bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product
Authors: Jones Nicholas , Ragheb Josette William , Ngo Brian , Uyeji David , Hii Anselm L.
Published: October 2019

For treatment of patients with multiple myeloma (MM), flow cytometry has become a widely used and valuable method for the evaluation of minimal residual disease (MRD) in bone marrow. Use of an optimized single-tube, 10-color flow cytometry panel for assessment of MM MRD has shown to be beneficial in patient monitoring and has shown correlation to the acknowledged EuroFlow 8-color, 2-tube method. In order to further correlate levels of MM and relapse in patients, validation of a sensitive MM MRD assay that can be applied to testing for residual disease in apheresis product prior to autologous stem cell transplant, a standard treatment for patients with multiple myeloma. This approach can exhibit great value in patient monitoring and treatment. As new combination therapies are developed for treatment of multiple myeloma in concert with autologous stem cell transplantation, evaluation of MRD in apheresis will likely continue to grow in importance. The purpose of this study is to show the validation and sensitivity of a flow cytometry assay designed to detect Multiple Myeloma (MM) cells in G-CSF and other mobilized apheresis samples from human Multiple Myeloma patients. Using GSM-mobilized apheresis product, spiked with different levels of patient de-identified MM plasma cells, validation of a 10-color MM MRD panel can be evaluated, intended to mimic patient apheresis at varying levels of residual disease post treatment. As with any minimal residual disease assessment, a high number of events are required to ensure sensitivity and precision. Typically, acquisition of 3-5 x 107 events is required to ensure precision of MM MRD levels to 0.001%.

Disclosures

No relevant conflicts of interest to declare.
Genomic Sequencing Reveals Complex and Actionable Molecular Profiles across Hematologic Malignancies
Authors: Galanina Natalie , Jamieson Catriona , Kurzrock Razelle
Published: October 2019

The translation of genomic discoveries to the clinic is the cornerstone of precision medicine. However, incorporating next generation sequencing (NGS) of hematologic malignancies into clinical management remains limited. We describe 235 such patients who underwent integrated NGS profiling (406 genes) and analyze the alterations and their potential actionability. Overall, 227 patients (96.5%) had adequate tissue. Most common diagnoses included myelodysplastic syndrome (22.9%), chronic lymphocytic leukemia (17.2%), non-Hodgkin lymphoma (13.2%), acute myeloid leukemia (11%), myeloproliferative neoplasm (9.2%), acute lymphoblastic leukemia (8.8%) and multiple myeloma (7.5%). Most patients (N = 197/227 (87%)) harbored ≥1 genomic alteration(s); 170/227 (75%), ≥1 potentially actionable alteration(s) targetable by an FDA-approved (mostly off-label) or an investigational agent. (Table 1) Altogether, 551 distinct alterations were seen, most commonly involving TP53 (9.3% of patients), TET2 (4.2%), DNMT3A (3.3%), ASXL1 (2.5%), MLL (2.5%), and KRAS (2.2%). (Figure 1 and Figure 2) The median tumor mutational burden (TMB) was low (1.7 alterations/megabase); 12% of patients had intermediate or high TMB (higher TMB correlates with favorable response to anti-PD1/PDL1 inhibition in solid tumors). In conclusion, 96.5% of patients with hematologic malignancies have adequate tissue for comprehensive genomic profiling. Most patients had unique molecular signatures, and 75% had alterations that may be pharmacologically tractable with gene- or immune-targeted agents.

Disclosures

No relevant conflicts of interest to declare.
Survival Among Minority Populations with Chronic Myeloid Leukemia: A Surveillance Epidemiology and End Results Database Analysis
Authors: Agbedia Owhofasa , Ngwa Julius , Darbari Deepika S. , Oneal Patricia
Published: October 2019

Introduction: Cancer affects people of all races in the U.S.; however, the burden is greater for minority populations. This is influenced by factors such as demographics, behavioral factors, and access to medical services. The disparity is well documented in some solid tumors. However, little information exists on a racial disparity in survival among minority populations diagnosed with chronic myeloid leukemia (CML) especially in the era of targeted therapies. A better understanding of the population impact of CML will drive further research into approaches to improve overall outcomes.

Methods: All cases of CML diagnosed between 1973 and 2017 and with available follow-up data reported in the Surveillance, Epidemiology, and End Results database were reviewed. We performed a population-based study of CML to evaluate survival by race and calendar year of diagnosis: 1975-1989 (the era of cytotoxic therapy; busulfan and hydroxyurea), 1990-2000 (the era of Allogeneic hematopoietic stem cell transplantation or interferon-alfa ± cytarabine), and 2001-2015 (the era of targeted therapy; Tyrosine Kinase Inhibitors).

Results: A total of 14572 (56.4% were females) patients diagnosed with CML were included in our analysis. The racial distribution was 83.4% white, 10% black, 0.8% American Indian/Alaska Native and 5.9% Asian or Pacific Islander. 5314 (36.5%), 3725 (25.5%) and 5544 (38%) cases of CML were diagnosed during the 1975-1989, 1990-2000 and 2001-2015 eras respectively. The median 5-year survival improved by race with each era, with the greatest improvement observed among patients diagnosed during the 2001-2015 era. Although a trend of improvement in the median 5-year survival is seen across all age categories, patients 65 years and older continue to experience only a modest survival benefit in the era of targeted therapies (Table 1). No significant differences in survival by race was observed in the era of targeted therapies (Figure 1).

Conclusions: This survival analysis includes a longer follow-up duration (Diagnosis Years: 1973-2015) for patients with CML compared to prior studies. Although an overall improvement in median survival is seen across all age groups and by each era, survival benefits are lagging in certain populations (black and American Indian/ Alaska Native) older than 65. SEER database captures information from metropolitan counties in the United States and this may not truly reflect the health of the entire US population.

Disclosures

No relevant conflicts of interest to declare.
Pharmacologic Targeting of LIN28/Let-7 in Acute Myeloid Leukemia
Authors: Roos Martina M , Li Michelle , Amara Pang , Chute John P
Published: October 2019

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy with high relapse rates and mortality due to the outgrowth of chemotherapy-resistant leukemic stem cells (LSCs). Thus, the development of novel therapeutic strategies capable of eradicating human AML represents a major area of unmet medical need. The RNA binding protein, LIN28, is a known driver of many cancer stem cells, AML included, wherein overexpression of LIN28 correlates with reduced patient survival. LIN28 blocks the function of the let-7 microRNA family, which exert tumor suppressive effects by repressing oncogenes and cell cycle regulators including MYC, RAS and CyclinD. Thus, LIN28 is an attractive mechanistic target for the purpose of inhibiting AML LSCs. Using a targeted high-throughput screen, we identified a class of small molecules which selectively block the LIN28/let-7 interaction (Roos et al., ACS Chem Biol, 2016). Preliminary studies demonstrate that a lead small molecule markedly impairs the proliferation and clonogenic capacity of human AML cell lines and primary patient AML samples. In vivo, systemic administration of a lead small molecule LIN28/let-7 inhibitor decreases leukemic tumor burden, reduces LSC numbers and significantly improves animal survival. Mechanistic studies revealed that targeted inhibition of the LIN28/let-7 axis restores let-7 microRNA levels in AML LSCs and subsequently inhibits a panoply of key oncogenic driver genes, including the NF-ĸB pathway, a hallmark for LSC proliferation. Furthermore, AML cell lines and primary patient cells treated with the LIN28/let-7 small molecule inhibitor showed a block at the G1/S phase interface and significantly decreased cell cycle progression. Consequently, LIN28/let-7 inhibition leads to LSC differentiation and ultimately leukemic cell death. In summary, we demonstrate for the first time the drugability of the LIN28/let-7 axis in vivo and reveal a novel pharmacological means to suppress a multitude of oncogenic driver genes in human AML. These results suggest that small molecule inhibition of LIN28/let-7 has high therapeutic potential as a new class of targeted therapies for AML.

Disclosures

No relevant conflicts of interest to declare.
Engineering Precision Medicine to Increase Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors
Authors: De Oliveira Satiro N. , Truscott Laurel C , Kao Roy L , Chiou Tzu-Ting
Published: October 2019

Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure. Trials using autologous T-cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the cells is transient, limiting efficacy. Our hypothesis is modification of hematopoietic stem cells (HSC) with CAR will lead to persistent production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory.

Design/Methods: Using CD19 as target, we generated second-generation CD28- and 4-1BB-costimulated CAR constructs for modification of human HSC for assessment in vitro and in vivo, using third-generation lentiviral vectors. Additionally, co-delivery of suicide gene systems was tested to allow ablation of gene-modified cells.

Results: Gene modification of HSC with anti-CD19 CAR using lentiviral vectors did not impair differentiation or proliferation, and led to functional CAR-expressing cell progeny, at 40-50% transduction efficiency and engineered antigen-dependent cytotoxicity in myeloid, NK and T-cells. In vivo studies using humanized NSG engrafted with CAR-modified HSC demonstrated similar levels of humanization to non-modified HSC, with multilineage CAR-expressing cells present in bone marrow, spleen, blood and thymus in stable levels up to 44 weeks of life. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. Ex vivo cells presented antigen-dependent cytotoxicity against targets. Mice engrafted with CAR-modified HSC had decreased CD19+ populations and successfully presented tumor growth inhibition and survival advantage at tumor challenge (55-60%). CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues.

Conclusions: CAR modification of HSC for cancer immunotherapy is feasible. This approach can be applied to different cancers by adjusting target specificity and could be employed in the context of HSC transplantation to augment the anti-lymphoma activity. It also bears the possibility of decreased morbidity and mortality and offers alternative treatment for patients with no available sources for bone marrow transplantation, benefiting ethnic minorities. These results also give proof of principle for CAR-modified HSC regulated by suicide gene; further studies are needed to enable clinical translation.

Disclosures

No relevant conflicts of interest to declare.
Mitochondrial Regulation Is Essential for Erythroid Nuclear Clearance
Authors: Liang Raymond , Menon Vijay , Lin Miao , Nowak Roberta , Fowler Velia M. , Girnun Geoffrey , Ghaffari Saghi
Published: October 2019

The terminal steps of red blood cell (RBC) generation involve an extensive cellular remodeling. This encompasses alterations of cellular content through five erythroblast stages that result in the expulsion of the nucleus (enucleation) followed by loss of mitochondria and all other organelles, and a transition to anaerobic glycolysis. Whether there is any link between the erythroid removal of the nucleus and the function of any other organelle including mitochondria remains unknown. Here we show that mitochondria are essential for nuclear clearance. We first demonstrate through high-throughput single-cell imaging and confocal microscopy that as mouse bone marrow erythroblasts mature (Gate 3: TER119+,CD44low, FSClow), mitochondria migrate to one end of the cell, aggregate and trail behind the nucleus as it extrudes from the cell, a prerequisite for enucleation to complete. We further show that mitochondrial localization behind the nucleus has similar kinetics as nuclear polarization. This process is also conserved in mouse fetal liver erythroid cells as well as in primary human CD34+-derived erythroblasts in culture. Notably, kinesin inhibition disrupts mitochondrial motility and localization and reduces significantly erythroblast enucleation rate in the absence of any impact on dynein or tubulin. These results suggest that mitochondria function as necessary chaperones during erythroblast enucleation. Furthermore, mitochondrial activity distinguishes erythroblasts on the verge of enucleation from others at the same erythroblast stage (Gate 3). We show that active mitochondrial respiration facilitates nuclear condensation and is required for nuclear extrusion. To our surprise however, metabolite profiling revealed that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation primarily through extracellular pyruvate but independently of glucose oxidation or anapleorotic reactions of amino acids. 13C-labeled metabolite tracing also confirmed pyruvate incorporation into mitochondria while glycolysis was minimal in orthochromatic erythroblasts. Thus, we provide evidence for the first time of a link between erythroid enucleation and mitochondrial metabolism. The process described establishes a model of mitochondrial compartmentalization within the cell for providing essential metabolites in a precise spatial and temporal manner. These findings are likely to improve the in vitro production of RBC and might be relevant to anemias of congenital mitochondrial disorders and aging.

Disclosures

No relevant conflicts of interest to declare.
Therapeutic Effect of Platelet Transfusion in Patients with Acute Lymphocytes Treated with CART Therapy
Authors: Yan Liu , Jue Xie , Hu Yongxian , Huang He
Published: October 2019

Background: CART therapy is the most effective method for the treatment of acute lymphoblastic leukemia (ALL) right now, which accompanied with severe cytokine storms (CRS) during treatment, resulting in severe platelet destruction and reduction. So the treatment of platelet transfusion is particularly important to the CART therapy. However, there is no clinical consensus on when given platelet transfusion can patients obtain the maximum benefit.

Methods: In the study, 14 ALL patients were enrolled for the CART treatment. The patients with platelet transfusion refractoriness (PTR) were excluded for non-immune factors such as large spleen and high fever. Blood routine and cytokines such as IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A were monitored during treatment. Body temperature should be less than 38 degrees before transfusion of platelets. Analyze the efficacy of platelet transfusion in patients before the CART treatment, the time with CRS, and after the CRS. The platelet transfusion effect was determined by 24-hour corrected count increment (CCI) (greater than 7.5 considered as effective, otherwise as invalid).

Results: All the patients in the study were treated with platelet transfusion before CART treatment. There was a CSR-3 level in every patients during the treatment. Four of them didn't get platelet transfusion as the lowest platelet count is more than 10×109/L without bleeding tendency. Ten of them received platelet transfusion treatment during CRS and the average of the cytokine were higher than before (IL-2 (12.65±21.08 pg/ml), IL-6 (8540.57±6791.91 pg/ml), IL-10 (1547.46±2939.45 pg/ml), IFN-γ (2846.49±2480.88 pg/ml),P<0.01). The 24 hour CCI of the 10 patients before CART treatment were 10.91±3.27, during CRS were 4.18±4.28, and after CRS were 8.72±2.93 (P<0.01). During the CRS, 24 hour CCI is higher in patients with platelet count below 5×109/L than patients with platelet count between 5-10×109/L(P<0.01).

Conclusion : CRS during CART treatment accelerated platelet consumption, resulting PTR. Platelet transfusion could not be given to patients when they were experiencing the most serious CSR without bleeding tendency, but it can be considered when the platelet count is less than 5×109/L,

Key Words: acute lymphoblastic leukemia, severe cytokine storms, platelet transfusion.

Fig 1: the relationship between 24 hour CCI and the highest IL-6:

The IL-6 is so high that we reduced it by one thousand times in the figure. The 24 hour CCI is negatively correlated with IL - 6, P<0.01, R=-0.92.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Second Primary Malignancies in Peripheral T-Cell Lymphoma, Not Otherwise Specified; A SEER Database Analysis
Authors: Kunwor Ranju , Nepal Mahesh , Ho Dominic , Ghimire Krishna
Published: October 2019

Background: Development of second primary malignancies (SPM) in Peripheral T-cell lymphoma, not otherwise specified (PTCL- NOS) patients is not well studied. This study was conducted to evaluate SPM in PTCL- NOS patients using data from the US Surveillance, Epidemiology and End Results (SEER) cancer registries.

Methods: We analyzed the Surveillance, Epidemiology, and End Results (SEER*Stat) 18 database. We compared secondary cancer rate among adult PTCL-NOS patients ≥ 20 years of age during the period of 2000 - 2015. We used SEER MP-SIR session and Graph pad scientific software to calculate p value and Observed/Expected (O/E) ratio and excess risk per 10,000 compared to general population.

Results: The total number of adult PTCL-NOS patients reported was 3,321. Among them 211 patients developed ≥1 SPM, 75% of SPM occurred ≤ 5 yrs from the diagnosis of PTCL-NOS. All site secondary cancer (n=224) incidence was significantly higher among PTCL-NOS patients compared to general population with O/E: 1.73, p value < 0.05 with excess risk of 93.94 per 10,000.

Most common SPMs were Non-Hodgkin's lymphoma (NHL) with O/E: 9.53, P-value <0.05, excess risk 46.28; all leukemia O/E: 5.0, P value <0.05, excess risk 14.31; melanoma O/E: 2.29, p value<0.05, excess risk 7.27. The risk of all SPMs was increased within 5 yrs from the diagnosis of PTCL-NOS. However, risk of NHL as SPM continues after 5 yrs of diagnosis of PTCL-NOS. Sub group analysis by age and sex showed the similar pattern in the incidence of SPM.

Conclusions: This study showed that overall risk of second malignancies among PTCL- NOS patients is higher compared to general population. The risk of leukemia, lymphoma and melanoma is significantly increased during the first five years of diagnosis of PTCL-NOS.

Disclosures

No relevant conflicts of interest to declare.
IL2 and Rapamycin Enhance Persistence of Adoptively-Transferred Ex-Vivo Expanded T Regulatory Cells
Authors: Furlan Scott N , Lopez Christina , Tkachev Victor , Betz Kayla , Hunt Daniel J , Hibbard James , Blazar Bruce R. , Trapnell Cole , Kean Leslie
Published: October 2019

As the field of regulatory T cell (Treg) adoptive therapy develops, questions concerning how to best to deploy these cells in patients must be addressed. Prominent among these questions is which adjunctive therapies will pair most synergistically with the transferred cells. Most ideally, a therapy that could prolong persistence and stabilize function of the cells is sought.

Prior work has shown that mechanistic target of rapamycin (mTOR) inhibition can afford both stability and a degree of increased persistence to ex-vivo expanded thymic Tregs (tTregs) upon adoptive transfer. In this study, we investigated whether the addition of low-dose interleukin-2 (IL2) to the mTOR inhibitor rapamycin could impart additional persistence to ex-vivoexpanded tTregs after adoptive transfer. Using a non-human primate model of CFSE-labeled autologous tTregs, we found that the addition of IL2 to rapamycin supported a near 10-fold increase in the half-life of adoptively transferred tTregs, effectively doubling the cells in the tTreg compartment for the first month after adoptive transfer.

Using a combination of single cell approaches, we were then able to show that transferred tTregs, in the setting of IL2 and rapamycin adjunctive therapy, retain high levels of Treg-specific genes, including FOXP3, after adoptive transfer. Additionally, we found that adoptively transferred tTregs are remarkably homogenous and become more transcriptionally similar to endogenous tTregs with time in vivo. Together these preclinical data support the use of combination IL2 and rapamycin as adjunctive therapy forex-vivo expanded adoptively transferred tTregs.

Disclosures

Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Kean:Regeneron Pharmaceuticals, Inc.: Research Funding.
SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application
Authors: C Abisha Crystal , Thangavel Saravanabhavan , Velayudhan Shaji Ramachandran , Srivastava Alok , Nath Aneesha , Srinivasan Saranya , Venkatesan Vigneshwaran
Published: October 2019

Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process.

The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+).

Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively.

Disclosures

No relevant conflicts of interest to declare.
Improving Hematologists' Skills in Caring for Patients Transitioning to Adult Care:the Effectiveness of Online Case-Based CME
Authors: Hurst Simi , Warren Charlotte
Published: October 2019

BACKGROUND: Hemophilia is a lifelong condition that requires ongoing evaluation of treatment and management strategies to meet the unique needs of each patient. However, these needs, whether they are physical or social, change as a patient moves from childhood, through adolescence and into adulthood and older adulthood. The goal of this study was to use continuing medical education (CME) to improve hematologists' knowledge, competence, and confidence in transitioning young adults to an independent hemophilia treatment plan.

METHODS: To address identified practice gaps among healthcare providers caring for patients with hemophilia, an online, case-based activity was developed. Featuring an 18-year old boy with hemophilia preparing for college and living away from home for the first time, the 15-minute CME/CE-certified activity utilized a multimedia approach that combined video vignettes that portrayed physician-patient communication scenarios with detailed commentary and perspective from a recognized faculty expert. The expert commentary was complemented by key supporting evidence in text and slides, as needed. The activity launched on June 30,2018 and was made available to healthcare providers without charge.

Educational effectiveness was assessed with a repeated-pairs pre-/post-assessment study design, in which each individual served as his/her own control. Responses to three multiple-choice, knowledge questions and 1 self-efficacy confidence question were analyzed. A chi-squared test assessed changes pre- to post-assessment. P values <0.05 are statistically significant. Effect sizes were evaluated using Cramer's V (<0.05 modest; 0.06-0.15 noticeable effect; 0.16-0.26 considerable effect; >0.26 extensive effect). Data were collected through 07/30/2018.

RESULTS: Data from the subset of hematologists/oncologists (n=33; hem/oncs) who answered all pre-/post-assessment questions during the study period were analyzed. Significant improvements were observed overall (P=.0193; V=.166) and in several specific areas of assessment (Table). Following activity participation, 34% of hem/oncs indicated increased confidence with regard to helping teenagers with hemophilia transition to adult care. Lastly, the findings uncovered educational needs that are the focus of ongoing interventions.

CONCLUSIONS: Case-based learning has been recognized for its benefits in modeling real-life problems, integration of knowledge into practice, and self-evaluation and reflection. Participation in this online education significantly improved hematologists' knowledge with regard to addressing the needs of patients at different points in their journey.

Disclosures

No relevant conflicts of interest to declare.
Preemptive mitigation of CD19 CAR T-cell cytokine release syndrome without attenuation of antileukemic efficacy
Authors: Gardner Rebecca A. , Ceppi Francesco , Rivers Julie , Annesley Colleen , Summers Corinne , Taraseviciute Agne , Gust Juliane , Leger Kasey J. , Tarlock Katherine , Cooper Todd M. , Finney Olivia C. , Brakke Hannah , Li Daniel H. , Park Julie R. , Jensen Michael C.
Published: October 2019

Gardner et al report that early intervention with tocilizumab and steroids at the first signs of mild cytokine release syndrome (CRS) following CD19 chimeric antigen receptor (CAR) T-cell infusion for B-cell acute lymphocytic leukemia reduces the development of life-threatening severe CRS without having a negative impact on antileukemic effect.
The microenvironmental niche in classic Hodgkin lymphoma is enriched for CTLA-4- positive T-cells that are PD-1-negative
Authors: Patel Sanjay S. , Weirather Jason L. , Lipschitz Mikel , Lako Ana , Chen Pei-Hsuan , Griffin Gabriel K. , Armand Philippe , Shipp Margaret A. , Rodig Scott J.
Published: October 2019

Key Points

CTLA-4 is enriched in the microenvironmental niche in cHL, and identifies a T-cell population largely distinct from PD-1 CD86, the natural ligand for CTLA-4, is enriched in the niche and expressed on HRS cells and a subset of TAMs that also express PD-L1
Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.
Authors: Kim Chaekyun , Dinauer Mary C.
Published: October 2019

Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.
Transplanted Human Bone Marrow Contributes to Vascular Endothelium.
Authors: Jiang Shuguang , Walker Luke , Afentoulis Michael , Anderson Daniel A. , Jauron-Mills Linda , Corless Christopher L. , Fleming William H.
Published: October 2019

Accumulating evidence indicates that bone marrow is a source of endothelial progenitor cells. Recently our laboratory has shown that functional endothelial cells (EC) can be clonally derived from phenotypically defined hematopoietic stem cells. To determine the endothelial cell potential of human hematopoietic cells, blood vessels in skin and gut biopsies from sex-mismatched allogeneic transplant recipients were examined. H&E sections were initially reviewed to ensure tissue integrity and the absence of significant graft versus host disease. Coded biopsy specimens from 12 sex-mismatched transplant recipients and 6 sex-matched female transplant recipients were evaluated in a blinded fashion. Endothelial cells were identified using a combination of immunohistochemistry (IHC) and XY interphase FISH. Tissues sections were first evaluated by IHC for EC markers (CD31 or vWF) and for the pan-hematopoietic marker CD45 then assayed by dual-color interphase FISH for donor derived cells (XY). Tissue samples from each recipient were assayed at least 3 times by sequential IHC/FISH. The frequency of CD31+, CD45−, Y+ cells and vWF+, CD45−, Y+ cells in biopsies from the 18 transplant recipients was evaluated by 2 independent observers. 9 of 12 sex-mismatched recipients showed evidence of donor-derived Y chromosome positive EC. The remaining 3 recipients did not exhibit hematopoietic engraftment at the time of biopsy. Donor derived endothelial cells were detected in the skin and the gut of transplant recipients with a mean frequency of 2.3% (range 0.9–3.9). Donor derived hematopoietic cells including intraepithelial lymphocytes and tissue macrophages were excluded by CD45 expression. None of the >4,000 endothelial cells examined had >2 sex chromosomes, consistent with an absence or very low incidence of cell fusion. In addition, Y chromosome signals were not detected in sex matched female recipients, thereby excluding vertical transmission of male cells. Furthermore, none of the recipients evaluated prior to hematopoietic engraftment had detectable donor derived endothelial cells, indicating a close linkage between the recovery of hematopoiesis and endothelial cell outcomes. Our results suggest that the close association between hematopoiesis and the vascular system originally identified during early development persists into adult life. Consequently it will be important to study the potential therapeutic role of bone marrow derived endothelial cells in the setting of hematopoietic and vascular disease.
Primary Central Nervous System Lymphoma in Children: A Report of 12 Cases.
Authors: Abla Oussama , Sandlund John T. , Blaser Susan , Brock Penelope , Corbett Rob , Kirov Ivan , Griffin Tim , Sung Lillian , Weitzman Sheila
Published: October 2019

We report the treatment and outcome of 12 children with primary central nervous system lymphoma (PCNSL) treated at 6 pediatric oncology centers from 1995 to 2003. The main purpose is to determine whether childhood PCNSL can be cured with chemotherapy alone without cranial radiation therapy (CRT). The clinical charts of 8 immunocompetent and 4 immunodeficient children with PCNSL were retrospectively reviewed. The children were diagnosed from 4 to 17 years of age. All cases were non-Hodgkin’s lymphomas: 7 (58.3%) were phenotypically B-cell (6 mature B, one B-precursor), 3 (25%) were T-cell and 2 (16.6%) were indeterminate-cell type. The histologic subtyping (REAL classification) showed 4 diffuse large cell, 4 anaplastic large cell, 2 immunoblastic large cell, 1 Burkitts and 1 high-grade lymphoma of uncertain lineage. Two patients were t(2;5) positive. Two patients had congenital immunodeficiency and 2 were HIV+. Immunologic work-up was either normal or not done in the remaining 8. EBV was positive in 2 patients, negative in 3 and not assessed in the rest. PCNSL presented as a single lesion in 4 patients and multifocal in 8. The most frequent tumor locations were frontal and parietal lobes. Nine of 12 children received chemotherapy alone, event free survival (EFS) at 4 years was 74±16%. Three had chemo plus CRT (3900–5000 cGy), EFS was 33±27% (P=0.1). The most frequently used drugs at the 6 centers were high dose (HD) MTX (5 to 8 g/m2), HD Ara-C (2 to 3 g/m2), dexamethasone, vincristine and cyclophosphamide. Three children died, 2 of whom were HIV+; 1 died after local relapse while the other died secondary to an opportunistic infection. Two other patients relapsed, one after chemotherapy alone and one after chemotherapy plus CRT. The patient who relapsed after chemotherapy alone is in second CR at 6+ months after chemotherapy, CRT and ABMT; the other patient died from progressive disease. Nine of 12 patients (75%) are alive at a median follow-up time of 72 months (range 18–105 months) in survivors. Six of the 8 children who are in CCR received chemotherapy alone, one received chemotherapy plus ABMT and one received chemotherapy plus CRT. Two of the long-term survivors (in CCR at 88+ and 105+ mo) had congenital immunodeficiency and were treated with chemotherapy alone. It appears that immunocompetent and immunodeficient children with PCNSL may be cured with chemotherapy alone without CRT, avoiding long-term radiation toxicity. Although retrospective, involving a small cohort, it is the largest pediatric series of PCNSL reported to date. Multicentre prospective studies are clearly needed in children with this rare lymphoma, whose frequency seems to be increasing especially in immunocompetent patients.
A Phase II Study of the TNF-α Inhibitor Etanercept and Thrice Weekly Rituximab: Evidence of Clinical Activity in the Absence of del(17p13.1) Genomic Abnormalities.
Authors: Lin Thomas S. , Heerema Nyla A. , Swenor Jennifer , Lucas Margaret S. , Moran Mollie E. , Cheney Carolyn M. , Lucas David M. , Young Donn , Caligiuri Michael A. , Grever Michael R. , Byrd John C.
Published: October 2019

The monoclonal anti-CD20 antibody rituximab (Rituxan) induces the death of chronic lymphocytic leukemia (CLL) cells in part through apoptosis. Tumor necrosis factor (TNF)-α and other cytokines up-regulate bcl-2 and other anti-apoptotic proteins, thereby inhibiting apoptosis. In addition, TNF-α may play a role in the cytokine release syndrome and infusion toxicity associated with monoclonal antibody therapy. Therefore, antagonists of TNF-α represent potential therapeutic agents that may be able to decrease infusion toxicity and enhance rituximab-induced apoptosis. We initiated a phase II trial of etanercept in combination with rituximab in patients (pts) with relapsed CLL or small lymphocytic lymphoma (SLL), with the goals of increasing clinical activity of rituximab and reducing infusion toxicity through neutralization of TNF-α. Pts received etanercept 25 mg subcutaneously (SC) twice weekly during weeks 1–5 and rituximab 375 mg/m2 intravenously (IV) three times weekly during weeks 2–5. Stepped up dosing of rituximab was used with the first two rituximab treatments. From 12/02 to 10/03, 19 pts (14 male) with a median of 2 prior therapies (range 1–8) were enrolled. Therapy was generally well tolerated, although 5 pts developed grade 4 neutropenia and 5 pts developed grade 3–4 infection or neutropenic fever. Four of 19 pts achieved an NCI 96 partial response, with no responses in 8 pts with del(17p13.1), corresponding to loss of the p53 gene. In contrast, 4 of 11 pts without del(17p13.1) achieved a PR, including 1 pt with a complex karyotype. This study was stopped for interim analysis and has reopened, with the exclusion of patients with a del(17p13.1) based upon the response outcome above and a previous preliminary report by our group demonstrating that rituximab was ineffective for del(17p13.1) CLL (Cancer Res63:36, 2003). We conclude that the combination of rituximab and etanercept has clinical activity in heavily treated, relapsed CLL pts without del(17p13.1), irrespective of prior rituximab treatment. Accrual to this study continues, and the results will be updated at the 2004 ASH meeting.
Evidence on Chromosome 11 for a Quantitative Trait Locus Influencing Levels of Activated Protein C-Protein C Inhibitor Complex.
Authors: Vossen Carla , Hasstedt Sandra , Callas Peter , Scott Bruce , Long George , Rosendaal Frits , Bovill Edwin
Published: October 2019

Earlier we showed high heritability for the variation in levels of activated protein C-protein C inhibitor complex in a large kindred with type I protein C deficiency (Vossen et al. 2004). To identify genomic regions, which might harbour a gene with a mutation that accounts for a difference in the levels of activated protein C-protein C inhibitor complex, we performed a variance component linkage analysis. Family members were genotyped for 375 autosomal markers at the Marshfield Medical Research Foundation with an average marker spacing of 9.4 cM (range 0–18 cM) and an average marker heterozygosity of 75% (range 42–89%). Levels of activated protein C-protein C inhibitor complex were measured using commercial paired antibody sets from Affinity Biologicals Inc. (Ancaster, Ontario, Canada). We estimated the probability of Identity By Descent (IBD) using the multipoint IBD method in Simwalk2 (Sobel & Lange 1996), in which the proportion of alleles shared identical by descent at marker loci is used to estimate IBD sharing at arbitrary points along the chromosome for each relative pair. Then, variance component linkage analysis was performed using SOLAR (Almasy & Blangero 1998) to test whether a proportion of the genetic variance in the levels of activated protein C-protein C inhibitor complex could be attributed to specific genomic locations. The levels of activated protein C-protein C inhibitor were log-transformed to reduce skewness (from 3.0 to 0.2) and kurtosis (from 11.6 to 0.4) and were assumed to distribute as a multivariate normal density with correlation =h2K + c2H + q2B + e2I, where matrix K contains the kinship coefficients, H contains 1 for pairs from the same household and 0 otherwise, B contains the IBD probabilities, and I represents the identity matrix. The parameters for heritability (h2), household effect (c2), and heritability contributed to a specific genomic location (q2) as well as the effects of the covariates were estimated simultaneously using maximum likelihood analysis. Lod scores were computed as the log10 likelihood for q2 estimated to q2=0. A lod score of 3.3 was used as cut-off point for statistical evidence for significant linkage (as suggested by Lander and Kruglyak 1995), and a lod score above 1.9 but below 3.3 for suggestive linkage evidence. We found suggestive linkage evidence for levels of activated protein C in complex with protein C inhibitor in 121 tested family members (without a history of venous thrombosis) for a region on chromosome 11 (marker D11S969, 146 cM) with a LOD-score of 2.6, with age added to the model. As no obvious candidate genes were available under the peak, and because the linkage region is too wide to be certain of linkage, we are currently finemapping the peak by adding microsatellite markers to the genome scan to narrow the region on chromosome 11.
A Community Cancer Center Experience of the Efficacy and Safety of the Use of Intravenous Iron Dextran in Chemotherapy Induced Anemia.
Authors: Sen Kaushik , Okoro Ngozi O. , Hedman Theresa R.
Published: October 2019

An adequate level of hemoglobin is essential during chemotherapy in order to maintain an acceptable quality of life (QOL) and to prevent cancer related fatigue. The role of hematopoitic growth factors like Erythropoitin or Darbepoitin alfa in chemotherapy induced anemia is now well-accepted with the recommended goal being a hematocrit value of 33% approximately. It is also established that an iron-replete state is essential for proper response to the erythropoitic growth factors and IV iron supplementation has been in routine clinical use in long term hemodialysis patients. Unfortunately many cancer patients are nutritionally depleted and at the same time do not tolerate oral iron replacement. Intravenous iron preparations such as iron dextran or sodium ferric gluconate (Ferrlecit) are good alternatives because of their quick and predictable effect on erythropoisis. However due to heightened concern about infusion related serious side effects their use has not been widespread in community clinical practice. We present our experience of treating 30 patients (7 lung cancer, 6 breast cancer, 3 lymphoma, 6 colorectal, 4 ovarian, 4 others) in our center with documented chemotherapy induced anemia with supplemental intravenous iron. None of the patients had any history of untreated pretreatment iron deficiency anemia. Utilizing a premedication protocol of hydrocortisone 100mg, benadryl 25 mg and ranitidine 50 mg IV prior to iron infusion we could record only 2 cases with grade I/II (mainly nausea and dizziness) and none with grade III/IV acute infusion related toxicity. Only one patient required treatment interruption and no one required hospitalization due to adverse events. There was no death associated with this therapy. The pretreatment average hematocrit was 29.26% (range 26.4–32.3%). The average posttreatment hematocrit was 35.33% (range 33.1–44.6%) within an average of 4 weeks. All patients were treated with Erythropoitin (40,000 unit/wk) or Darbepoitin alfa (200 mg/14 days) during the period of observation. All patients received their respective chemotherapy for the primary cancer without interruption. The effect persisted over the next 10–14 weeks during which the primary therapy was continued. No long term adverse effect was observed during this period of observation.

Conclusion- This small observational study indicates that intravenous iron infusion is a safe procedure in the community cancer center setting. It has the potential of significantly augmenting the effect of erythropoitic growth factor therapy for chemotherapy induced anemia. Further studies are required to evaluate the routine use of intravenous iron in chemotherapy induced anemia similar to its use with erythropoitin in patients on chronic hemodialysis.
Cocaine’s Direct Effect on Either Sickle or Normal Erythrocytes Does Not Increase Phosphatidylserine Exposure, Calcium Pumping, or Scramblase Activity.
Authors: DeJong Kitty , Hagar Robert
Published: October 2019

Hypothesis: Cocaine directly affects the exposure of phosphatidylserine, calcium pumping, and scramblase activity in sickle cells.

Background: Cocaine is highly associated with pulmonary hypertension in both normal and sickle cell disease patients. Given the growing interest in pulmonary hypertension as a major morbidity factor, the causes and determinants of pulmonary hypertension need to be elucidated to allow rational study designs. Phosphatidylserine (PS) exposure is known to occur in sickle cells and appears to be relevant for vascular damage. Although the mechanism underlying PS exposure is poorly understood, signal transduction processes appear to play a role. We wondered if cocaine could activate these processes and cause PS exposure, or interfere with the proteins involved in the transbilayer movement of PS.

Methods: Sickle and normal erythrocytes were exposed to cocaine HCl (from a 10% topical solution, Roxane Labs) at concentrations between 100 and 1000 ng/ml, which is the range of determined plasma concentrations after cocaine use. PS exposure after cocaine treatment was measured by labeling with fluorescently conjugated annexin V (AV) and analysis by flow cytometry. Alterations in scramblase activity were determined by assessing the percentage of PS-exposing cells following loading of the cells with 0.1 mM calcium using calcium ionophore. Calcium influx and Ca-ATPase-mediated calcium efflux were monitored using the fluorescent probe Fluo4, and flippase activity was assessed using NBD-PS followed by analysis with flow cytometry.

Results: Sickle cells differ from normal cells with respect to most of the measured parameters, such as having more PS-exposing cells, lower flippase activity, and alterations in calcium kinetics. However, cocaine did not have any effect on PS exposure, calcium-induced scrambling, calcium influx, calcium pump activity, or flippase activity. The cell scatter patterns did not show any gross changes in red cell shape or density after cocaine treatment.

Discussion: The lack of effect implies that cocaine is working through other mechanisms than effects on red cell membranes. Therefore, future studies should focus on other vascular cell types and membrane pathways. Alternatively, cocaine metabolites should be evaluated for their activities on red cells.