. In contrast, NSG recipients of HSC + CD8+/TCR−CD56dim/− FC exhibited durable donor chimerism in PB and showed significantly higher levels of donor chimerism in spleen (13%) and BM (9.43%) compared to recipients of HSC alone. To evaluate the function of CD8+/TCR−/CD56dim/− FC in vitro, HSC were incubated with CD8+/TCR−CD56dim/− FC for 18 hrs and then cultured in methylcellulose for 14 days in colony-forming cell assay. HSC plus CD8+/TCR−CD56dim/− FC generated significantly more colonies compared with HSC alone (P = 0.0038), suggesting that human CD8+/TCR−/CD56dim/− FC have a direct effect on the clonogenicity of HSC (Figure). In summary, our data indicate that (1) human CD8+/TCR− FC are a heterogeneous cell population; (2) CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo and promote HSC clonogenicity in vitro; and (3) recipients of CD8+/TCR−/CD56dim/− FC maintain a durable donor chimerism in BM and spleen.

Disclosures:

Bozulic: Regenerex, LLC: Employment. King:Regenerex, LLC: Employment, Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.
Macrophages Prevent Human Red Blood Cell Reconstitution In NOD/SCID and NOD/SCID/γc−/− Mice.
Authors: Hu Zheng , Yang Yong-Guang
Published: October 2019

Abstract 3723

An animal model supporting human erythropoiesis will be highly valuable for assessing the biological function of human RBCs under physiological and disease settings, and for evaluating protocols of in vitro RBC differentiation from human embryonic stem cells. Although immunodeficient mice on the NOD background have been widely used to study human hematopoietic stem cell function in vivo, the successful use of these mice in the study of human erythropoiesis and RBC function has not been reported. We have previously shown that co-transplantation of human fetal thymic tissue (under renal capsule) and CD34+ fetal liver cells (FLCs; i.v.) in NOD/SCID or NOD/SCID/γc−/− mice results in the development of multilineage human hematopoietic cells. Here, we analyzed human RBC reconstitution in these humanized mice. Although a large number of human erythrocytes, which consisted predominantly of immature nucleated erythrocytes, were detected in the bone marrow of human fetal thymus/CD34+ FLC-grafted mice, human RBCs were undetectable in blood of these mice, even in those with nearly full human chimerism in peripheral blood mononuclear cells (PBMCs). Recipient mouse macrophage-mediated rejection is, at least, one of the major mechanisms responsible for the lack of human RBCs in these mice, as human RBCs became detectable in blood following macrophage depletion and disappeared again after withdrawal of treatment. Furthermore, treatment with human erythropoietin (EPO) and human IL-3 significantly increased human RBC reconstitution in mice that were depleted of macrophages. Like the human RBCs developed in the humanized mice, exogenously injected normal human RBCs were also rapidly rejected by macrophages in NOD/SCID mice. Taken together, our data demonstrate that human RBCs are highly susceptible to rejection by macrophages in immunodeficient mice. Thus, strategies for preventing human RBC rejection by macrophages are required for using immunodeficient mice as an in vivo model to study human erythropoiesis and RBC function.

Disclosures:

No relevant conflicts of interest to declare.
Organ-Specific Protection from CD8+ T Cell-Mediated Acute Skin GVHD by Bortezomib Administration Correlates with Decreased IL-6.
Authors: Sun Kai , Sckisel Gail , Tietze Julia , Li Minghui , Sayers Thomas , Blazar Bruce R. , Murphy William J.
Published: October 2019

Abstract 3735

We have previously demonstrated that while early administration of bortezomib after murine allogeneic bone marrow transplantation (allo-BMT) can prevent acute graft versus host disease (GVHD), delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity (PNAS 2004, Blood 2005). This was primarily mediated by donor CD4+ T cells in fully MHC-mismatched allo-BMT (Blood 2008). In the current study, we assessed continuous bortezomib administration on CD8+ T cell-mediated acute GVHD. Using an MHC-matched, mHAg-mismatched model (C3.SW donor cells into lethally irradiated C57BL/6 recipients) in which acute GVHD is primarily induced by CD8+ T cells, we confirmed that significant skin and liver GVHD was observed after CD8+ T cell transfer. Histological evaluation of acute liver GVHD displayed severe lymphocyte infiltrates and severe subacute bile duct hepatitis consistent with hepatic GVHD. Severe acute skin GVHD was noted by persistent fur loss and skin ulcerations. Cutaneous histopathological assessment exhibited severe inflammatory infiltrates, epidermal hypertrophy, loss of hair follicles, satellite cell necrosis and dermal fibrosis. Notably, after allo-BMT, 6 weeks or thereafter, a significant increase in serum IL-6 levels was observed in the mice exhibiting marked skin damage, suggesting organ-specific correlation of higher serum IL-6 expression with acute cutaneous GVHD associated injury. We next investigated the effects of continuous treatment with bortezomib in this GVHD model. Surprisingly, we found that continuous bortezomib administration at the conventional dose of 0.75–1 mg/kg twice per week resulted in fractional GVHD- dependent lethal toxicity, exacerbating lymphocyte infiltration in liver, gut and lung as evidenced by pathological examination. However, skin GVHD pathology was markedly improved. Due to the limitations of conventional doses of bortezomib for treatment of acute GVHD, we then performed a low-dose bortezomib treatment at 0.125 mg/kg, twice per week. We found that mice receiving low-dose bortezomib were protected from lethal toxicity and displayed significant amelioration of skin GVHD pathology. Significant decreases in serum IL-6 levels were also consistently observed consistent with the amelioration of skin GVHD. Interestingly, while skin GVHD was markedly improved, there was no difference in liver, gut and lung pathology compared to control GVHD recipients indicating an organ-specific protection. These results demonstrate that continuous bortezomib can be applied in CD8+ T cell-mediated acute GVHD but necessitates lower dosing and organ-specific protection is observed.

Disclosures:

No relevant conflicts of interest to declare.
CD19 Fc-Fusion Protein for Detection of Cells Expressing Anti-CD19 Chimeric Antigen Receptors.
Authors: Oliveira Satiro N. De , Wang Jiexin , Hollis Roger P. , Kohn Donald B.
Published: October 2019

Abstract 3756

Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain that is capable of activating T cells. CARs are displayed on the surface of transduced cells, performing non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of modified mature T lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. We developed a specific reagent for the detection of anti-CD19 CAR expressed on cells, a soluble fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (19sIg). Genes encoding 19sIg fusion proteins were constructed by fusing either Exons 1 to 3 (19sIg1-3) or Exons 1 to 4 (19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The 19sIg proteins were produced from 293T cells by stable lentiviral vector transduction and purification from culture medium. ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule 19sIg1-4, but no binding to 19sIg1-3. Conjugation of the 19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays. Large-scale production and stable conjugation of this fusion molecule will provide a useful reagent to detect anti-CD19 CAR derived from any monoclonal antibody.

Disclosures:

No relevant conflicts of interest to declare.
Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.
Authors: Clemmensen Stine Novrup , Roervig Sara , Wren Jonathan , Illemann Martin , Glenthoj Andreas , Mora-Jensen Helena , Cramer Elisabeth , Larsen Maria Torp , Jacobsen Lars C. , Cowland Jack B , Borregaard Niels
Published: October 2019

Abstract 3775

Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules.

Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB.

Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not.

Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils.

Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist.

Disclosures:

No relevant conflicts of interest to declare.
Real-World Analysis of Venous Thromboembolism Rates and Thromboprophylaxis Prescribing In US Patients with Cancer.
Authors: Amin Alpesh N , Lin Jay , Thompson Stephen , Wiederkehr Daniel
Published: October 2019

Abstract 3802

Background:

Deep-vein thrombosis (DVT) is a frequent complication of cancer and treatment of cancer. Among clinical guidelines, there is broad consensus regarding the importance of thromboprophylaxis in hospitalized cancer patients, including prolonged prophylaxis in high-risk patients. The objective of this analysis was to assess the real-world use of prophylaxis for DVT, as well as symptomatic rates of DVT and pulmonary embolism (PE) in cancer patients, both during hospitalization and after hospital discharge.

Methods:

Data were extracted from the US Premier Perspective(tm)-i3 Pharma Informatics linked database for non-surgical cancer patients who were admitted to hospital from January 2005 to November 2007. Included patients were aged ≥ 18 years and had ≥ 6 months' continuous plan enrollment. Patients were excluded if they were discharged to an acute-care facility, had length of hospital stay ≤ 0 or > 30 days, had missing/unknown gender or age data, or if they were diagnosed with atrial fibrillation during index hospitalization. Clinical rates of DVT/PE, as well as thromboprophylaxis status and duration were evaluated for during hospitalization and post-discharge.

Results:

Of the 3,759 cancer patients analyzed, 51.9% received inpatient pharmacological and/or mechanical prophylaxis, and 2.8% received outpatient pharmacological prophylaxis in the 14 days following discharge. During index hospitalization 40.0% of patients received mechanical prophylaxis and 21.1% pharmacological prophylaxis, with 9.3% of patients receiving a combination of mechanical and pharmacological prophylaxis. Mean (± standard deviation) duration of prophylaxis was 1.3 (± 2.3) days for inpatients and 0.7 (± 4.5) days post-discharge among all cancer patients. Symptomatic DVT/PE occurred in 2.4% of patients during hospitalization. In the 30 days following hospital discharge, 1.7% of patients was either rehospitalized for DVT/PE or treated for DVT/PE in the outpatient setting.

Conclusions:

This real-world analysis demonstrates considerable symptomatic rates of DVT/PE in hospitalized cancer patients, with approximately half of patients not receiving any thromboprophylaxis. The risk of DVT/PE persisted into the outpatient setting, but few patients received anticoagulants post-discharge. Further efforts are needed to ensure that patients with cancer receive appropriate thromboprophylaxis and that it is of adequate duration to reduce the large burden of DVT/PE.

Acknowledgment: This study was funded by sanofi-aventis U.S., Inc. The authors received editorial/writing support in the preparation of this abstract provided by Tessa Hartog, PhD of Excerpta Medica, funded by sanofi-aventis U.S., Inc.

Disclosures:

Amin: sanofi-aventis US Inc.: Honoraria, Speakers Bureau. Lin:sanofi-aventis US Inc.: Employment, Research Funding. Thompson:sanofi-aventis US Inc.: Employment. Wiederkehr:sanofi-aventis US Inc.: Research Funding.
Publication Patterns of Cancer Cost-Effectiveness Studies Presented at the American Society of Hematology: Timeliness of Publication, Quality and Possible Bias.
Authors: Mozessohn Lee , Chan Kelvin KW , Cheung Matthew C
Published: October 2019

Abstract 3816

Background:

The timely publication of cancer cost-effectiveness analyses (CEA) is essential to inform decisions regarding new drug adoption by relevant policy makers and stakeholders. This study examined the publication pattern and quality of CEA presented in the annual meetings of the American Society of Hematology (ASH).

Methods:

ASH abstracts from 1997 to 2007 were reviewed. Abstracts with a malignant focus and that reported primary outcomes of incremental cost per life-year-gained or quality-adjusted-life-year (QALY) were included. Data including ICER (adjusted to USD) and author affiliation to the pharmaceutical industry was extracted. Quality indicators associated with well-performed CEAs were derived from the literature (Weinstein MC et al., JAMA 1996;276:1253-1258) and used to determine abstract quality. A search for subsequent publication of the abstract findings was conducted using Medline. The primary outcome of time-to-publication was determined using Kaplan-Meier statistics. Predictor variables were tested using the log-rank statistic.

Results:

29 abstracts met inclusion criteria. Only 13 were published (overall rate of 44.8%). The actuarial 1 and 3 year publication rates were 24.1% and 41.4% respectively (see figure). All but 1 abstract presented at ASH had an ICER less than $100 000/QALY (median $33 000/QALY) with 55.2% disclosing author affiliation to the pharmaceutical industry. Specific to abstracts published after 2001, when ASH instituted a policy of disclosure reporting, the proportion of abstracts reporting pharmaceutical affiliation was 72.7%. No variable or quality indicator predicted for time-to-publication though there was a trend for abstracts reporting dominant ICERs to yield more timely publications (p = 0.075). In terms of quality indicators, the reporting of a societal perspective, lifetime horizon, or a sensitivity analysis was noted in only 37.9%, 24.1% and 62.1% of abstracts, respectively.

Conclusions:

The publication rate of CEA abstracts from ASH was low and not timely for open discussion among stakeholders. Although there was no direct evidence of bias in abstracts selected for presentation, most reported highly favorable ICERs and consistent author affiliation with the pharmaceutical industry. Overall, the quality of abstracts appeared suboptimal.

Disclosures:

No relevant conflicts of interest to declare.
Toll-Like Receptors 2 and 4 Mediate the Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells to Support the Proliferation and Differentiation of CD34+ Cells In a Non-Synergistic Way.
Authors: Wang Xingbing , Cheng Qiansong , Wang Jian , Xia Liang , Zhang Xuhan , Ding Kaiyang , Sun Zimin
Published: October 2019

Abstract 3850

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent nonhematopoietic progenitor cells, which can differentiate into osteoblasts, adipocytes, chondrocytes and other tissues. The most important function of BM-MSCs is to support hematopoiesis. Toll-like receptors (TLRs) are a conserved family of receptors that can be activated by both pathogen components and mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. In the past a few year, several studies reported that TLRs are expressed in hematopoietic and non-hematopoietic to modulate their biological functions. We hypothesized that MSCs are equipped with TLRs that enable them to dynamically change hematopoiesis-related cytokines expression pattern and level by sensing correspondent agonists, thus efficiently supporting hematopoiesis.

In this study, BM-MSCs were analyzed for mRNA expression of TLR 1–9 by reverse transcription-polymerase chain reaction. TLR 1–6, but not TLR 7–9 were expressed by MSCs. The expression of TLR2 and TLR4 was also confirmed by flow cytometic assay. We further explored the role of TLR2 and TLR4 in mediating the capacity of MSC to support the proliferation and differentiation of CD34+ cells. The pre-stimulation with TLR2 agonists (Pam3Cys) or TLR4 agonists (LPS) enable MSCs to enhance CD34+ cells proliferation and promote CD34+ cells differentiation towards the myeloid lineage (CD33+, CD11b+), as well as granulocyte colony formation by those cells. The production of interleukin 8 (IL-8), IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF were also increased by stimulated MSCs. Interestingly, although Pam3Cys and LPS displayed different inductive magnitudes, they have no synergistic effect on MSCs. We hypothesized there may be some antagonistic effect between TLR2 and TLR4 intracellular signal conductive pathway, or they can downregulate the expressive level of the TLRs on MSCs.

Together, our findings suggest that TLR2 and TLR4 signalings may indirectly regulate hematopoiesis by modulating MSCs' functions. The increased haemopoietic proliferation and myeloid lineage differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production.

Disclosures:

Wang: National Natural Science Foundation (30700329): Research Funding; Anhui Provincial Outstanding Young Investigator Program (08040106810): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.
MT1-MMP Plays a Critical Role In the Modulation of Hematopoiesis.
Authors: Nishida Chiemi , Heissig Beate , Tashiro Yoshihiko , Seiki Motoharu , Nakauchi Hiromitsu , Hattori Koichi
Published: October 2019

Abstract 3851

Specific niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. In the recent study, activation of the fibrinolytic pathway via matrix metalloproteinases (MMPs) including MMP-9 resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate MMP-9 in the process of mutual activation of MMP. It has already known that BM myeloablation with irradiation or anti-cancer drug induces MT1-MMP expression, but the role of MT1-MMP in hematopoiesis is not well-understood. We examined MT1-MMP deficient mice (MT1-MMP-/-) 12 days after birth. MT1-MMP-/- were suffering from pancytopenia, and are reduced numbers of bone marrow mononuclear cells (BMMCs), splenocytes and number of hematopoietic progenitor cells in the BM although the number of HSCs in BM showed no significant difference. BM cytospins from MT1-MMP-/- mice showed mild erythropoietic disturbance and severer impairment of myelopoiesis. Interestingly, a powerful hematopoietic factor, Kit-ligand (KitL) levels were significantly lower in MT1-MMP-/- mice than wild type. MT1-MMP knockdown by shRNA or/and siRNA impaired KitL expression and secretion in transfected stroma cells compared to Mock controls, demonstrating that reduced KitL plasma levels were due to impaired release/production and not due to reduced numbers of stromal cells in MT1-MMP-/-. Similarly, impaired proliferation and differentiation of MT1-MMP-/- BMMCs in vitro could be restored by exogenous sKitL. Others and we reported that BM ablation induces the production of stromal cell-derived factor-1 (SDF-1; CXCL12), which plays a key role in stem cell homing and B-cell lymphopoiesis. Reduced SDF-1 expression was observed in BMMCs of MT1-MMP-/- mice and genetic knockdown of MT1-MMP resulted in lower SDF-1 expression both on a transcriptional and protein level. BMMCs of MT1-MMP-/- showed a decrease in the percentage of mature B cells compare to controls. Knocking down of MT1-MMP in stromal cell reduced the number of adherent hematopoietic cells, but addition of rec. SDF-1 could reverse the phenotype. These results suggested stromal-derived MT1-MMP was functionally important to maintain HSC function in long-term cultures of WT HSCs. Thus, MT1-MMP is a critical modulator of hematopoiesis, as it alters the growth factor output of niche cells.

Disclosures:

No relevant conflicts of interest to declare.
Role of Polycomb Repressive Complex 1 (PRC1) In Runx1 Mediated Gene Regulation
Authors: Yu Ming , Mazor Tali , Huang Hui , Huang Emily , Kathrein Katie , Woo Andrew , Zon Leonard I. , Fraenkel Ernest , Cantor Alan B
Published: October 2019

Abstract 3863

The transcription factor Runx1 is required for the generation of all definitive hematopoietic stem cells (HSCs), and for normal megakaryocyte, lymphocyte and granulocyte terminal maturation. Runx1 and its cofactor CBF-β are also the most common targets of chromosomal translocations in human leukemias. Somatic and germline point mutations in Runx1 occur in myelodysplastic syndrome and undifferentiated leukemias, and are associated with a poor prognosis. Despite the key roles that Runx1 plays in normal and malignant hematopoiesis, its transcriptional mechanisms remain incompletely understood. In this study, we purified Runx1 containing multiprotein complexes from megakaryocytic cells and identified several associated chromatin-remodeling complexes, including Polycomb Repressive Complex 1 (PRC1), NuRD, SWI/SNF and MLL/TrxG. Interactions were validated by independent biochemical assays and demonstrate a direct interaction between Runx1 and the PRC1 component Bmi1. ChIP-seq studies identified a large overlap between Runx1/CBF-β and Ring1b (another PRC1 core component) occupied sites, with 45% of the peaks at these genes < 200 bp from each other. ShRNA mediated gene knockdown of CBF-β shows differential gene expression of many of the co-occupied genes. Among the direct CBF-β/Ring1b co-occupied targets are other key hematopoietic transcription factors including FOG-1, SCL and Lyl1, and a number of cell adhesion related genes. ShRNA knockdown of Ring1b impairs megakaryocyte endomitosis, partially phenocopying Runx1 deficient megakaryocytes. Morpholino mediated knockdown of Ring1b or Bmi1 in zebrafish embryos reduces the number of phenotypic definitive HSCs, also partially phenocopying Runx1 morphants. We also show that Runx1/CBF-β interact with Ring1b in the human T cell line Jurkat, and that Ring1b occupies Runx1/CBF-β bound sites of key direct target genes in primary murine thymocytes, including CD4, TCRβ, and Th-POK. Surprisingly, we did not find enrichment for histone 2A monoubiquitination at most of the megakaryocytic and T-lymphocyte co-occupied sites examined, suggesting that PRC1 acts through alternate mechanisms at these genes. Collectively, these data provide evidence for a broad role of PRC1 in Runx1 mediated gene regulation.

Disclosures:

Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Cantor:Amgen, Inc: Consultancy.