bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-Hodgkin Lymphoma and American Indians/Alaska Natives: A SEER Population Study from 2000 to 2012
Authors: Westin Gustavo Figueiredo Marcondes , Go Ronald S.
Published: October 2019

Background: American Indians and Alaska Natives (AI/AN) currently represent approximately 2% of the US population. However, population-based cancer studies are generally limited. Utilizing the Surveillance, Epidemiology and End Results Program (SEER), we investigated the incidence, major subtype distribution, and survival of non-Hodgkin lymphomas (NHL) in the AI/NA population.

Methods: We queried the SEER 2000-2012 database for AI/AN patients with NHL using ICD-O-3 codes 9590-9823. We calculated their incidence rate (case/1,000,000) and trend as well as risk ratios compared to other races, age-adjusted to the US 2000 standard population. Patient level data were extracted to perform summary statistics and calculate overall survival (OS) using STATA/IC 12.1.

Results: We identified 1,135 AI/NA patients with NHL. The average age was 59.6 years (range, 20-85) and 53% were males. The distribution of the major NHL subtypes resembles that seen among the rest of the population and is as follows: diffuse large B-cell (DLBCL; 28%), marginal zone (MZL; 19%), follicular (FL; 13%), small lymphocytic/chronic lymphocytic leukemia (SLL/CLL; 10%), NHL not-otherwise specified (NOS, 4%), mantle cell (MCL; 2%), anaplastic large cell (ALCL; 2%), lymphoplasmacytic (LPL; 1%), and peripheral T-cell (PTCL; 1%). The overall incidence of NHL is 26.4 and did not significantly change over the 12-year study period (Annual Percent Change 0.0, 95% CI [-0.3, 0.3]). Compared to other races, AI/NA have lower incidence of NHL including most of the major subtypes (Table). The median OS for all types of NHL combined is 4.16 years. AI/AN OS is statistically inferior compared to Whites(Figure).

Conclusions: The NHLincidence in AI/NA is lowest among the major race groups and has remained stable in the past 12 years. However their OS is inferior when compared to Whites. The distribution of major NHL subtypes and OS are similar to the rest of the population.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Activation of C/EBPa Myeloid-Specific Transcription Factor By NAMPT/SIRT1-Triggered Deacetylation
Authors: Samareh Bardia , Nasri Masoud , Zimmer Inna , Klimenkova Olga , Keller Leonie , Kanz Lothar , Welte Karl , Skokowa Julia
Published: October 2019

Previously, we described new mechanism of G-CSF-triggered granulocytic differentiation of hematopoietic stem cells (HSCs) via activation of the enzyme Nicotinamide Phosphorybosyltransferase (NAMPT) leading to NAD+ production and activation of NAD+ -dependent protein deacetylase sirtuin 1 (SIRT1). We found, that upon stimulation of HSCs with NAMPT, SIRT1 bound to the key myeloid transcription factor C/EBPα followed by transcriptional induction of C/EBPα target genes G-CSFR and G-CSF and granulocytic differentiation.

In the present work we investigated the mechanism of NAMPT/SIRT1-triggered deacetylation of C/EBPα. We found that C/EBPα is acetylated at the position Lys 161, which is evolutionarily conserved. Lys 161 is localized in the transactivation element III (TE-III) of the transactivation domain (TAD) of C/EBPα protein, which is responsible for recruitment of SWI/SNF and CDK2/CDK4. Western blot and DUOLINK analysis using rabbit polyclonal antibody specifically recognizing acetyl-Lys 161 of C/EBPα revealed predominantly nuclear localization of acetylated C/EBPα protein in acute myeloid leukemia cell lines NB4 and HL60 as well as in primary HSCs. Induction of myeloid differentiation of HSCs by treatment with G-CSF as well as ATRA-induced differentiation of NB4 cells resulted in the deacetylation of C/EBPα. NAMPT inhibition in NB4 and HL60 cell lines using specific inhibitor FK866 led to the dramatically elevated levels of acetylated C/EBPα and reduced amounts of total C/EBPα protein, which was in line with diminished mRNA expression of C/EBPα target genes (G-CSF, G-CSFR and ELANE). Interestingly, treatment of acute myeloid leukemia cell line HL60 with NAMPT or transduction of HL-60 cells with NAMPT-expressing lentiviral construct induced myeloid differentiation of these cells even without addition of ATRA. This was in line with time- and dose-dependent increase of total C/EBPα protein levels upon NAMPT treatment. Therefore, NAMPT overcomes transcriptional repression of C/EBPα in HL-60 cells by activation of positive CEBPA autoregulation.

Taken together, we described a new mechanism of regulation of C/EBPα activities in hematopoiesis and leukemogenesis by its post-translational modification via NAMPT/SIRT1-triggered de-/acetylation.

Disclosures

No relevant conflicts of interest to declare.
Primary Cutaneous Marginal Zone B-Cell Lymphoma Treated with Intralesional Rituximab: Experience of a Single Center
Authors: Domingo-Domenech Eva , Salas Queralt , Climent Fina , Aguilera Carmen , Pomares Helena , González-Barca Eva , Mercadal Santiago , Lucas Anna , Fernandez de Sevilla Alberto , Sureda Anna , Servitje Octavio
Published: October 2019

BACKGROUND

Primary cutaneous marginal zone B-cell lymphomas (PCMZL) are low-grade lymphomas with an indolent course, but with a high rate of skin relapses. Surgical excision, local radiotherapy, or combinations of both are the first-line treatment in solitary or localized disease. Therapeutic options in cases of multifocal disease or relapse, include steroids or intravenous Rituximab in monotherapy or in combination with intravenous or oral chemotherapy. The effectiveness of intralesional Rituximab (ILR) in primary cutaneous B-cell lymphomas has been described in a small multicenter series of patients.

OBJECTIVE

To evaluate the effectiveness and safety of ILR in patients with PCMZL.

METHODS

In our center ILR is used since 2010 as fist-line treatment for patients with PCMZL with single facial lesions, multifocal disease that cannot be irradiated or in relapsed disease. The treatment regimen consist of a course of three injections of 10 mg ( days 1, 3 and 5) in a single week at monthly intervals. Oral paracetamol and dexclofeniramire were administrated 30 minutes before the procedure. Courses of ILR were monthly repeated until response or in cases of relapse. The primary outcome measure of the study was the rate of remissions (complete and partial remissions).

RESULTS

Eleven patients with PCMZL have been treated so far: 55% as first-line treatment and 45% in the relapse setting. The relapsed-group had previously received a median of 3 previous treatments (range, 1-6) including radiotherapy in 5 cases, surgery in 1 case, intravenous chemotherapy in 2 cases, and topic steroids in 2 cases. Clinical characteristics of the 11 patients are shown in the table below.

A total of 40 courses or ILR have been completed. All patients responded, with 73% complete responses, without evidence of progression or relapse in the site of ILR treatment.

Four patients experienced transitory mild perilesional erythema that did not require medication and resolved in less than 4 hours. No systemic toxicities or infections were observed; the tolerance was excellent in all cases.

CONCLUSIONS

In our preliminary experience, ILR is an effective and safe therapeutic approach for selected PCMZL patients with single facial lesion, multifocal disease than cannot be irradiated or in relapsed cases. Further controlled studies are needed to confirm these findings.

Disclosures

Sureda: Takeda: Consultancy, Speakers Bureau.
Downregulation of Glycosyltransferase a Expression By miRNA-331-3p Is Mediated By the Inhibition of the Transcription Factor SP1
Authors: Kronstein-Wiedemann Romy , Bugert Peter , Seifried Erhard , Tonn Torsten
Published: October 2019

More than 100 ABO subgroup-related variations were detected in the coding region of glycosyltransferases, which may be causative for a weak blood group antigen A or B expression. Most variation in expression is explained genetically by mutations that reduce transferase activity. However, a substantial number of weak variants have not yet been explained by current methods. We recently postulated a role of miRNA in the regulation of blood group A antigen expression levels. By using different approaches, including gene array analysis, luciferase reporter assay and overexpression of glycosyltransferase specific miRNAs in primary hematopoietic stem cells (HSCs), we found that miR-331-3p directly targets glycosyltransferase A and B mRNA. Now we have been further embarking on the underlying mechanisms of miRNA and glycosyltransferase interactions and show that the effects of miR-331-3p are mediated by inhibition of transcription factor SP1, which is a major regulator of the ABO gene, thereby resulting in downregulation of blood group A antigen expression in A2O individuals by up to 80% and in A1O individuals by up to 50%. Using microRNA target prediction tools we also identified Sp1 as a potential target gene for miR-331-3p. Western blot analyses of glycosyltransferase protein expression showed that overexpression of miR-331 led to a decreased glycosyltransferase and SP1 protein expression. Further approaches with the SP1 inhibitor, mithramycin A, revealed similar results. Analysis of Aw04/O1v genotypes revealed elevated miRNA levels, thus confirming microRNAs as regulators of blood group glycosyltransferase expression. Our findings extend our understanding of blood group regulation in carriers of weak blood group variants. Involvement of miRNAs in the downregulation of ABO blood group antigen expression may also provide an explanation for observed changes of ABO antigen expression in abnormal processes such as tumorigenesis, pregnancy and aging. Furthermore, this pathway may play a role in the regulation of other blood group variants (for instance Rhesus, KELL, amongst others).

Disclosures

No relevant conflicts of interest to declare.
Impaired DNA Damage Repair in Severe Congenital Neutropenia Patients
Authors: Mir Perihan , Klimiankou Maksim , Dannenmann Benjamin , Zeidler Cornelia , Schulze-Osthoff Klaus , Kanz Lothar , Welte Karl , Skokowa Julia
Published: October 2019

Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1(mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation.

We first investigated differences in DNA damage susceptibility of CD34+ and CD33+ bone marrow cells from CN-ELANE (n = 3) and CN-HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair (BRCA1 and RAD51), mismatch repair (MSH2 and PCNA) as well ascell cycle regulation (CHEK2 and CDKN2C) in CD33+ of both CN groups as compared to healthy individuals. Interestingly, CN-ELANE and CN-HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B, RAD50 and ATR expression in the CN-HAX1 group only and of MRE11A in the CN-ELANEgroup only.

Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation.

Disclosures

No relevant conflicts of interest to declare.
Engineered Exosomes Expressing HLA and Co-Stimulatory Molecules to Generate Antigen-Specific CD8+ T Cells for Adoptive Cell Therapy
Authors: Kim Sueon , Sohn Hyun-Jung , Lee Hyun-Joo , Sohn Dae-Hee , Hyun Seung-Joo , Cho Hyun-Il , Oh Il-Hoan , Kim Tai-Gyu
Published: October 2019

S.K and H.-J.S. contributed equally for this works

Dendritic cell-derived exosome (DEX) has been known as an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to broad utility for immunotherapy. In this study, we engineered K562 cells expressing triple-co-stimulatory signals (CD80, 4-1BBL, and CD83) with HLA-A2 as an AAPC. Specifically, CD137L (4-1BBL) is an ideal signaling molecule for long-term propagation of CD8+ T cells, and the addition of other co-stimulatory molecules, such as CD80 and CD83, is used to support the expansion of naive T cell subsets. DC-derived exosomes display immunologically important molecules such as HLA and co-stimulatory molecules. Likewise, CoEX-A2 expressed high levels of HLA-A2, CD80, CD83, and CD137L (41BBL) and mediate strong, antigen-specific CD8+ T lymphocyte activation. The stimulation of freshly isolated peripheral CD8+ T cells with the appropriate antigen specificity observed here was likely made possible by the use of the sensitive ELISPOT assay. Viral or tumor protein-pulsed exosomes can directly stimulate CD8+ T cell proliferation and differentiation into CTLs in vitro. In addition, exosomes can be taken up by both CD8+ T cells and K562 cells. Meanwhile, K562 cells that have taken up exosomes can also stimulate CD8+ T cells, which may be due to the higher levels of HLA-A2, CD80, CD83, and 41BBL expression observed on exosomes. Therefore, the CD8+ T cell antigen-specific expansion observed in our cultures is likely the result of coated CoEX-A2s working directly or in a cross-dressed manner. The results suggest that these novel exosomes may provide a crucial source to generate antigen-specific CD8+ T cells for adoptive cell therapy against viral infection and tumors.

Disclosures

No relevant conflicts of interest to declare.
A Large-Scale Prospective Cohort Study on Factor XIII Deficiency in Southeast of Iran
Authors: Naderi Majid , Dorgalaleh Akbar , Peyman Eshghi , Malek Fatemeh , Karimi Mehran
Published: October 2019

Background: Congenital factor XIII (FXIII) deficiency is an extremely rare bleeding disorder with an estimated incidence of 1 per 2 million which has the highest global incidence in the southeast of Iran. Hence, this cohort study was designed to assess the prevalence of FXIIID, its molecular basis and clinical manifestations of FXIII deficiency (FXIIID) in Sistan and Baluchestan Province, southeast Iran.

Methods: All data about the prevalence of this disorder, its genotype and phenotype were collected prospectively from 2010 to 2016.

Results: In total , 410 patients were identified within the study period, a prevalence of 1 per ~ 7,000 in this area. The mean age of patients was 12 years (range, 1 month to 55 years), of which 51% (n: 209) were female. The main clinical presentation leading to the diagnosis of FXIIID was umbilical cord bleeding (82%). Three hundred and eighty patients were receiving regular prophylaxis of 10 IU/Kg of FXIII concentrate (Fibrogammin) every 28 days. This therapeutic dose was successful in the management of major and minor bleeding and the prevention of life-threatening bleeding in this cohort. But despite receiving prophylaxis, 63 minor bleeding episodes were recorded: 70% lower extremity ecchymoses, 25% oral cavity bleeding and 5% of menorrhagia. The prophylaxis interval for 5 patients was reduced to 21 days due to these bleeding events. Molecular analysis showed that 407 of the patients had a unique Trp187Arg mutation in exon 4 of the FXIII-A subunit gene, while the three remaining patients did not have a detectable underlying mutation.. During the study period, 70 patients underwent minor and major surgeries that were managed by 10-50 IU/Kg Fibrogammin without any significant bleeding. Furthermore, 32 successful deliveries were reported, consisting of 17 normal deliveries and 14 cases of Caesarian section, with a dose of 10-25 IU/Kg. No blood-borne disease was detected in patients receiving Fibrogammin.

Discussion: FXIIID has the highest global incidence in the southeast of Iran. The Trp187Arg mutation im the FXIII-A gene is the sole underlying mutation in this area due to the founder effect. Fibrogammin is a safe and suitable therapeutic choice for prophylaxis and management of major and minor bleeding.

Disclosures

No relevant conflicts of interest to declare.
Ribavirin Treatment for Respiratory Syncytial Virus in Immunocompromised Hosts
Authors: Nouvini Rosa , Kiner Bonnie , Yarovikova Margarita , Strachan Paul
Published: October 2019

Intro: Respiratory syncytial virus (RSV) is a common virus that can cause infection in the immunocompromised population.. In patients with hematologic malignancies, mortality rates have ranged from 7% to 83%. Most studies have focused on allogeneic stem cell transplant patients; however, those that have studied hematologic malignancies have shown equivalent RSV related mortality rates. Mortality is seen in the setting of lower respiratory tract infections (LRTI) and RSV progresses to lower respiratory tract infection in 40-50% of infected patients. In this study, we reviewed the charts of 35 immunocompromised, hospitalized patients diagnosed with RSV and treated with oral Ribavirin +/- IVIG to evaluate clinical outcome.

Methods: A retrospective chart review was conducted on 35 patients who tested positive for RSV via polymerase chain reaction (PCR). Data was collected between January 2010 and July 2016. These patients were treated with oral Ribavirin 10mg/kg loading dose on day 1, 400mg q8h on day 2 followed by 600mg q8h for the duration of the therapy from day 3 forward with or without IVIG 500mg/kg weekly as per treating physician.

Results: The cohort consisted of 20 males and 15 females. There were13 allogeneic stem cell transplant patients, 1 autologous stem cell transplant patient, and 21 patients with hematologic malignancies actively undergoing chemotherapy. Thirty-three patients were tested via nasopharyngeal swab and two were tested via BAL. Twenty were diagnosed with LRTI and 15 diagnosed with upper respiratory tract infection (URTI). Twenty one patients developed worsening hypoxemia with increased oxygen requirement. Two patients were intubated. Thirty patients were treated with Ribavirin and IVIG and 5 patients with Ribavirin alone. Sixteen patients started treatment within 2-3 days of positive RSV PCR and the rest began treatment after 3 days. All patients showed clinical improvement within 14 days of treatment. No patients died during the treatment period.

Conclusion: In our cohort,oral ribavirin with or without IVIG was an effective treatment for RSV infection in immunocompromised patients with hematologic malignancies. Further studies are needed to confirm this result.

Disclosures

No relevant conflicts of interest to declare.
Rabbit ATG Plus Cyclophosphamide with or without Fludarabine Is Superior Conditioning Regimen for Patients with Severe Aplastic Anemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Ahmed Parvez , Satti Tariq Mahmood , Chaudhry Qamar Un Nisa , Mehmood Kamran , Shahbaz Nighat , Khan Mehreen Ali , Ghafoor Tariq , Iftikhar Raheel , Satti Humayoon , Hanif Andleeb
Published: October 2019

Objective: To compare rabbit antithymocyte globulin (ATG) containing conditioning regimen (group A) with horse antilymphocyte globulin (ALG)-based regimen (group B) for sibling allogeneic hematopoietic stem cell transplant (HSCT) outcome, disease free survival, rejection and complications in severe aplastic anemia patients.

Methods: We analysed 205 aplastic anemia patients undergoing allogeneic HSCT from HLA matched sibling donors from July 2001 to April 2016. Group A (n=169) received conditioning with ATG plus Cyclophosphamide (CY) 200 mg/Kg with Fludarabine (Flu) 120mg/m2 (n=100) or without Flu (n=69), whereas group B comprised of CY plus ALG (n=36). The stem cell source was bone marrow (43.9%); PBSC (8.3%) and bone marrow plus PBSC (47.8%). Cyclosporin (CsA) was given as GVHD prophylaxis in 129 patients, while methotrexate (MTX) was added to CsA in 76 cases. Chi-square test was used to compare categorical variables. Kaplan Meier survival curves with log rank test was applied to compare the groups for survival analysis.

Results: Overall survival was 78.7% in Group A compared to 69.4% in group B (p=0.23). Patients in group A had significantly better disease free survival (DFS) (75.1%) than group B (55.6%) (p=0.018). The incidence of acute GVHD was 18.3% in Group A compared to 22.2% in Group 2 (p=0.64), while chronic GVHD was significantly higher in Group 2 (22.2%) as compared to Group A (9.5%) (p= 0.04). Likewise frequency of Mucositis was high in Group B (30.5%) compared to Group A (10.7%) (p = 0.006). Moreover, patients receiving ALG-based conditioning had more frequent infections (86%) compared to ATG-based group (67.4%) (p=0.03). There was no significant difference within group A with respect to use of fludarabine as part of conditioning.

Conclusion: Rabbit ATG containing conditioning regimen is associated with less complications and better survival in patients with severe aplastic anemia undergoing allogeneic HSCT.

Disclosures

No relevant conflicts of interest to declare.
Immunoglobulin Recovery after ASCT for Patients with Multiple Myeloma: Impact on Overall Survival and Progression-Free Survival
Authors: Jimenez-Zepeda Victor , Duggan Peter , Neri Paola , Chaudhry Ahsan , Tay Jason , Bahlis Nizar J.
Published: October 2019

Introduction Immunoparesis, defined as suppression of uninvolved immunoglobulins (Igs), has been described as one of the most common indicators of immune dysfunction in patients with multiple myeloma (MM). The role of immunoglobulin recovery, however, has not been properly evaluated, in particular in the setting of immunomodulatory therapy. In the present study, we aimed to assess the impact of immunoparesis and Igs recovery after ASCT on Overall and Progression-Free survival (OS and PFS).

Methods All consecutive patients undergoing ASCT at our center from 01/2004 to 03/2016 were evaluated. All patients received induction chemotherapy before undergoing auto-SCT. Patients received conditioning with either HDM at 200 mg/m2 (or adjusted as per renal failure) or HDM with Bortezomib (Bor-HDM). Definitions of response and progression were used according to the EBMT modified criteria. Immunoglobulin recovery was assessed at 12 months post-ASCT in those cases where immunoparesis was noted at diagnosis.

Results Clinical characteristics are shown in Table 1. Decrease of uninvolved Ig's was noted in 94% of cases. At the time of analysis, 69.2% of patients are alive and 54.9% have already progressed, respectively. Median OS did not differ between patients with or without immunoparesis (p=0.851). With regards to Ig recovery at 12 months post-ASCT, median OS was longer for those patients with complete or partial normalization of Ig's compared to those with no recovery at all (Estimate median OS of 97, 77 and 69 months, respectively, p=0.008) (Fig1a). In addition, median PFS was longer for those patients with normalization or partial recovery of Ig's at 12 months post-ASCT (median of 40.3 and 38 months, compared to 24.6 months for those without Ig recovery, p=0.002) (Fig1b). Furthermore, 121 patients receiving lenalidomide maintenance were evaluated, 49% of cases exhibited complete or partial recovery of Ig's at 12 months post-ASCT, compared to 40% for those cases that were not treated with any maintenance strategy (p=0.6).

In conclusion, Immunoglobulin recovery at 12 months post-ASCT is an important factor to predict for better progression-free and overall survival. The use of lenalidomide as a maintenance strategy did not seem to affect the process of Ig recovery. Based on our observations, the use of immune enhancement strategies in the post-transplant setting seem appealing and requires further investigation.

Disclosures

Jimenez-Zepeda: Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene, Janssen, Amgen, Onyx: Honoraria. Neri:Celgene and Jannsen: Consultancy, Honoraria. Bahlis:Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; BMS: Honoraria; Onyx: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau.
CD34+ Selected Cells "Boost" for Poor Graft Function Post Allogeneic Stem Cell Transplantation
Authors: Mohty Razan , Ruggeri Annalisa , Battipaglia Giorgia , Malard Florent , Brissot Eolia , Belhocine Ramdane , Eder Sandra , Giannotti Federica , Dulery Remy , Mohty Mohamad
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is widely used in the treatment of a variety of hematologic diseases. However, allo-HSCT can be associated with many complications, including poor graft function early after transplant requiring long-lasting supportive care. In the literature, the incidence of poor graft function post allo-HSCT has been reported to range from 4 to 27%.

Here, we retrospectively studied 10 patients (male/female: 4/6, median age: 45 years, range 19 to 67) who received a boost of CD34+ selected cells for poor graft function after allo-HSCT (of whom 4 cases of haplo-identical allo-HSCT with post-Cy prophylaxis), between January 2014 and January 2016. Patients' disease and transplant characteristics are summarized in the below table.

Patients were selected for the CD34+ cells "boost" therapy after eliminating other causes that could explain a poor graft function (eg. drug toxicity, infections, disease relapse, etc.) The same original allo-HSCT donor was used to collect the CD34+ cells after mobilization with G-CSF and positive selection. The patients did not receive any prior conditioning therapy prior to CD34+ cells boost infusion. At time of the boost, all patients were in full donor chimerism. The number of infused CD34+ cells differed from one patient to another ranging from 2.91 to 7.99 x106 cells/kg recipient body weight. The median day of infusion post- allo-HSCT was 120 (range, 76-352). Among these 10 patients, 7 patients had full counts recovery at a median of 15 days (range, 7-30) post-infusion, while 3 patients had an incomplete response with persistent anemia and/or thrombocytopenia. None of the patients experienced clinically significant GVHD symptoms after the boost. At last follow-up, 7 patients were alive whereas 3 patients died of severe infections after 1, 6 and 13 months post-boost.

Based on these results, we concluded that boost therapy can be used in the treatment of poor graft function post-allogeneic HSCT, including in those patients who received a haplo-transplant.

Disclosures

No relevant conflicts of interest to declare.
Adenosine Signaling-Mediated Metabolic Reprogramming Regulates Erythropoiesis
Authors: Liu Hong , Zhang Yujin , D'Alessandro Angelo , Nemkov Travis , Couturier Jacob , Zhao Shushan , Hansen Kirk C , Lovering Andrew T. , Roach Robert , Kellems Rodney E. , Eltzschig Holger K. , Blackburn Michael R. , Xia Yang
Published: October 2019

Erythropoiesis is an extremely dynamic process finely regulated by cytokines, hormones, and growth factors at transcriptional and translational levels. Stress-induced erythropoiesis is defined as a stimulated basal erythropoiesis with expansion of the erythroid progenitor pool, associated with reticulocytosis and splenomegaly. Stress erythropoiesis is stimulated under the condition of insufficient oxygen availability such as high altitude, blood loss, infection, and anemia. Thus, stress erythropoiesis is an important stress adaptive response for survival. Although stress erythropoiesis has been long speculated to be linked with increased metabolic requirements, until recent two years with innovative metabolomics profiling and state of art isotopically labelled metabolic flux approaches, the filed has evolved and revealed that enhanced glucose and glutamine metabolism is essential for stress erythropoiesis. However, molecular basis underlying metabolic reprogramming to enhance glucose metabolism and subsequently stress erythropoiesis remains unclear.

To address this question, we conducted both human and mouse studies. First, we found that plasma adenosine is rapidly induced and associated with stress erythropoiesis features including increased hematocrit (HCT), hemoglobin (Hb) mass and reticulocytes in healthy human volunteers at high altitude and in mice exposed to hypoxia mimicking high altitude. Follow-up mouse genetic studies showed that activation of adenosine signaling via erythroid ADORA2B promotes the survival and expansion of proerythroblasts both in spleen and bone marrow and in this way contributes to hypoxia-induced stress erythropoiesis independent of erythropoietin. Using unbiased high-throughput metabolic profiling, we identified that erythroid ADORA2B contributes to an overall hypoxia metabolic reprogramming with substantial increased glycolysis in proerythroblast progenitors in mice. Finally, using primary human CD34+ hematopoietic stem cells culture, we showed that adenosine analogue and ADORA2B agonist promote the survival and expansion of erythroid progenitors in a time and dose-dependent manner. Taken together, both human and mouse studies identify that adenosine ADORA2B is a previously unrecognized purinergic signaling underlying hypoxia-induced erythropoiesis by facilitating expansion and survival of proerythroblasts, and highlight that enhancing this pathway is a potential strategy to induce erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
Sargramostim Accelerates Leukocyte Recovery and Improves Mortality Rate at Day 60 in a Non-Human Primate Model of Hematopoietic Acute Radiation Syndrome When Administered 48 h after Total Body Irradiation
Authors: Clayton Nicholas P. , Charpentier Eric J.J. , LaCasse Emily R. , Khan-Malek Richard C. , Keutzer Joan M.
Published: October 2019

Background: Hematopoietic acute radiation syndrome (H-ARS) occurs in individuals that are exposed to high levels of radiation over a short period of time. The destruction of bone marrow leads to pancytopenia, which increases the incidence of infections and accounts for the majority of morbidity and mortality. Sargramostim (yeast-derived rhGM-CSF) is a leukocyte growth factor that promotes differentiation, maturation and activation of granulocytes, monocytes and macrophages.

Methods: We evaluated the efficacy of repeat administrations of sargramostim (7 μg/kg/day) in a blinded, GLP study in total body irradiated non-human primates (NHPs) when administered 48 h post-irradiation with minimal supportive care (e.g., no blood products or individualized antibiotics). The primary objective was to assess the efficacy of sargramostim versus vehicle on mortality rate at Day 60 in irradiated male and female NHPs at LD50-60/60 (n=36/group). Secondary objectives included the efficacy of sargramostim on overall survival and its effect on hematology parameters. The study included an exploratory arm of male and female NHPs that were irradiated at a LD70-80/60(n= 18/group) and received the same treatments.

Results: Sargramostim decreased the mortality rate at Day 60 by 36% in the LD50-60/60 group (p=0.0018) and by 44% in the LD70-80/60 group (p=0.0076). Additionally, neutrophil, platelet, lymphocyte, and white blood cell levels in survivors demonstrated accelerated recovery in the sargramostim-treated NHPs in the LD50-60/60 and LD70-80/60 groups.

Conclusion: Treatment with sargramostim (7 μg/kg/day) beginning at 48 h, in the absence of blood products and individualized antibiotics, suggests that the use of sargramostim is a viable therapeutic strategy for H-ARS in a mass casualty event.

Funding: This project has been funded in whole or in part with Federal funds from the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201300005I.

Disclosures

Clayton: Sanofi Genzyme: Employment. Charpentier:Sanofi: Employment. LaCasse:Sanofi Genzyme: Employment. Khan-Malek:Sanofi: Employment. Keutzer:Sanofi Genzyme: Employment.
L-DEP Is an Effective Treatment for Adult and Adolescent Patients with Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis
Authors: Lai Wenyuan , Wang Yini , Wang Jingshi , Wang Zhao
Published: October 2019

Epstein-Barr virus-associated hemophagocytic lymphohistocytosis (EBV-HLH) is the most frequent subtype of secondary HLH with infectious triggers. In addition to meet the HLH-2004 diagnostic criteria, the diagnosis of EBV-HLH requires the evidence of EBV infection and exclusion of primary HLH and lymphoma-associated HLH. The prognosis of EBV-HLH is generally dismal. To assess the efficacy of current treatment protocols in adult and adolescent patients with EBV-HLH, we retrospectively analyzed 171 HLH patients presented at our institution from March 2009 to April 2016. Of those, 133 patients were EBV-HLH; 35 patients were lymphoma-associated HLH; 3 of 68 patients who underwent genetic test were diagnosed with primary HLH including one with homozygous mutation in UNC13D, one with homozygous mutation in RAB27A, and one with hemizygous mutation in SH2D1A. The rest of the 68 patients exhibited various levels of genetic defects. Of the 133 EBV-HLH patients, 91 male and 42 female cases (2.2:1) were aged between 14 - 77 years with a median age of 26 years. EBV-DNA load on admission was at a median of 6.6 × 105 copies/ml (range: from 5.0 × 103 - 7.8 × 109 copies/ml). One-year mortality rate for them was 66.4%. One hundred and twelve cases were initially treated by HLH-94 or HLH-2004 regimen. Of those, 52 cases (46.4%) had response including 22 cases (19.6%) of complete response (CR) and 30 cases (26.8%) of partial response (PR). Six patients received L-DEP regimen as initial treatment, of which 5 cases had response consisting 3 cases of CR and 2 cases of PR. The overall response rate (ORR) was 83.3%. The rest 15 patients received initial treatment without etoposide, 5 patients (33.3%) achieving PR and 10 patients (66.7%) showing no response. Sixty-nine refractory or relapsed cases received DEP or L-DEP regimen, ORR of which was 79.7%. For those cases, 26 cases received DEP regimen, of which 17 cases (65.4%) had response including 5 cases of CR and 12 cases of PR. Forty-three patients received L-DEP as salvage therapy, of which 38 cases (88.4%) had response including 13 cases of CR (30.2%) and 25 cases of PR (58.1%). Compared to DEP treatment, who received L-DEP had a higher effective rate (65.4% versus 88.4%, p=0.031). Of 133 EBV-HLH patients, only 36 patients received allogeneic hematopoietic stem cell transplantation, the survival rate was 61.1%. In conclusion, EBV-HLH is a fatal disease. Our results indicate DEP or L-DEP is an effective salvage therapy. Additionally, L-DEP may be a more effective therapy than HLH-94/2004 regimen to be used as an initial treatment. Finally, allogeneic hematopoietic stem cell transplantation is an effective approach for the treatment of EBV-HLH.

Disclosures

No relevant conflicts of interest to declare.
Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy
Authors: Tyler Paul M , Servos Mariah M , Klebanov Boris , Kashyap Trinayan , Shacham Sharon , Landesman Yosef , Dougan Michael , Dougan Stephanie K
Published: October 2019

Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016).

Disclosures

Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.
Impaired Metabolic Fitness in T Cells in Chronic Lymphocytic Leukemia
Authors: van Bruggen Armando , Endstra Sanne , van der Windt Gerritje J.W. , Kater Arnon P.
Published: October 2019

Quiescent T cells primarily use oxidative phosphorylation (OXPHOS) to generate ATP, while in response to activation T cells switch to high rates of aerobic glycolysis, also known as the Warburg effect. While less efficient in overall ATP production, this switch to aerobic glycolysis is essential to produce cellular biomass needed for proliferation, and is required for T cell effector functions. In chronic lymphocytic leukemia (CLL) acquired T cell dysregulation occurs independent of treatment with functional impairment and exhaustion of T cells. As tumor-imposed metabolic restrictions in mouse models can mediate T cell hyporesponsiveness during cancer, we hypothesized that in the context of CLL T cell metabolism might be altered.

Comparison of gene expression profiles of T cells from patients with CLL and age-matched healthy donors (HD) revealed a highly significant increase in the expression of genes in the OXPHOS pathway (P=3.4*10-15) in the CD8 T cell compartment. In corroboration with these array results, we found that in naïve CD8 T cells in CLL, mitochondrial mass and respiration were significantly increased. In addition, increased mitochondrial ROS production was observed in the naïve CD8 T cell subset.

Using Seahorse EFA technology on sorted CD8 T cells from CLL and age-matched HD, we found increased oxygen consumption rates in CLL derived CD8 T cells, indicating increased OXPHOS, while the spare respiratory capacity was lower in these cells, indicating impeded ability to cope with cellular stress. Extracellular acidification rates (ECAR; indicating glycolysis), and uptake of fluorescently labeled glucose were similar in CD8 T cells from CLL patients and HD.

Next, we studied the metabolic plasticity of CLL derived T cells by stimulation of PBMCs from CLL patients and age-matched HD using anti-CD3/CD28 antibodies. Two days after stimulation, CLL derived T cells had diminished expression of activation markers CD25 and CD38, which correlated with reduced uptake of fluorescently labeled glucose. Moreover, preliminary Seahorse analysis of the immediate response to stimulation with anti-CD3/CD28 showed a reduced increase in ECAR in CLL derived CD8 T cells, indicating an impairment of the glycolytic switch in these cells.

Taken together, these results demonstrate that the metabolic fitness of CD8 T cells is impaired in CLL at resting state and after activation. Boosting T cell metabolism in CLL might therefore improve existing immunotherapies in CLL such as CAR-T cell therapy.

This work is funded by VENI and VIDI grants from the Dutch Organisation for Scientific Research, and a Marie Curie Career Integration Grant from the European Union.

Disclosures

Kater: Celgene: Research Funding.
A Novel Monoclonal Antibody Against A2 Domain of Von Willebrand Factor Reduces Its Proteolysis By ADAMTS13
Authors: Zhang Lulu , Su Jian , Shen Fei , MA Zhenni , Zhao Yiming , Yu Ziqiang , Ruan Changgeng
Published: October 2019

Introduction:ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. A minimal region as a functional substrate for ADAMTS13 consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and deletion of the E1660-R1668 region (the C-terminal α helix of VWF A2 domain) led to the loss of cleavage by ADAMTS-13. This suggested that the C-terminal α helix of VWF A2 domain contributes to ADAMTS13 binding to substrate. We will explore whether murine monoclonal antibody (mAb) against the C-terminal α helix of human VWF A2 domain affects the susceptibility of VWF to proteolysis by ADAMTS13 in vitro.

Methods:Balb/c mice were immunized with R1659-R1668 of the VWF polypeptide. Monoclonal antibodies (mAbs) were developed by standard hybridoma technology and identified with ELISA. The epitope of mAbs were mapped using recombinant VWF A2 and a series of its truncated mutants with western blot analysis. The effect of mAbs to VWF on its proteolysis by ADAMTS13 was investigated under static/denaturing condition by 1.3% agarose gel electrophoresis and immunologic analysis. The effect of mAbs to GST-VWF73-His on its proteolysis by recombinant ADAMTS13 was measured by 15% SDS-PAGE under reducing condition and western blot analysis.

Results: One mAb, designated SZ-179, was found to have high affinity (50 ng/ml) with both a synthetic peptide encompassing residues 1659-1668 and purified pVWF. SZ-179 has isotype of IgG1. It was shown that SZ-179 reacted specifically with recombinant VWF A2 and GST-VWF73-His, respectively, but not with recombinant VWF A1 nor with recombinant VWF A3. Epitope mapping experiments revealed that SZ-179 bound to the distal portion of the VWF A2 domain (E1660-L1666). SZ-179 inhibited proteolytic cleavage of GST-VWF73-His by recombinant ADAMTS13 in a concentration-dependent manner, with a maximal inhibition at concentrations of 50 ug/ml. SZ-179 also reduced degradation of high molecular-weight (HMW)-VWF-multimers under static/denaturing condition dose-dependently, with a maximal inhibition at concentrations of 1 ug/ml.

Conclusion:SZ-179, a novel mAb to the A2 Domain of VWF, inhibits the interactions between VWF and ADAMTS13, and prevents excessive degradation of HMW-VWF multimers. This mAb might be useful for the development of therapeutic options to treat bleeding episodes among hemorrhagic diseases.

Disclosures

No relevant conflicts of interest to declare.
TOP2A a New Predictive Marker of Response to Carfilzomib in Multiple Myeloma
Authors: Reale Antonia , Khong Tiffany , Mithraprabhu Sridurga , Reynolds John , Ramachandran Malarmathy , Savvidou Ioanna , Bergin Krystal , Hocking Jay , Spencer Andrew
Published: October 2019

Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib (CZ), a second-generation proteasome inhibitor (PI) currently used in clinical settings, could be identified.

Materials and Methods: Nine human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive or resistant to carfilzomib. Subsequently, the gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs were compared. GEP-identified differences were validated on a panel of 17 HMCLs with q-RT-PCR and siRNA knockdown assays. Topoisomerase 2-alpha (TOP2A) immunohistochemistry was performed on a panel of fully annotated trephine biopsy specimens acquired prior to treatment with PIs.

Results: 206 genes were differentially expressed between the sensitive and resistant HMCLs. Gene ontology analysis identified two pathways that were significantly different: pathogenic Escherichia Coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A has also been noted to be overexpressed in the 'proliferation cluster' of published MM GEP profiles and associated with poor outcome. Following suppression of TOP2A by siRNA, carfilzomib-resistant HMCLs were re-sensitized to carfilzomib, moreover, the combination of carfilzomib with topoisomerase inhibitors known to target directly TOP2A, demonstrated synergistic cytotoxic effects against HMCLs. Trephine-derived TOP2A protein expression levels were shown to be higher in patients treated with PIs including bortezomib, carfilzomib, ixazomib, marizomib, failing to achieve a response when compared to responding patients. Finally, logistic regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.764, p=0.048).

Conclusion: TOP2A status may be used as a predictive factor for patient response to PIs including carfilzomib.

Disclosures

No relevant conflicts of interest to declare.