bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Immunotherapeutic Approaches for Hodgkin Lymphoma
Authors: Savoldo Barbara , Grover Natalie S , Ramos Carlos A. , Dotti Gianpietro
Published: November 2019

The outcome of patients with relapsed/refractory Hodgkin Lymphoma (HL) for whom salvage therapy or stem cell transplant have failed is poor. HL tumor cells (HRS) cells universally express CD30, which is an excellent target for directed therapy such as the antibody drug conjugate brentuximab vedotin (BV). Patients with relapsed HL can also respond to checkpoint inhibitors, emphasizing the susceptibility of this tumor to T-cell-mediated immune control. The adoptive transfer of chimeric antigen receptor T (CAR-T) cells, which combine antibody-mediated antigen specificity with the effector function and replication potential of T lymphocytes, offers the opportunity to infuse large numbers of T cells with defined antigen-specificity and MHC-independent tumor targeting. In two parallel and simultaneously conducted phase 1/2 dose-escalation studies, we administered CD30-specific CAR-T (CD30.CAR-T) cells after lymphodepletion to patients with HL that had relapsed after multiple lines of therapy, including BV, checkpoint inhibitors and stem cell transplant. We observed an excellent safety profile, with modest and self-limited cytokine release syndrome and no neurologic toxicity. The overall response rate was 78%, including 18 patients (67%) with complete responses, and the 1 year progression free survival was 47% (95% CI: 25% - 67%). Despite these promising results, unsolved obstacles include CAR-T cell trafficking and persistence in the tumor site. HL is characterized by a unique tumor microenvironment that creates a physical barrier and a hostile niche for CD30.CAR-T cells. Expression of PD-1 on CD30.CAR-T cells indicates that these cells likely remain susceptible to inhibition by PD-L1 in the tumor, and suggests that the combination of CD30.CAR-T cells with checkpoint blockade should be investigated. Enhancing CAR-T cell trafficking to tumor sites should also tip the delicate balance between effector and inhibitory mechanisms towards the former. Since the tumor microenvironment of HL is rich in TARC (thymus and activation regulated chemokine/CC chemokine ligand 17), we have initiated a clinical study in which CD30.CAR-T cells are further modified to co-express the cognate receptor (CCR4) for TARC to improve their tumor homing (NCT03062157).

Disclosures

Savoldo: Baylor College of Medicine: Patents & Royalties: CAR.CD30 patent; Bluebirdbio: Other: research agreement; Cell Medica: Other: Research Agreement; Bellicum: Other: Research Agreement. Grover:Seattle Genetics: Consultancy. Ramos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding.
Sensing and Sensibility: Programming Smarter Chimeric Antigen Receptor T Cells
Authors: Lim Wendell
Published: November 2019

CAR T cells have shown remarkable outcomes when targeting specific hematologic cancers. But targeting solid cancers has proven difficult: it is difficult to identify unique antigens, to traffic to tumors, and to mount a strong response in the solid tumor microenvironment. To overcome these issues, we are using the approaches of synthetic biology to engineer next-generation T cells. We are engineering cells that utilize new sets of sensors and modular control circuits that enable them to execute new recognition and response programs with higher precision and robustness. These new capabilities may allow us to develop therapeutic T cells that are far more effective and safe against solid tumors.

Disclosures

Lim: Cell Design Labs: Other: founder of Cell Design Labs; acquired by Gilead in 2017; Allogene: Consultancy.
Activation of the Receptor Tyrosine Kinase (RET) By GDNF/GFRα1 Improves Cord Blood-Derived HSC in Vitro expansion and In Vivo engraftment
Authors: Grey William , Chauhan Rakhee , Garcia-Albornoz Manuel , Huerga Encabo Hector , McDonald Neil , Bonnet Dominique
Published: November 2019

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171).

Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRα1), significantly improves the outgrowth of HSCs invitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice.

The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (~1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (~1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRα1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRα1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRα1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRα1 or SR1/UM171 treatments alone.

RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRα1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFκB activation and increased CD44 cell surface expression, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRα1.

Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation.

Disclosures

No relevant conflicts of interest to declare.
Prophylactic Donor Lymphocyte Infusion for Relapse Prevention; A Meta-Analysis
Authors: Poonsombudlert Kittika , Kewcharoen Jakrin , Prueksapraopong Chattip , Nath Limpruttidham
Published: November 2019

Objective: relapsed hematologic malignancies after standard hematopoietic stem cell transplant (HSCT) is one of the most challenging diseases to treat, therefore ongoing research are aiming at relapse prevention and minimizing the transplant related side effects. Prophylactic donor lymphocytes (pDLI) had been proposed as a valuable strategy for relapse prevention but early study results had been discouraging due to association with acute graft-versus-host disease (aGVHD). We attempted to establish the efficacy of pDLI for relapse prevention and define its association with aGVHD.

Method: we searched for titles of articles in MEDLINE (PubMed), Cochrane library, EMBASE database and Google Scholar in May 2019 that evaluated the association between pDLI, relapse and aGVHD. We conducted a random effect meta-analysis of 9 studies involving a total of 748 participants and reported the pooled odd ratio (OR) for incidence of relapse and aGVHD between patients in the pDLI group and the control group.

Result: we found a significantly decreased risk of relapse in the pDLI group versus the control group with a pooled OR of 0.42, 95% CI 0.30-0.58, I2=0% while there is no significantly increased in the risk of aGVHD with a pooled OR of 0.98, 95% CI 0.56-1.72, I2=0.8%

Conclusion: there is significantly decreased risk of relapse in the pDLI group compared to the control group but there is no statistically significant increased risk of aGVHD. We concluded that pDLI is a valuable method of post-transplant relapse prevention for hematologic malignancies and further research is encouraged in a larger population.

Disclosures

No relevant conflicts of interest to declare.
A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo
Authors: Vega Mario I , Shi Yijiang , Frost Patrick , Huerta-Yepez Sara , Lichtenstein Alan
Published: November 2019

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model.

Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831

Disclosures

No relevant conflicts of interest to declare.
A Distinct Metabolic Signature in DNMT3A-Mutated Leukemia
Authors: Dai Yu-Jun , Hu Fang , He Si-Yuan , Tian Xiao-Peng , Li Hong-Hong , Qin Ze-Yuan , Chen Siliang , Liang Yang
Published: November 2019

Background: Acute myeloid leukemia (AML) is a type of cancer that consists of a group of hematological malignancies with high heterogeneity. AML patients with DNMT3A mutations often have a poor prognosis. Metabolic alterations have long been recognized in cancer cells. Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change the cellular metabolism, priming cells for malignancy. It is well known that 2-hydroxyglutarate (2-HG) is an oncometabolite resulting from mutations of the isocitrate dehydrogenase 1 and 2 (IDH1and IDH2) genes and isa strong prognostic predictor independent of other clinical and molecular features. Alpha-ketoglutarate-dependent dioxygenase encodedby the TET2 gene could catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), thereby regulating DNA methylation and inducing hematopoietic malignancies.

Methods: Metabolomic profiles of all serum samples were achieved using the GC-TOF MS platform. CCK-8 assays were used to examine cell viability and cytotoxic activity of 2-ketoisocaproic acid (2-KA) to leukemia cells. Methyl-Cellulose colony-forming cell assays and flow cytometry were performed to assess cell proliferation and cell differentiation. The pIpC was administrated in the Dnmt3aR878H/WTMx1Cre+mice at the time of weaning (4 weeks old) to activate transgene expression in vivo. The expressions of genes/proteins were detected by RNA sequencing, quantitative RT-PCR and western blot.

Results: We report a metabolomics study with a total of 171 newly diagnosed AML patients and identified a distinct leucine metabolic signature in AML patients with DNMT3A mutations. The prognostic value of 2-KA was demonstrated in cytogenetically normal AML patients, and a low 2-KA level predicted a poor overall patient survival. We further compared the gene expression patterns of the blast cells in bone marrow (BM) between AML groups with and without a DNMT3A mutation; these patterns correlated well with those of the leucine metabolic pathways and exhibited enhanced ACAA1 and decreased ALDH7A1 gene expression in DNMT3A-mutated leukemic cells. In vitro results demonstrated that a high 2-KA level could inhibit the proliferation of DNMT3A-mutated cells but did not affect the differentiation of these leukemic cells.

Conclusions:Our data suggest that 2-KA isa unique feature of AML with a DNMT3A mutation and might be a metabolic marker of early-stage hematopoietic malignances.This study also suggests that ACAA1 and ALDH7A1 were aberrantly expressed in DNMT3A mutant leukemic cells and may be potential targetsfor AML therapy. We also hypothesized that a combination of the metabolic inhibitor and chemotherapeutic agent may be a possible treatment strategy for DNMT3A-mutated AML.

Disclosures

No relevant conflicts of interest to declare.
ST2 and Rg3a As a Biomarker for Predicting of Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Goker Hakan , Aladag Elifcan , Buyukasik Yahya , Chao Nelson J. , Akman Umit , Demiroglu Haluk
Published: November 2019

Background:Graft-versus-Host Disease (GvHD) is a crucial complication after leading to significant morbidity and mortality allogeneic hematopoietic stem cell transplantation (AHSCT) which occurs in approximately half the transplant recipients. Although there are some studies conducted, a biomarker which can be shown to accurately predict acute GVHD has not been well established, yet. Suppression of tumorigenicity 2 (ST2) and Regenerating islet-derived 3-alphamight be important biomarkers to predict acute GVHD or GVHD with acute exacerbations.

Material and Methods: In the present study, blood samples were collected from 17 patients with acute GVHD and 12 control patients after allogeneic stem cell transplantation. All patients save informed consent in accordance with declaration of Helsinki. ST2 and Reg3a were measured in plasma samples and we compared the ST2 and Reg3a levels of patients with acute GVHD patients and controls.

Results:The median age of the study population was 42 (range,19-49). As compared with the patients with acute GVHD and controls, the ST2 levels had the significant association with Acute GVHD (mean 9794 ng/dl vs 2646 ng/dl, p:0.008 )

Conclusion:The ST2 level could be used as a significant biomarker for predicting acute GVHD. However, further studies with larger patient populations are needed.

Disclosures

No relevant conflicts of interest to declare.
Population-Level Trends in Early Mortality and Overall Survival of Patients with Multiple Myeloma. Are We Facing Stagnation?
Authors: Costa Luciano J. , Bal Susan , Chhabra Saurabh
Published: November 2019

Background: Therapeutic advances in multiple myeloma (MM) in the last two decades resulted in population-level reduction in early mortality (EM) and improvements in overall and relative survival when consecutive cohorts are compared. We utilized updated information from the Surveillance Epidemiology and End Result (SEER-18) program to test whether prior improvement trends have persisted in more recent years.

Methods: Patients diagnosed with MM as first malignant neoplasm between the years of 2001 and 2015 were included in the analysis. Survival data are available up to the end of 2016. We analyzed year-by-year EM, herein defined as death from any cause within the first year of diagnosis of MM. Updated 5-year OS was available for patients diagnosed 2001-2011 and analyzed year-by-year. We also analyzed outcomes using age brackets (<65, 65-74 and 75+ years) and race/ethnicity subgroups [non-Hispanic White (NHW), non-Hispanic Black (NHB) and Hispanic].

Results: There were 54,475 patients included in the EM analysis and 37,452 in the 5-year OS analysis. For the EM analysis, 42.7% of patients were <65, 29.0% were 65-74 and 28.4% were 75+. NHW were 62.0%, NHB 19.7%, Hispanic 11.4% , and 54.6% of patients were male. We observe a year-by-year decline in EM between 2001 (31.0%) and 2012 (20.5%), however no significant reduction in EM was noted between 2013 (19.5%) and 2015 (18.5%) (P= 0.24). Such plateau in EM was also noted in each of the age brackets (Figure 1) and race/ethnicity subsets, with no significant difference seen in the last 3 years of data. For patients diagnosed in 2015, EM remained high at 11.6% for patients <65, 17.1% for patients 65-74, 31.5% for patients 75+, 19.5% for NHW, 18.8% for NHB and 16.8% for Hispanics. Five-year OS improved between cohorts of patients diagnosed in 2001 (29.3%) and patients diagnosed in 2008 (43.3%) (P<0.001), but remained stable for patients diagnosed between 2009 (44.4%) and 2011 (44.2%) (P=0.86). Similar pattern was seen for all age (Figure 2) and race/ethnicity subsets.

Conclusions: These data indicate possible stagnation in risk of EM and 5-year survival for patients diagnosed with MM in the US beyond the introduction of proteasome inhibitors and immunomodulatory agents. The absolute risk of EM remains unacceptably high for all ages pointing to an unmet need in MM care. Future data will indicate whether the introduction of monoclonal antibodies and other new classes of drugs will have sufficient impact to change population-level outcomes in MM.

Disclosures

Costa: Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding; Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; Janssen: Research Funding, Speakers Bureau; Abbvie: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Dasatinib and Nilotinib As Upfront Therapy for Chronic-Phase Chronic Myeloid Leukemia (CML-CP) in Qatar: A Cost Effectiveness Analysis (CEA)
Authors: Adel Ahmad M , Abushanab Dina , Hamad Anas , Abdulla Mohammad Abdul-Jaber , Yassin Mohamed A
Published: November 2019

Objectives: Many tyrosine kinase inhibitors (TKIs) are approved for treating the chronic phase of chronic myeloid leukemia (CML-CP). This study aims at identifying the optimal upfront TKI treatment strategy; considering cost-effectiveness for CML patients from Qatar healthcare system perspective. Evaluated strategies included the newer generation TKIs; dasatinib 100 mg once daily and nilotinib 300mg twice daily. We evaluated both treatments considering their molecular and cytogenetic response outcomes at 12 months follow up as well their respective acquisition costs.

Methods: A decision tree model was developed linking clinical effectiveness (defined as major molecular response MMR and/or complete cytogenetic response CCyR), utility, and cost data over a 12-months period. The choice of 12 months period is due to the evidence that the degree of short-term molecular and cytogenetic responses at certain follow- up times is correlated with CML prognosis. Patients who achieve a CCyR are shown to have high remission rates as well overall survival (OS) rates. Patients with newly diagnosed CML are adopted from Qatar Cancer Registry.The probability of primary clinical outcome is calculated from DASISON (dasatinib) and ENESTnd (nilotinib) trials. Direct healthcare costs were derived from the national healthcare payer system, whereas ADR data were derived from local incident reporting system.

Results: In the first-line treatments of CML patients, with greater MMR (39% nilotinib vs 12% dasatinib) and CCyR (24% nilotinib vs 16% dastinib) response outcomes, and more adverse drug reactions (ADR) than dasatinib (13.3% vs 4%), nilotinib use is more cost effective with annual costs (QAR 232,102) and after 12 months follow up. Despite the lower acquisition annual cost of dasatinib (QAR 217,125), the incremental cost effectiveness ratio (ICER) of nilotinib versus dasatinib per MMR/CCyR achieved was 56,506 QAR per year. There were no cases in both arms that progressed to accelerated or blast phase. At a threshold of 3 times GDP per capita of Qatar and according to WHO recommendation, the nilotinib use is still cost-effective.

Limitations: For the surrogate outcome, we only consider the percentage of patients with or without MMR / CCyR response at 12 months. Nevertheless, we do not consider the depth and the duration of the MMR or CCyR.

Conclusions: Upfront therapy of CML-CP patients by nilotinib plan appears to be more cost-effective than dasatinib. Although local real-life data for the effectiveness of these drugs are still warranted. The results of this study implying that nilotinib would be advantageous as first-line treatment for CML-CP patients from Qatar healthcare system perspective.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Ogata, Red and Combined Score of Flow Cytometry in the Study of Myelodysplastic Syndrome
Authors: Serrano Juan Carlos Carlos , Velez Graciela , Varon Carlos Roberto
Published: November 2019

Background

Flow cytometry (CF) is an important diagnostic tool for myelodysplastic syndrome (MDS). Different analysis systems have been developed, the "Ogata score" is a simple two-color protocol based on surface expression SSC, CD45 and CD34, which has been validated in a multicentric study showing sensitivity of 69% and specificity of 92% in low risk MDS. Scoring based on erythroid markers by CF (Red) uses the coefficient of variation of CD36 and CD71 in combination with the fluorescence intensity of CD71 and the percentage of CD117 + in erythroid progenitors.

Goals.

To analyze the usefulness of the "Ogata, Red score and combined system" in the diagnosis of MDS of a patient population in Cucuta, the north of Santander, Colombia.

Methods

Case-control study, conducted at the "Unidad Hematológica Especializada" of Cucuta, a total of 32 patients with MDS and 28 control patients were included estimating the "Ogata and Red score", we followed protocols of Euroflow that includes processes of dyeing, analysis and washing using as a Lysis / fixation solution, but it deferred from Leukemia-Net standards. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prevalence were established in our cohort, in addition we estimated a combined scoring system that integrated "Ogata and Red score"

Results

In "Ogata score" the sensitivity was 47% (95% CI 0.30-0.64) the specificity 90% (95% IC 0.77-1.03), PPV 88% (95% IC 0, 73-1.03), NPV 51% (95% IC 0.35-0.68), prevalence 62%. Red score showed sensitivity of 38% (95% IC 0.21- 0.54), specificity of 85% (95% IC 0.69-1.01), PPV 80% (95% CI 0.60-1.00), NPV 46% (95% IC 0.30-0.62) and prevalence of 62% Combined score of "Ogata plus Red", obtained sensitivity of 62.50% (95%CI 43.69 -78.90), specificity 90.62% (95%CI, 74.98 -98.02), PPV 86.96 (95% CI 68.72-95.29), NPV 70.73% (95%CI 60.38 -79.30) with a prevalence of 50% (95% CI 37.23 -62.77).

Conclusions

In our study, low sensitivity was found in the "Ogata and Red" score, but "Ogata" score showed lower sensibility than the published studies. Both scores had a high specificity and negative predictive value, in accordance with literature. Lysis method could interfere in this results. The Combined Score in FC helped increase the sensitivity of the test for MDS. It is necessary to implement strategies for the harmonization of FC techniques in different laboratories, in order to become a coadjutant tool that contributes to the diagnosis of MDS along with morphology, cytogenetics and molecular biology.

Disclosures

No relevant conflicts of interest to declare.
A State Level Retrospective Analysis of Newborn Screening for Hemoglobinopathies
Authors: Gbadamosi-Akindele Maryam , Aurit Sarah , Johnson Amissabah , Nester Alex
Published: November 2019

Introduction: Screening for variant hemoglobin is essential in identifying newborns born with hemoglobinopathies, including sickle cell trait (SCT). Screening allows families the opportunity for patient education as well as genetic counseling. This study aimed to better understand the trends in newborn screening of variant hemoglobin in the state of Nebraska over a period of eight years.

Methods: Using the Department of Health and Human Services state-level screening information,the crude incidence rate of SCT was calculated as the screened positives divided by the total number of screened newborns within the state of Nebraska from 2011 to 2018. Rates were further delineated by variant. A Daniel's test was utilized to determine if the crude incidence rates exhibited characteristics of a trend. Rates were also examined with joinpoint regression to determine annual percent change (APC). Excel v 1808 and Joinpoint Trend Analysis v 4.5 software packages were used for all analyses; p < 0.05 was considered significant.

Results: We identified 3,416 newborns with SCT among a total of 211,697 live births, which is an incidence rate of 161.4 cases per 10,000 screened newborns. There was no evidence of a trend in the overall incidence rate from 2011 to 2018 (p = 0.493). However, the incidence of FA Barts significantly increased over the examined interval (p = 0.015) with a 4.4% APC (95% CI: 0.7% to 8.3%; p =0.026)

Conclusion: The data obtained from this study showed no significant change in the incidence of SCT in the state of Nebraska from 2011 to 2018. However, there was a significant increase in the incidence of Hemoglobin FA Barts. Hemoglobin FA Barts is associated primarily with alpha thalassemia, which is most prevalent in southeast Asia, but is also common in the Mediterranean, Africa, Middle east, Central Asia and India. This finding is of interest and likely related to the changing migration patterns in the state of Nebraska. For the healthcare system to continue to provide appropriate care, future studies should aim to further elucidate this trend and patient demographics of this increasing patient population in the state of Nebraska.

Disclosures

No relevant conflicts of interest to declare.
The Frequency of Cytogenetic and FISH Abnormalities Is Higher in Female Than in Male Multiple Myeloma (MM) Patients
Authors: Cantu Eduardo S , Szymanska Jadwiga , Watson Alan R , Trevizo Sandy L , Rostami Sassan
Published: November 2019

Sex determination in Homo sapiens is generally dependent on the inheritance of sex chromosome combination of XX (♀) or XY (♂). One could surmise that clinical manifestations may also be sex-dependent and detectable by genetic testing. Therefore, this laboratory examined whether different cytogenetic and FISH results occur that are sex-dependent in multiple myeloma (MM) patients.

De-identified cytogenetic and FISH records of 146 MM patients (03/03/17 to 04/19/19) were retrospectively reviewed. There were 58 females (age range 40 to 88 years) and 88 males (age range of 38 to 90 years) in this survey. Data was collected from these cases and formatted into an Excel spreadsheet in order to use the TTest for statistical analyses.

Cytogenetic studies showed an abnormal-to-normal (A:N) ratio of 20:38 (0.53) in females and an A:N ratio of 10:78 (0.13) in males. This ratio for females was significantly higher than for males (P=0.003244). By FISH studies the A:N ratio was 43:15 (2.87) in females, while in males it was 50:38 (1.32) and again significantly higher in females (P=0.023312). The number of females tested was considerably less than for males (58:88=0.66), however the detection rate of abnormalities was much higher in females by both these testing methods. Specific numerical (+1 & -13) and structural abnormalities (1p & 17p modifications) detected by cytogenetics showed over-representation in females than in males. Also, FISH studies indicated that +1q and +7 were noticeably higher in females. With the exception of +7, these are genetic markers associated with poor clinical outcomes. These results are different than expected for equal distribution of abnormalities between the two genders.

The data collected showed that: 1) there are fewer females tested than males (either a true prevalence or underdiagnosed MM females), 2) the frequency of abnormalities detected by cytogenetics and FISH is higher in females than in males especially for certain markers, and 3) the 1p and -13/13q- (cytogenetics) and +1q abnormalities (FISH) found in females (but not as high in males) is not only different but may be more consequential clinically. These data suggest that there are fewer "XX" MM females (or perhaps underdiagnosed) with a higher risk of a more serious clinical outcome. By contrast, there are a greater number of "XY" MM males with a less frequent severe phenotype. MM is a multifactorial clinical entity that presently eludes a complete understanding. However, the genetic basis for MM includes sex chromosome influences that manifest chromosome abnormalities differently in females than in males.

Disclosures

No relevant conflicts of interest to declare.
Blockade of CD47-Sirpα Interactions Improved Myeloid Effector Cell Mediated Lymphoma Cell Killing By the HLA-DR Antibody Apolizumab
Authors: Petersen Judith , Rösner Thies , Kölling Max , Klausz Katja , Kellner Christian , Humpe Andreas , Weiner George J , Schewe Denis Martin , Peipp Matthias , Valerius Thomas
Published: November 2019

Background:Apolizumab (APO) is a humanized IgG1 antibody targeting a HLA-DRβ chain specific epitope, which is expressed by approx. 50 % of B cell lymphomas. The antibody's clinical development was hampered by toxicity, potentially mediated by its strong CDC activity. However, previous studies demonstrated that APO efficiently triggered tumor cell killing by myeloid cells, especially by granulocytes. Myeloid effector cell engagement is strongly regulated by CD47-SIRPα interactions, and blockade of this axis has been demonstrated to improve the efficacy of therapeutic antibodies. Hence, our goal was to characterize the influence of CD47-SIRPα blockade on myeloid cell engagement by APO.

Methods: Expression of the APO epitope, CD20 and CD47 on different B lymphoma cell lines was measured by flow cytometry. The capacity of APO and Rituximab (RTX) to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) by mononuclear (MNC) or polymorphonuclear (PMN) effector cells was analysed by 51Cr release assays. Antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages was analysed by fluorescence microscopy. PMN-mediated ADCC and macrophage-mediated ADCP experiments were performed in the presence or absence of the CD47 blocking antibody 5F9-IgG2σ to analyse the impact of CD47-SIRPα blockade on APO-mediated lymphoma cell killing by myeloid cells.

Results: As anticipated we observed binding of APO on approx. 50% of the tested B lymphoma cell lines, including Raji (Burkitt's lymphoma), MINO (mantle cell lymphoma) and Carnaval (double hit DLBCL). Focusing on these cell lines, APO's CDC activity was more potent (Raji) or similar (Mino) compared to RTX. Furthermore, APO significantly induced tumor cell lysis by PMN or MNC in 51Cr release assays and achieved higher lysis rates in comparison to RTX. Blocking CD47-SIRPα interactions by 5F9-IgG2σ resulted in significantly increased APO-induced ADCC by PMN against all tested B cell lymphoma lines (Mino: 28.8 ± 10.2 % vs. 41.8 ± 13.3 %; Carnaval: 37.9 ± 17.9 % vs. 55.6 ± 17.6 %; Raji: 23.6 ± 11.7 % vs. 52.4 ± 19.4 % for APO alone vs. APO + 5F9-IgG2σ, respectively, p < 0.05 by ANOVA). However, no ADCC by PMN and RTX was observed - even in the presence of CD47-SIRPα blockade. Moreover, APO mediated similar or even stronger ADCP by macrophages compared to RTX, which could be further enhanced by 5F9-IgG2σ (Figure 1).

Conclusions: APO's potent myeloid cell activation could be further enhanced by disruption of CD47-SIRPα interactions. Our observations suggest to generate isotype variants of APO with lower CDC but preserved or enhanced ADCC activity. Subsequently, a combination of APO and CD47-SIRPα blocking strategies may improve B cell lymphoma immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
Iron Catalyzed Hydrogen Sulfide and Its Prevalence during Oxidative Events in Sickle Cell Disease
Authors: Yang Jie , Bithi Nazmin , Link Christopher , Saunthararajah Yogenthiran , Hine Christopher
Published: November 2019

In sickle cell disease (SCD), red blood cell (RBC) oxidation-reduction (redox) status is a critical determinant of hemolysis and RBC adhesion/vaso-occlusive crises (VOC). Iron, while essential for hemoglobin and RBC production, also is an important catalyst in processes that produce reactive oxygen species (ROS) which damage RBCs and ultimately other tissues. This has necessitated mechanisms for iron sequestration to control its activity, primarily achieved through binding of iron to heme, hemoglobin, and ferritin. In hemolytic disorders such as SCD, however, there is overload of protein-free, unbound iron, which by increasing ROS, significantly contributes to disease pathology. There is hence a need for novel druggable targets and treatments to attenuate this pathway of ROS production and pathophysiology. Previously, we determined a novel catalytic role of iron in which hydrogen sulfide (H2S) gas is produced both in vitro and in blood ex vivo by iron- and Vitamin B6- mediated catalysis of cysteine. We additionally determined that the bound versus unbound status of iron as well as the integrity of RBCs dictates this non-enzymatic capacity for H2S production. H2S serves as a potent regulatory redox factor throughout the body, and here we hypothesize that excess H2S in blood produced via iron catalysis protects against oxidative stress generated during SCD-related hemolytic crisis and VOC. In the current study, we further show that H2S produced in vitro from iron and Vitamin B6 catalysis of cysteine not only exists as a gas in the headspace, but itself reacts with iron in the solution. This was determined by the formation of acid labile iron sulfide, which formed a light yellow precipitate. The addition of a strong acid removed the yellow colored precipitate and resulted in further release of H2S in to the headspace. This indicates that H2S was interacting with free iron (Fe3+) and formed the iron sulfide product, which may have decreased capabilities for the formation of tissue- and cellular-damaging ROS. On-going work will examine the inhibition of H2S on iron-induced ROS generation in vitro and test the therapeutic potential of applying D-/L-cysteine and/or Vitamin B6- supplemented dietary interventions to enhance iron/B6 catalyzed H2S production in blood to lessen oxidative stress and VOC events in SCD mice in vivo. Free-iron in SCD is conventionally considered only to be a liability. However, here we exploit iron as a catalyst for beneficial H2S production in blood that can protect against oxidative stress and SCD-related pathologies. This recently discovered pathway has the potential to create novel opportunities and approaches to modulate the devastating pathophysiology of SCD.

Figure

Disclosures

Saunthararajah: Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties.
Combination of Gatekeeper Mutations and Cysteine 481 Replacement Causes Super Resistance to the Irreversible BTK Inhibitors Ibrutinib, Acalabrutinib and Zanubrutinib
Authors: Estupiñan Velasquez Hernando Yesid , Shi Yuye , Mohammad Dara K. , Wang Qing , Vihinen Mauno , Zain Rula , Berglöf Anna , Smith Edvard CI
Published: November 2019

Resistance to the irreversible Bruton´s tyrosine kinase (BTK) inhibitors is the main cause of disease progression in patients with Chronic Lymphocytic Leukemia (Quinquenel et. al. Blood 2019). Cysteine to serine substitution at the position 481 in BTK, is the most common resistance mutation. Other less frequent mutations like the constitutively active phospholipase C-gamma 2 (PLCG2) variant also occur (Woyach et. al. J. Clin. Oncology 2017). Different from many other tyrosine kinase inhibitors, BTK mutations less frequently affect the gatekeeper residue in the kinase domain (Maddocks et. al. JAMA Oncology 2015).

In this study, we have performed mutation scanning with substitutions replacing the gatekeeper residue. We have generated all the possible amino acid substitutions requiring a single nucleotide change in the gatekeeper and several variants requiring 2 or 3 nucleotide substitutions. Selected variants were also combined with substitutions at the C481, which is the binding site of irreversible BTK inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib.

Our results show unexpected, super-resistant variants and demonstrate that concomitant mutations, such as cysteine 481 to serine combined with threonine 474 to isoleucine or methionine, enhanced the resistance to irreversible BTK inhibitors. On the other hand, reversible BTK inhibitors displayed different inhibitory responses against the super-resistant mutants. Binding of the BTK inhibitors was subjected to molecular dynamics predictions, which correlated with the experimental binding data. Based on the available clinical and experimental results, the mechanisms underlying the spectrum of resistance mutations in BTK are presented.

Disclosures

No relevant conflicts of interest to declare.
Targeting Transcriptional Co-Activator PGC-1a for the Treatment of Sickle Cell Disease
Authors: Habara Alawi , LE Cuong , Chui David H.K. , Steinberg Martin H. , Cui Shuaiying
Published: November 2019

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is a FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). Transcriptional co-activator PGC-1α has recently been showed to play a crucial role in globin gene regulation and erythropoiesis by our group. Loss of function in the PGC-1α knock out mice significantly reduced the expression of murine embryonic εy- and βh1-globin genes; both of them are homologues of the human ε- and γ-globin genes. Forced overexpression of PGC-1α in vitroby adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. These results directly validate a role for PGC-1α in human γ-globin induction and provide significant support for the hypothesis that modulating PGC-1α activity, or the signaling pathways that it regulates, in SCD patients may be an effective approach that could therapeutically benefit individuals with SCD. Here we report the effects of PGC-1α in inducing HbF in human primary erythoid progenitor CD34+cells. We infected CD34+cells with lentivirus carrying PGC-1α and found that over-expression of PGC-1α resulted in significantly increased F-cells in the culture, from 1.6% up to 41.1%. We further discovered several PGC-1α agonists that are highly selective for PGC-1α. We found that one of the PGC-1α agonists, Compound SR-18292, induces PGC-1α mRNA expression in a dose dependent manner (2.6-fold at 1 μM, 5.7-fold at 10 μM) in cultured CD34+cells without effecting cell viability. Consistent with the increase of PGC-1α levels, the percentage of F-cells increased from 2.76% (vehicle control) to 32.1% when cells exposed to 1 µM SR-18292, which is comparable to that of 25 µM HU (32.2%). The mean fluorescence intensity of F-cells in SR-18292 treated cells was higher than in controls, suggesting that the treatment not only increased the number of F-cells but also increased the concentration of HbF in F-cells. The increased levels of PGC-1α reactivates the expression of fetal γ-globin genes resulting in significant HbF accumulation in adult erythrocytes, suggesting that the transcriptional co-activator PGC-1α comprises a new molecular target for possible therapeutic intervention in treating SCD.

Disclosures

No relevant conflicts of interest to declare.
Protein Phase Separation in Hematopoietic Stem Cell Aging
Authors: Maneix Laure , Iakova Polina , Moree Shannon , Sweeney Melanie , Moka Nagaishwarya , Yellapragada Sarvari Venkata , Catic Andre
Published: November 2019

Imbalanced protein homeostasis (proteostasis) is a driver of aging and a vulnerability for long-lived cells such as hematopoietic stem cells. To determine critical proteostasis factors, we analyzed the proteome of hematopoietic stem and progenitor cells and found prolyl isomerases to be the dominant cytosolic chaperones. Genetic removal of prolyl isomerases led to accelerated aging in the stem cell compartment. We identified intrinsically disordered proteins as common substrates of these chaperones, including several key players that control phase transition. Phase separation allows the formation of supramolecular membrane-less organelles that regulate DNA and RNA biology as well as protein translation. Using microscopy and biochemistry, we show that prolyl isomerases promote phase separation and thereby increase cellular resistance to stress. Our research links a ubiquitously expressed chaperone family to phase transition and identifies macromolecular condensation dynamics as a driver of blood stem cell aging.

Disclosures

Yellapragada: Novartis: Employment, Other: Spouse Employment ; Celgene: Research Funding; BMS: Research Funding; Takeda: Research Funding.
Mesenchymal COX2-Derived PGD2 Activates an ILC2-Treg Axis to Promote Proliferation of Normal and Malignant HSPCs
Authors: Wu Limei , Lin Qiqi , Ma Zhilin , Chowdhury Fabliha , Mazumder Md Habibul , Du Wei
Published: November 2019

Cyclooxygenase (COX)-dependent production of prostaglandins (PGs) is known to play important roles in tumorigenesis. PGD2 has recently emerged as a key regulator of tumor- and inflammation-associated functions. We previously reported that mesenchymal stromal cells (MSCs) from patients with acute myeloid leukemia (AML) overexpressed COX-2 and secreted high levels of PGs including PGD2. Since little is known about the role of PGD2 in normal and malignant hematopoiesis, we prioritized this mesenchymal source of PG for further investigation. We observed that AML MSCs or normal MSCs overexpressing COX-2 promotes proliferation of co-cultured hematopoietic stem and progenitor cells (HSPCs), which can be prevented by treatment with COX-2 knockdown or TM30089, a specific antagonist of the PGD2 receptor CRTH2. Mechanistically, we demonstrate that PGD2-CRTH2 signaling acts directly on type 2 innate lymphoid cells (ILC2s), potentiating their expansion and driving them to produce Interleukin-5 (IL-5) and IL-13. We further show that IL-5 but not IL-13 expands CD25+Foxp3+ IL5Ra+ T regulatory cells (Tregs) and promotes HSCP proliferation. Disruption of the PGD2-activated ILC2-Treg axis by specifically blocking the PGD2 receptor CRTH2 or IL-5 impedes proliferation of normal and malignant HSPCs. Conversely, co-transfer of Lin-CD127+CRTH2+ ILC2s and CD4+CD25+IL5Ra+ Tregs promotes malignant HSCP proliferation and accelerates leukemia development in xenotransplanted mice. Collectively, these results indicate that the mesenchymal source of PGD2 promotes proliferation of normal and malignant HSPCs through activation of the ILC2-Treg axis. These findings also suggest that this PGD2-activated ILC2-Treg axis may be a valuable therapeutic target for cancer and inflammation-associated diseases.

Disclosures

No relevant conflicts of interest to declare.
3D Telomeric Fingerprint of Advanced Cutaneous T-Cell Lymphoma
Authors: Bienz Marc Nicolas , Taylor-Kashton Cheryl , Benlimame Naciba , Petrogiannis-Haliotis Tina , Pehr Kevin , Mai Sabine , Knecht Hans
Published: November 2019

Introduction

Cutaneous T-cell lymphomas (CTCLs) are characterized by dermal and epidermal infiltration of skin homing clonal CD4+ memory T-cells. Little is known about the oncogenic events driving either the progression of skin-limited disease such as Mycosis Fungoides (MF) to a leukemic form or Sézary Syndrome (SS), and there are no histologic means to predict evolution. Genetic instability is a hallmark of malignancy progression and telomere remodeling has been shown to play a role in the progression of hematological malignancies. Thus, the aim of this study is to characterize the three-dimensional (3D) telomeric organization in early and advanced CTCLs.

Methods

We performed 3D telomeric quantitative fluorescent in situ hybridization (3D Telo-Q-FISH) of 5mm skin tissue slides of 10 patients with MF and SS and of CD4+ lymphocytes of 3 healthy controls (Figure 1). Using the program TeloView (Vermolen et al., 2005), the proportion of telomeres of low intensity (TLI) (<5000 u), the nuclear volume and the total number of telomeric signals per cell were calculated. Patients were stratified based on CD30 expression (CD30 high, n=3 versus CD30 low, n=7) and clinical stage (early stages I-IIA, n=6 versus advanced stages IIB-IV, n=4).

Results

TLI represented 27% of telomeres in CTCL cells compared to 16% in control lymphocytes (p<0.0001). The highest proportion of TLIs was found in tumors CD30 high (34%), compared to 22% in tumors CD30 low (p<0.0001). Similar findings were observed when stratifying for advanced and early clinical stages (30% vs 24%, p<0.0001). Nuclear volume and total number of telomeric signals increased in CTCL cells expressing CD30 (both p<0.0001), and in CTCL cells associated with advanced clinical stages (p=0.0021 and p=0.001, respectively). However, as expected, nuclear volume and total number of telomeric signals were higher in control cells compared to CTCL cells (both p<0.0001).

Conclusion

We report clear evidence that CTCLs with either CD30 expression or advanced clinical stage are associated with loss of telomeres and telomeric signal intensity. These very small telomeres, termed 't-stumps', are a hallmark feature in many tumor cells. Analysis of the nuclear volume and total number of telomeric signals suggest that CTCL cells undergo in a first step telomere shortening and loss compared to healthy controls. In a second step further telomeric shortening associated with chromosomal rearrangements and bridge-breakage-fusion cycles may be involved in the progression of CTCL.

Disclosures

Pehr: Merck: Consultancy, Other: Clinical research on Alzheimers Drug; Eli Lilly: Consultancy, Other: Clinical research on Alzheimers Drug; galderma: Consultancy, Other: tactupump Forte; Actelion: Consultancy; valeant: Consultancy; CeraVe: Consultancy; Janssen-Cilag: Other: Train the trainer.