bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified-CHOP with Reduced Dose of Non-Pegylated Liposomal Doxorubicin ± Rituximab in First Line Treatment for Elderly Patients with Aggressive Lymphoma Non Suitables for Standard Chemotherapy.
Authors: Gimeno Eva , Sánchez Blanca , Alvarez Alberto , Pedro Carme , Abella Eugenia , Gomez Miquel , Saumell Silvia , Comin Josep , Besses Carles , Salar Antonio
Published: October 2019

Abstract 4752

BACKGROUND

CHOP ± Rituximab ( R) is the standard regimen for elderly patients (pts) with aggressive lymphoma but many of them are not suitable for it due to severe associated comorbidities. The aim was to evaluate retrospectively the efficacy and safety of a modified-CHOP (with reduced dose non-pegylated liposomal doxorubicin (NPLD) ± R in elderly pts with clinically aggressive lymphoma not tributary to standard anthracycline-containing chemotherapy.

PATIENTS AND METHODS

Retrospective analysis of 30 pts (16M/14W). Median age 76 years (60-88). Stage III-IV: 19 pts (58%). IPI 3-5: 16 pts (49%). Median baseline left ventricular ejection fraction (LVEF): 63% (33-80). Median NT-proBNP determination: 1431 ng/L (60-9120), 14 patients had NT-proBNP>900. All patients had one or more severe comorbidities. Schedule: NPLD 30mg/m2 (d1), cyclophosphamide 750mg/m2 (d1), vincristine 1.4mg/m2 (d1), prednisone 100mg/d (d1-5) ± R 375mg/m2 (d1) + Pegfilgrastim (d2) every 21 days.

RESULTS

Median follow-up time was 18 months. Median number of cycles was 4 (range 1-6). Complete response (CR/uCR): 24 pts (73%), Partial response: 4 pts (12%). Two pts progressed during chemotherapy and 10 pts relapsed during follow-up (5 of them dying with active disease). Overall Survival (OS) at 12 and 24 months was 76% (95%CI 61-91) and 70% (95%CI 49-91), respectively. Progression-free survival (PFS) at 12 and 24 months was 65% (95%CI 49-81) and 55% (95%CI 32-78), respectively. A total of 154 cycles were administered. 52% of patients showed grade III-IV neutropenia and 33% of them required hospital admission for febrile neutropenia. LVEF neither NT-proBNP value was significantly different before and after treatment with one patient showing an important improvement in his LVEF. Multivariate analysis recognized NT-proBNP determination >900ng/L as the most negative important factor in OS and PFD.

CONCLUSION

Disclosures:

No relevant conflicts of interest to declare.
Non Hodgkin Lymphoma: Experience of a Referral Hospital.
Authors: Castellanos-Sinco Humberto , Rivas-Vera Silvia , Argote Victoria E. Ferrer , Martínez-Murillo Carlos , Peñafiel Christian Omar Ramos
Published: October 2019

Abstract 4774

Introduction

Non-Hodgkin lymphomas corresponds a heterogeneous group of neoplasms deriving of lymphocytes, in development countries represents a great cause of morbility and mortality. The General Hospital of Mexico is a referral centre localized in Mexico City, whose first cause of primary hematology attention in NHL.

Material and methods

Is an observational, descriptive and retrospective study, based on medical records of patients treated between 1992-2009.

Objectives

Determine the response, overall survival (OS) and event free survival (EFS) (progression, death or last visit) associated with treatment by CHOP as first line treatment without rituximab and four cycles of DEP as a second line treatment (dexamethasone 40 mg/m2 IV daily, days 1-4, VP-16 300 mg/m2 IV daily, days 1-2 and cysplatin 100 mg/m2 IV day 1), in cycles of 21 days each one.

Results

In a period of 17 years, 303 of 632 patients were eligible. The average age was 52 years, with male:female relation 1:1.1. Thirty four percent of the sample were older 60 years. The most frequent NHL was diffuse large B cell lymphoma (DLBCL) (N=146 treated), 60% were IPI low plus intermediate low; the most frequent indolent NHL were follicular lymphoma (N=20 treated), 71% were FLIPI low plus intermediate. The resting 94 cases were treated with CHOP, too.

Conclusion

The global trend is the addition of rituximab to all of CHOP-based regimens. In our country, because the economics limitations, the use of rituximab is low. The CHOP regimen remains the first line treatment; and the second line treatments are based on the experience of the institutional protocols. DEP is the institutional protocol for second line treatment and their response, OS and EFS are comparable to more expensive protocols (ESHAP, DHAP). Concluding that DEP is a low toxic and cheaper second line regimen and this could be an alternative for development countries.

Disclosures:

No relevant conflicts of interest to declare.
Response of Patients with Aggressive Non Hodgkin's Gastro Intestinal Tract Lymphoma to Upfront Rituximab Based Chemotherapy: A Single Institution Experience.
Authors: Ciobanu Bety , Sreenivasappa Shylendra B , Shah Mousami , Yim Barbara , Catchatourian Rosalind
Published: October 2019

Abstract 4791

Background

Response and factors affecting response in patients with aggressive Non Hodgkin's Gastro Intestinal (GI) tract lymphoma to upfront Rituximab based chemotherapy is not well characterized.

Methods

41 consecutive pts diagnosed and treated with GI tract lymphoma in 2002 to 2009 were studied as a retrospective cohort for clinical presentation, prognostic characteristics and long term survival. Prognostic characteristics and survival was analyzed using logistic regression and Cox Proportional Hazards model. Multiple imputation methods were used for missing data.

Results

41 pts, median age at presentation was 58 yr (28-80). 10 (24.4%) were African Americans, 22 (51.2%) were Hispanics and 10 (24.4%) were Caucasians. 35 (85.4%) had gastric lymphoma and 6 (14.6%) had intestinal lymphoma. 33 pts had aggressive NHL. 31 had Diffuse large B cell lymphoma, 2 Mantel cell lymphoma and 1 Burkitt's lymphoma. 6 (17.6%) presented with stage IE, 4 (11.8) stage II1, 6(17.6%) Stage II2, 7(20.6%) Stage IIE, and 10 (29.4%) Stage IV. 28(82.3%) pts were diagnosed by endoscope. 5 (14.7%) pts underwent surgery as they presented with obstruction or bleeding, none underwent curative resection. All pts received Rituximab based chemotherapy upfront. 29 received CHOPR, 2 CVPR and 2 Hyper CVAD R. Mean follow up was 23.7 months.

22 (64.8%) pts had a complete response, 6 (17.6%) partial response and 6 (17.6%) had progressive disease. 11 pts relapsed. The median disease free survival (DFS) was 13 months (1-105). Age was the sole significant prognostic factor that influenced DFS OR 1.047 CI 1.003-1.094). Age (OR 1.164 CI 1.045-1.296), stage (OR 1.743 CI 1.053-2.888) and performance status (OR 2.703 CI 1.030-7.097) were factors which significantly affect survival.

Survival function was 100%, 55.6% and 58.3% for low, intermediate and high risk group of stage modified IPI for gastric lymphoma. 100%, 70% 58.3% for low intermediate and high risk group as per the revised IPI but survival functions are not significant.

12 (35.3%) of pts tolerated chemotherapy with no grade 3 or 4 adverse events. 13 (38%) had g 3or 4 hematological toxicity. 5 (15%) had surgery post chemotherapy. 2 (6%) has GI perforation and 3 (9%) developed GI bleeding post chemotherapy. Surgical complications occurred after the 1st cycle of chemotherapy.

Conclusion

Aggressive Non Hodgkins GI lymphoma can be effectively treated with Rituximab based chemotherapy as upfront therapy with a response rate of 83%. Age, stage and performance status significantly effect survival. R IPI may need to be revised to predict survival in gastric lymphoma. 15% treated with chemotherapy upfront may need surgery for complications. Perforation rate was about 3% and most surgical complications occur after 1st cycle of chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy and Safety of Yttrium-90 Ibritumomab Tiuxetan in Older Patients with Indolent Non-Hodgkin Lymphoma.
Authors: Capalbo Silvana , Palumbo Gaetano , Dell'Olio Matteo , Franzese Maria Grazia , Guarini Attilio , Di Renzo Nicola , Spinosa Giuseppina , Lapietra Angela , V Elsa Pennese , Cascavilla Nicola
Published: October 2019

Abstract 4795

Introduction

Radioimmunotherapy (RIT) has emerged as an important treatment options for patients with non-Hodgkin lymphoma (NHL). Yttrium-90 ibritumomab tiuxetan (Zevalin®) consist of ibritumomab, a murine monoclonal antibody to CD20, conjugated to the metal chelator tiuxetan for retention of the beta emitter Yttrium-90. Clinical trials with this agent have demonstrated significant activity in indolent NHL with mild toxicity. The median age of NHL patients included in these trials is mainly < 65 years. Our aim was to evaluate the effectiveness of Zevalin as treatment option for patient > 65 years old with indolent NHL.

Patients and Methods

Between November 2005 to June 2009 fifteen patients, five males and ten females, median age 76 years (range 67-82), with indolent NHL (13 follicular and 2 small lymphocytic) were treated with Zevalin. Six patients had stage IV disease, five stage III and four stage II. All patients received an initial infusion of rituximab at a dose of 250 mg/m(e)2 on day 1 and a second infusion at same dose on day 8 followed by a weight-based dose of Zevalin (median dose 1006 MBq; range 668-1260). Eight patients perfomed Zevalin as consolidation after first line therapy with Rituximab plus chemotherapy (6 R-CHOP, 1 R-FN, 1 R-COMP): of these three were in complete remission (CR) and five in partial remission (PR). Seven patients perfomed Zevalin in relapse (four in first and three in second relapse).

Results

After RIT 13 of 15 patients were evaluable. Overall response rate was 92% (10 CR, 2 PR); in particular all patients in first line of treatment achieved CR. One patient had stable disease. At a median follow-up of 15 months (range 2-34), all patients are alive in persistent CR or PR. One of two patients in PR achieved CR after successive therapy. Treatment was well tolerated; transient thrombocytopenia (grade 3-4) was seen in 9 patients and transient neutropenia (grade 3-4 ) in 6 patients. Only one patient developed herpex-zoster virus infection.

Conclusion

In our experience, Zevalin produces high response rate (up to 90%) and durable remission without severe toxicity in older patients with indolent NHL. Notably, in first-line treatment, RIT resulted in PR-to-CR conversion in all five patients in PR after the R-chemotherapy. The favourable safety profile of this regimen makes it an effective consolidation treatment for older patients who, because of age and comorbidity, are not eligible for intensive treatment as high-dose therapy and stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Abbreviated, Dose Dense Fludarabine and Rituximab for Elderly Patients with Indolent B Cell Malignancies.
Authors: Ballester Gabriela , Frazer Lisa , Tirona Maria , Ballester Oscar F
Published: October 2019

Abstract 4796

Introduction

Therapy for indolent B cell malignancies is primarily palliative. Therefore limiting toxicities by reducing drug exposure is desirable, particularly in elderly individuals. The combination of fludarabine and rituximab has been extensively used in patients with these disorders. We sought to decrease the number of fludarabine cycles, attempting to maintain the high response rates of this combination by increasing the doses of rituximab and the dose density.

Patients and Methods

We designed an abbreviated, but dose dense regimen (addFR) consisting of fludarabine 25 mg/m2/d for 5 days on weeks 1, 6 and 11 plus rituximab at 375mg/m2 on weeks 2-5 and 7-10. Maintenance was offered to responding patients with rituximab for 4 weekly doses every 6 months (for 2 years). This schema represents a 50% decrease in the dosage on fludarabine with a 33% increase in the dosage of rituximab compared to the standard regimen. Twelve patients with a median age of 70 years, (range 43 to 85, 9 of the 12 patient are older than 65 years of age) have been treated. Nine patients had chronic lymphocytic leukemia (CLL, n= 5) or small lymphocytic lymphoma (SLL, n= 4). Three patients had marginal zone lymphoma (MZL). Three patients had received prior therapy. Responses were assessed by standard criteria 4 weeks after completion of the planned 11 weeks of therapy.

Results

Two patients are still completing their 11 weeks of therapy. Ten patients have completed the 11 weeks of addFR. They are evaluable for toxicity and responses. Hematological toxicities included neutropenia Grade >III in 2 patients with a single episode of neutropenic fevers requiring hospitalization. One patient with refractory disease was shown to have histological transformation from SLL to a diffuse large B cell lymphoma. All of the remaining 9 patients achieved a complete clinical remission, including 8 patients with CLL/SLL and 1 patient with MZL. With a median follow up of 13 months (range 10 to 23 months); these 9 patients have remained free of disease progression.

Conclusions

addFR seems to be well tolerated and effective even in patients older than 65 years of age. Experience with a larger number of patients and longer follow up is needed.

Disclosures:

No relevant conflicts of interest to declare.
Improved Immune Reconstitution After Bortezomib-Based Acute Graft-Versus-Host Disease Prophylaxis for HLA-Mismatched Unrelated Donor Reduced-Intensity Conditioning Allogeneic Transplantation.
Authors: Koreth John , Stevenson Kristen E. , Kim Haesook T. , McDonough Sean , Herrera Maria I. , Reynolds Carol G. , Murga Gorka , Ho Vincent T. , Cutler Corey , Armand Philippe , Soiffer Robert J. , Antin Joseph H. , Alyea Edwin P. , Ritz Jerome
Published: October 2019

Abstract 48

We recently reported on the use of the proteasome-inhibitor bortezomib as a novel strategy to control acute graft-versus-host-disease (GVHD). We evaluated bortezomib, tacrolimus and mini-methotrexate for GVHD prophylaxis after reduced-intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) with unrelated donors mismatched at 1-2 HLA loci (-A, -B, -C, -DRB1). The regimen was safe, with zero non-relapse mortality. There was a 13% incidence of grade II-IV acute GVHD.

We analyzed immune reconstitution in the bortezomib cohort (n=24) [Bort] vs. i) 8/8 matched-related donor (-A, -B, -C, -DRB1) RIC HSCT cohort (n=96) [MRD]; ii) 8/8 matched-unrelated donor RIC HSCT cohort (n=139) [MUD]; and iii) 1-2 HLA mismatched-unrelated donor RIC HSCT cohort (n=24) [MMUD]. Comparator groups received sirolimus, tacrolimus and mini-methotrexate GVHD prophylaxis. All received fludarabine/busulfan conditioning and G-CSF mobilized adult-donor peripheral blood stem cells.

All groups had rapid hematopoietic engraftment and >90% median donor-chimerism by day +45 post-HSCT. They were not different with regards to age (p=0.53), sex (p=0.39) and acute grade II-IV GVHD (p=0.63). There was a difference in the proportion of good-risk disease: [Bort] (46%), [MRD] (21%), [MUD] (14%) and [MMUD] (21%) (p=0.005). Chronic GVHD was also different: [Bort] (41%), [MRD] (39%), [MUD] (53%) and [MMUD] (67%) (p=0.03).

Peripheral blood cells incubated with directly conjugated monoclonal antibodies defining distinct lymphocyte populations were analyzed by flow cytometry. Lymphocyte subsets included T (CD3+, CD4+, CD8+) B (CD19+, CD20+) NK (CD56+CD16+) NK-T (CD56+CD3+) and regulatory-T (Treg; CD4+CD25+) cells.

Unadjusted analyses revealed no significant differences in B, NK, NK-T or Treg recovery. In contrast, CD3+, CD4+ and CD8+ T-cell reconstitution was significantly higher in the bortezomib group, especially at 3 months post-HSCT.

T-cell immune reconstitution parameters are improved after HLA-mismatched RIC HSCT and bortezomib-based GVHD prophylaxis. Importantly this can be accomplished with rapid hematopoietic engraftment and without increased GVHD incidence in patients without HLA-matched donors.

Disclosures:

Off Label Use: bortezomib for GVHD control.
HERG K+ Channels Promote Angiogenesis by Enhancing the Expression Level of VEGF in Leukemia Cells in Vitro.
Authors: Li Huiyu , Jie Shenghua , Tian Yuan , Gao Shaoyang , Guo Dongmei , Guo Linlin , Huang Shiang
Published: October 2019

Abstract 4818

Introduction

FOur previous studies demonstrated that HERG1 K+ channels are aberrantly expressed in leukemia cells. Recent studies also indicate the leukemia cells to have influence on angiogenesis. If there are any involvements of HERG1 K+ channels on the leukemia-induced angiogenesis are not yet investigated. In this study, the effect of HERG1 K+ channels in K-562 cells on angiogenesis was measured by the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), acknowledging that these are critical steps for angiogenesis. The possible mechanism of HERG K+ channels on leukemic cells in angiogenesis is also investigated by measuring expression level of VEGF in both K-562 cells and primary leukemic cells.

Methods

K-562 cells were cultured in the medium containing various concentration (0,10.20,40uM) of E-4031, a HERG1 K+ chnanel special inhibitor. HUVECs were seeded in the conditional medium and HUVEC proliferation was measured by the CCK-8 cell proliferation assay. HUVEC cell migration was performed by Boyden chamber (Trans-well). The HUVECs were seeded in the up-chamber and the conditional medium was in down-chamber. The angiogenesis of HUVECs cultured in conditional medium were measured by endothelial tube formation in Matrigel in vitro. The role of HERG K+ channels on VEGF secretion by leukemic cells was determined by ELISA and VEGF mRNA expression of leukemic cells were examined by RT-PCR.

Results

The results showed that blocking HERG K+ channels on K-562 cells could inhibit the capacity of K-562 cells to induce human HUVEC proliferation, migration and tube formation in a concentration manner in vitro. This suggests that HERG1 K+ channels could influence the cross-talk between the leukemic cells and vascular endothelial cells. Further analyses revealed that HERG1 K+ channels can increase the capacity of K-562 cells and primary leukemia cells to produce VEGF with up-regulation of VEGF mRNA. The similar results were also obtained from other tumor cells.

Conclusions

We demonstrated that HERG1 K+ channels on the membrane of leukemic cells have a promoting effect on angiogenesis. The mechanism is that HERG1 K+ channels can increase the level of VEGF expressions. This may be a critical step of HERG1 K+ channels on tumor induced angiogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Spherocytic Hemolytic Anemia Caused by Disruption of GATA1-FOG1 Interaction.
Authors: Shimizu Ritsuko , Hasegawa Atsushi , Yamamoto Masayuki
Published: October 2019

Abstract 775

Transcription factor GATA1 is essential for erythroid/megakaryocytic cell differentiation. Gene ablation studies have revealed that GATA1 plays essential roles in the differentiation of erythroid and megakaryocytic lineages. Two functional zinc finger domains have been identified within the GATA1 molecule, and N-terminal finger (NF) is required for the association with cofactor FOG1 and also DNA recognition. Several substitution mutations in the GATA1 NF that diminish association of GATA1-FOG1 have been identified in patients with thrombocytopenia and anemia. We previously established a genetically engineered mouse model, which mimics a human disease X-linked thrombocytopenia with inherited GATA1 mutation, utilizing the transgenic complementation rescue approach. GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of mutant GATA1 (GATA1V205G) and showed severe thrombocytopenia with impaired cytoplasmic maturation of megakaryocytes resembling the human disease. However, these mice did not show dyserythropoietic phenotype except for the attenuated erythropoiesis under stress conditions. On the contrary, GATA1-deficient mice rescued by transgenic expression of GATA1V205G at the level comparable with that of the endogenous GATA1 hardly survived though the postnatal stage. Accordingly, we analyzed rescued embryos during perinatal period. At 18.5 embryonic days, expected number of rescued embryos survived although they showed macroscopic anemia, indicating that embryonic erythropoiesis was not fully sustained by transgenic expression of GATA1V205G at the endogenous level. Surprisingly, rescued newborns suffered from severe anemia and jaundice. Serum indirect bilirubin level was significantly elevated and circulating erythrocytes showed abnormal morphology characterized as spherocytes by means of scanning electron microscopic observation. The expressions of Band3 gene/protein were strikingly diminished in the rescued erythroid cells, while expression of the other membrane related genes varied in individuals. Thus, these results indicate that hemolytic event associated with the lack of Band3 and dysfunction of erythrocyte membrane is induced during perinatal period, and GATA1 maintains the erythroid membrane homeostasis through interaction with FOG1.

Disclosures:

No relevant conflicts of interest to declare.
Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.
Authors: Sathyanarayana Pradeep , Pradeep Anamika , Licht Jonathan D. , Wojchowski Don M.
Published: October 2019

Abstract 777

The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation.

Disclosures:

No relevant conflicts of interest to declare.
Human Adenylate Kinase 2 Deficiency Inhibits Hematopoietic Cell Differentiation towards Neutrophil and T Lymphoid Lineages.
Authors: Six Emmanuelle M. , Lagresle-Peyrou Chantal , Demerens-deChappedelaine Corinne , Rieux-Laucat Frederic , Didati Andrea , Stolzenberg Marie-Claude , Cassinat Bruno , Chomienne Christine , Fischer Alain , Cavazzana-Calvo Marina
Published: October 2019

Abstract 78

Reticular dysgenesis (RD), an autosomal recessive form of human Severe Combined Immunodeficiency is characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of AK2 protein expression. AK2 is a mitochondrial protein which regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP and AMP. In order to understand the precise role of AK2 in hematopoiesis, we have developed a RNA interference strategy through lentiviral-mediated gene transfer of AK2 short hairpin RNAs (shAK2). The knock-down of AK2 in human or murine hematopoietic stem cells (HSC) inhibits their capacity to form granulocyte colonies in methycellulose assays and prevents them to generate mature polynucleated cells in liquid culture in the presence of G-CSF. We also determine the ability of shAK2-transduced HSC to differentiate along the T lymphoid lineage after co-culture on a OP9Delta1 stroma cell line. Our data demonstrated that the apparition of CD4+CD8+ cells was profoundly reduced in the presence of shAK2. To delineate the mechanism involved in this defect, we also studied the neutrophil differentiation of the HL60 promyelocytic cell line, following retinoic acid treatment. In this system, the absence of AK2 expression led to an arrest of neutrophil differentiation process, increased cell apoptosis and disrupt the mitochondrial membrane potential. All these data suggest a novel mechanism regulating hematopoietic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Longitudinal Analysis of Antibody Response to Immunization in Pediatric Survivors After Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Inaba Hiroto , Posner Meredith , Hartford Christine , Yang Jie , Pei Deqing , Hayden Randall , Srinivasan Ashok , Triplett Brandon , Pui Ching-Hon , Leung Wing
Published: October 2019

Abstract 795

Re-immunization is a common practice after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the optimal vaccine schedule and long-term response have not been established. We prospectively and longitudinally evaluated the antibody response to childhood vaccines in 210 survivors after allo-HSCT. Positive titer lasting for 5 years or more after immunization was observed more often for diphtheria (98.1%), tetanus (96.1%), rubella (92.9%), and polio vaccines (96.2% for type 1; 98.1% for type 2 and 3), but less often for pertussis (25.0%), measles (65.4%), mumps (64.3%), and hepatitis B (75.0%). As shown in the table below, vaccine failure was associated with patients who were older at the time of re-immunization, received a T cell depleted graft, had a high-risk hematological malignancy, lower IgG levels, higher IgM levels, chronic GvHD, positive recipient CMV status, and negative titers prior to immunization. These clinical factors are useful to formulate immunization and monitoring strategies for survivors after allo-HSCT. Patients at risk for vaccine failure should be monitored closely during long-term follow-up, and booster immunizations should be considered when there is no seroconversion.Table.Factors associated with negative titers after re-immunizationVaccineFactorOdds ratio95% confidence intervalp value DiphteriaT cell depleted graft5.521.14-26.820.034 TetanusOlder age at immunization1.541.05-2.220.026 Lower IgG level*1.031.00-1.050.033 MeaslesLower IgG level*1.011.00-1.020.022 MumpsHigh risk vs. standard risk2.631.02-6.670.045 RubellaHigher IgM level*1.121.03-1.230.012 High risk vs. standard risk16.71.56-100.000.02 PoliovirusType 1Older age at immunization1.431.05-1.920.021 Positive recipeint CMV titer3.181.58-6.410.001 Higher IgM level*1.081.01-1.150.027 Negative titer before immunization8.332.17-33.330.002 Type 2Older age at immunization1.411.04-1.920.027 Positive recipeint CMV titer2.651.36-5.200.004 Higher IgM level*1.061.00-1.140.04 Negative titer before immunization14.34.55-50.00<0.001 Type 3Older age at immunization1.411.04-1.890.027 Positive recipient CMV titer3.111.67-5.80<0.001 Higher IgM level*1.061.00-1.120.035 Chronic GVHD2.71.05-6.950.04 Negative titer before immunization5.881.92-20.000.002*Total IgG and IgM, not disease specific Ig levels

Disclosures:

No relevant conflicts of interest to declare.
Adoptive Transfer of Hexon-Specific T-Cells as a Treatment of Adenovirus Reactivation Following Allogeneic Stem Cell Transplantation.
Authors: Opherk Kathrin , Schuster Friedhelm R , Bethge Wolfgang Andreas , Bader Peter , Greil Johann , Stuhler Gernot , Albert Michael H , Sauer Martin G. , Handgretinger Rupert , Lang Peter , Feuchtinger Tobias
Published: October 2019

Abstract 796

In pediatric patients human adenovirus (HAdV) was identified as a common viral pathogen responsible for significant morbidity and mortality post allo SCT. Antiviral chemotherapy is often insufficient. Given that T-cell immunity is crucial for protection against adenoviral infection/reactivation, cellular immunotherapy is a promising therapeutic option. The capsid protein Hexon has been shown to contain immunodominant T-cell epitopes, with T-cell responses in the majority of the healthy population. Therefore a prospective phase I/II clinical study was performed analysing safety and feasibility of adoptive Hexon-specific T-cell transfer in patients after allogeneic SCT and HAdV infection refractory to Cidofovir treatment. Hexon-specific T-cells were isolated from the SCT-donor by using the IFNγ secretion system and small T-cell populations were immediately infused, without in vitro expansion steps. Fourty pediatric and adult patients with a mean age of 15 years were treated according to the study protocol after haploidentical, matched unrelated and matched sibling donor SCT between day 11 and 327 post SCT. The T-cell dose varied from 300-25000 T-cells/kg. No acute toxicitiy was observed. In two patients GvHD °I-°II of the skin occured within two weeks after administration of specific T-cells, one patient also developed GvHD of the gut. In vivo T-cell responses were absent in all patients before adoptive T-cell transfer and detectable in 70% of evaluable patients within the first weeks after adoptive transfer, associated with a clinical and/or virological response to the adoptive T-cell transfer. However, in patients with adenoviral disease response rate was lower and 6 of 14 evaluable patients succumbed with the infection within few days, in spite of adoptive immunotherapy. This lead to the assumption, that adoptive treatment in patients with severe infection related morbidity was to late during the course of infection. In conclusion we could show that adoptive immunotherapy is safe, feasible and a promising therapeutic option in patients with HAdV infection. Infusion of small IFNγ producing Hexon-specific T-cells populations resulted in an in vivo expansion of specific T-cells in the majority of cases. Emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment and Self-Renewal.
Authors: Notta Faiyaz , Doulatov Sergei , Dick John E.
Published: October 2019

Abstract 816

A fundamental tenet that has guided our insight into the biology of hematopoietic stem cells (HSCs) over the past 50 years is the principle that an HSC can only be assayed by functional repopulation of an irradiated host1. In its strictest definition, only a HSC can provide long-term reconstitution of all the major lineages following single cell transplantation. However, the existing strategies for human HSC isolation lack quantitation and do not submit to this rigorous standard, thus precluding further biological analysis. Here, we report the prospective and quantitative analysis of human cord blood (CB) HSCs transplanted into female NOD/SCID/IL-2Rgcnull mice. We identify integrin a6 (CD49f) as a novel marker of cord blood (CB) HSCs and report that single Lin-CD34+CD38-CD90+CD45RA-RholoCD49fhi cells can reconstitute myeloid, B-, and T-cell lineages for 18 weeks. 5 of 29 mice transplanted with single cells gave rise to human cells indicating that approximately 20% of cells in this fraction are HSCs. This advance finally enables utilization of near-homogeneous populations of human HSCs to gain insight into their biology and to harness them for stem cell-based therapeutics.

Disclosures:

No relevant conflicts of interest to declare.
HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.
Authors: Chapman Michael A , Brunet Jean-Philippe , Keats Jonathan J , Baker Angela , Adli Mazhar , Schinzel Anna C. , Ahmann Gregory , Christina Harview , Moore Anna , Shanmugam Vijayalakshmi , Perkins Louise M. , Auclair Daniel , Hahn William C. , Bernstein Bradley E. , Bergsagel Leif , Fonseca Rafael , Carpten John , Trent Jeffrey , Golub Todd R.
Published: October 2019

Abstract 832

We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Hematopoietic Stem Cell Transplantation (autoHSCT) in CLL: First Results of An EBMT Randomized Trial Comparing Autotransplant Versus Wait and Watch.
Authors: Michallet Mauricette , Dreger Peter , Sutton Laurent , Brand Ronald , Richards Sue , van Biezen Anja , Sobh Mohamad , Gratwohl Alois , Kolbe Karin , Dearden Claire E. , Hallek Michael , de Witte T. M. , Niederwieser Dietger W. , Leporrier Michel , Milligan Donald
Published: October 2019

Abstract 877

This phase-III randomized EBMT-intergroup trial studied the impact of a consolidating autoHSCT vs no consolidation for patients with CLL in Binet stage A progressive, B or C , in CR, nodular PR or VGPR after first or second line therapy. The primary objective was to show that autoHSCT increased the 5-year progression-free survival (PFS) by 30%. Although it had been calculated that 270 patients were to be randomized, the study was terminated by the steering committee in July 2007 due to poor accrual. Here we present a first analysis based on 69% of expected follow-up forms.

Results:

Between November 2001 and July 2007, 223 patients were enrolled (SFGM-TC/FCLLG n=98, MRC n=62, GCLLSG n=32, SAKK n=10, other EBMT centers n=17). There were 74% males and 26% females. Binet stages were progressive A 13%, B 67%, C 20%; 59% were in CR, and 41% in very good or nodular PR. Of note, SFGM-TC/FCLLG included only patients in CR. 82% of the patients were enrolled in 1st, and 18% in 2nd remission. Patients were randomized between group 1 (autoHSCT n=112) and group 2 (observation n=111) after an induction treatment which was left at the discretion of the investigators. Median PFS was 43 months in the observation group but not reached in the autoHSCT group; 5-year PFS was 48% and 65%, respectively (p=0.005). Accordingly, autoHSCT halved the relapse risk (5-year relapse incidence 25% vs. 51%; HR 0.4 [0.23-0.71], p=0.002). Cox modeling for randomization arm, Binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confirmed that autoHSCT significantly improved PFS (HR 0.41 [0.23-0.75] p=0.004). The beneficial effect of autoHSCT was stable over all contributing groups although patients accrued by SFGM-TC/FCLLG overall had a significantly better PFS than patients from other countries (HR 0.2 [0.08-0.55], p=0.001). At 5 years, the probability of OS was 92% and 91% for autoHSCT and observation, respectively. Significant differences in terms of non-relapse death were not observed. At the last follow up, among 205 evaluable patients, 186 are alive (147CR, 39 relapse), 19 died (14 from relapse and 5 from non-relapse causes) . In conclusion, in patients with CLL in first or second remission, consolidating autoHSCT reduces the risk of progression (PFS) by more than 50%, but has no effect on overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Detrimental Role of Excess Adenosine-Mediated 2,3-Diphosphoglycerate Induction in Erythrocyte Sickling and Novel Mechanism-Based Therapies.
Authors: Zhang Yujin , Dai Yingbo , Wen Jiaming , Kellems Rodney E. , Blackburn Michael R , Juneja Harinder S. , Xia Yang
Published: October 2019

Abstract 906

Introduction:

Sickle cell disease (SCD) is a devastating inherited hemolytic disorder due to a single missense mutation in the β-globin gene resulting in the production of hemoglobin S (HbS). Despite our knowledge of the molecular defect in HbS, we remain unable to control HbS polymerization and erythrocyte sickling under hypoxic conditions, which are central to the pathophysiology of the disease.

Methods and Results:

Here we report that adenosine, a cellular metabolite that is well known to be induced under hypoxic/ischemic conditions, is increased in the blood circulation of patients with SCD and in SCD transgenic (Tg) mice. To our surprise, we found that adenosine contributes to hypoxia-induced sickling of cultured erythrocytes from humans with SCD. Next, to evaluate the pathogenic role of increased adenosine in erythrocyte signaling in vivo, we took advantage of SCD transgenic (Tg) mice, a well accepted animal model of SCD. Intriguingly, treatment of SCD Tg mice with polyethylene glycol-modified adenosine deaminase (PEG-ADA) enzyme therapy to lower circulating adenosine levels reduced sickling and attenuated multiple tissue damage (including lung, kidney, liver and spleen) seen in SCD Tg mice. These findings revealed a previously unrecognized role for excess adenosine in sickling and the progression to multiple life-threatening complications. Next, by screening erythrocytes for small metabolites that are induced by adenosine and may contribute to sickling, we determined that 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific metabolite known to promote O2 release from Hb, is elevated in erythrocytes of both SCD Tg mice and humans. Using both pharmacological and genetic approaches, we demonstrated that adenosine-mediated 2,3-DPG induction is through A2B receptor signaling and that this signaling pathway is a major contributor to hypoxia-induced erythrocyte sickling.

Conclusion and Significance:

Overall, we have identified for the first time that elevated adenosine induces 2,3-DPG via A2BR signaling and contributes to sickling in both human and mouse with SCD. Thus, our studies provide strong support for the new concept that adenosine-mediated 2,3-DPG induction is beneficial for normal erythrocytes by promoting O2 release from Hb to hypoxic tissues. However, for SCD patients, the beneficial effect of adenosine-mediated 2,3-DPG induction becomes detrimental by inducing erythrocyte sickling due to 2,3-DPG mediated increased O2 release and increased deoxyHbS polymerization. Our findings reveal a novel therapeutic possibility to treat and prevent sickling and progression to multiple life-threatening complications by targeting on the adenosine signaling pathway.

Disclosures:

No relevant conflicts of interest to declare.
Crosstalk Between the Bone and Immune Systems: Osteoclasts Function as Antigen-Presenting Cells and Activate CD4+ and CD8+ T Cells.
Authors: Li Haiyan , Hong Sungyoul , Qian Jianfei , Yang Jing , Zhang Liang , Yi Qing
Published: October 2019

Abstract 918

Bone is a dynamic tissue that is constantly being remodeled by bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OB). Bone destruction, observed in patients with autoimmune disease rheumatoid arthritis and malignancies such as multiple myeloma, is believed to be caused by hyperactivation of OCs. OCs differentiate from hematopoietic monocytic precursors under stimulation by the cytokines RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor), which are produced primarily by bone marrow stromal cells, OBs, and activated T cells. As OCs are derived from monocytes/macrophages lineage similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with the stimulation of RANKL and M-CSF in vitro. DCs derived from monocytes with the stimulation of GM-CSF and IL-4 were used as positive controls. First, we examined the phenotype of OCs by flow cytometry, and OCs were detached by non-enzymatic cell dissociation solution. Results showed that OCs expressed MHC class I and II molecules and costimulatory molecules important for APCs such as CD80, CD86 and CD40. The expression of these molecules could be upregulated by LPS and IFN-γ. Second, we showed by PCR that OCs expressed IL-10, TGF-β, IL-6 and TNF-α, but not IL-12p35 and p40. Third, we examined the ability of OCs to present alloantigens and activate alloreactive T cells in a mixed lymphocyte reaction assay. OCs could present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells to proliferate as detected by [3H]thymidine incorporation and CFSE dilution assay. The activation was restricted by MHC class I and II molecules. Finally, we recruited tetanus toxoid (TT)-immunized healthy donors to test whether OCs could uptake exogenous soluble antigens and present them to CD4+ T cells. OCs pulsed with TT could activate autologous specific CD4+ T cells, which was MHC II molecule restricted. These findings indicate that OCs could function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight to the effect of OCs on the immune system especially T cells. This is not only important for a better understanding of the crosstalk between the bone and immune system but also may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems.

Disclosures:

No relevant conflicts of interest to declare.
Coordinate Loss of a MicroRNA Mir 145 and a Protein-Coding Gene RPS14 Cooperate in the Pathogenesis of 5q- Syndrome.
Authors: Kumar Madhu , Narla Anupama , Nonami Atsushi , Ball Brian , Chin Christine , Chen Cindy , Kutok Jeffery Lorne , Galili Naomi , Raza Azra , Attar Eyal C. , GIlliland D. Gary , Jacks Tyler , Ebert Benjamin L.
Published: October 2019

Abstract 947

Large hemizygous chromosomal deletions are among the most common molecular abnormalities in cancer, but the identification of critical haploinsufficiency disease genes within the deleted regions has been difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a well-studied chromosomal deletion syndrome characterized by a consistent clinical phenotype with macrocytic anemia and thrombocytosis. We have previously shown that while hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not account for the thrombocytosis. Evaluation of the effects of all protein coding genes in the CDR on hematopoietic differentiation showed no genes other than RPS14 altered the ratio of megakaryocytic to erythroid cells, either alone or in combination with RPS14. We therefore examined the 5q- syndrome CDR for non-coding RNAs and identified a microRNA, miR-145, which targets Fli-1, a transcriptional factor that regulates megakaryocyte development. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 in CD34+ cells decreases megakaryocyte production, while inhibition of miR-145 or overexpression of Fli-1 has the reciprocal effect. These findings have been validated in vivo using transgenic mice. Moreover, the combined loss of miR-145 and RPS14 cooperate to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate for the first time that coordinate deletion of a microRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.

Disclosures:

No relevant conflicts of interest to declare.
Lineage-Specific Activation of p53 in Response to Ribosomal Haploinsufficiency in Human Bone Marrow Cells.
Authors: Dutt Shilpee , Narla Anupama , Kutok Jeffery Lorne , Ebert Benjamin L.
Published: October 2019

Abstract 948

Haploinsufficiency for the ribosomal protein genes RPS14 and RPS19 have been implicated in the erythroid defect in the 5q- syndrome and Diamond Blackfan Anemia, respectively. However, the mechanism by which defective ribosome biogenesis causes erythroid failure is unknown. In this study, we found that shRNA mediated knockdown of RPS14 or RPS19 in primary human CD34+ cells stabilize TP53 by day 4 after infection with concomitant arrest of these cells at G1 stage of cell cycle. The levels of TP53 attained are comparable to the levels observed following gamma irradiation (5Gy) of the CD34+ cells. Using quantitative PCR, we confirmed that stabilized TP53 activates expression of downstream target genes MDM2, p21, Bax and Wig-1. Furthermore, treatment of the CD34+ cells with Nutlin-3 phenocopies RPS14 or RPS19 knockdown, suggesting that the mechanism of TP53 activation is mediated by MDM2 pathway. Conversely, treatment with pifithrin-alpha, which inhibits the transactivation activity of TP53, rescues the effects of RPS14 or RPS19 knockdown. The in vitro activation of TP53 in CD34+ cells was restricted to erythroid cell lineage, consistent with the clinical phenotype of RPS14 or RPS19 haploinsufficiency. Moreover, immunohistochemical analysis of bone marrow biopsies from patient with the 5q- syndrome demonstrated intense staining of TP53 that was restricted to erythroid progenitor cells. Taken together our study indicates that inhibition of ribosomal biogenesis causes TP53 activation selectively in erythroid progenitor cells. Clinically, TP53 staining of patient samples could be used as a diagnostic marker for some types of MDS.

Disclosures:

No relevant conflicts of interest to declare.