bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemotherapy Does Not Enhance CD34+ Cell Collection When Added to Growth Factor and Plerixafor in Patients Who Are Poor Mobilizers
Authors: Rosenbaum Eric R , Nakagawa Mayumi , Pesek Gina , Theus John W , Barlogie Bart , Cottler-Fox Michele H.
Published: October 2019

Abstract 4388

Background:

Many patients who are poor CD34+ cell mobilizers receive the CXCR4 inhibitor plerixafor to enhance mobilization. Here we examined whether chemotherapy followed by growth factor (GF) + plerixafor for mobilization improves CD34+ cell collection in these patients.

Materials and Methods:

We reviewed CD34+ cell collection data from January 2009 through June 2011 for all patients (n=152) at our institution who received plerixafor with GF ± chemotherapy. The primary disease in both groups was multiple myeloma, with 14 non-myeloma patients in the chemotherapy group and 8 non-myeloma patients in the no-chemotherapy group. There were 82 collections preceded by mobilization with chemotherapy (53 male, 29 female), and 80 collections without chemotherapy (51 male, 29 female). Several patients collected on more than one occasion. Age range in the chemotherapy group was 36–76y (median 64) and in the no-chemotherapy group was 42–83y (median 63). Our standard protocol is to collect with plerixafor for 4 consecutive days using a large volume leukapheresis (30L processed). Of the total 162 collections, CD34+ cells collected on each of the 4 consecutive days were recorded. If during the collection process a day was skipped, if less than 4 days of collection were performed, or a collection event on a particular day processed any blood volume other than 30L, all days including and subsequent to such events were excluded from analyses. CD34+ cells were quantified by flow cytometry. Mean CD34+ cells collected on days 1–4 were recorded separately for both groups and compared using two-tailed student’s t-tests.

Results:

Mean number of CD34+ cells collected for the chemotherapy group on days 1–4 were 2.65 (n=82), 2.27 (n=69), 1.60 (n=47), and 1.25 × 106 CD34+ cells/kg (n=38), respectively. Mean number of CD34+ cells collected for the no-chemotherapy group on days 1–4 were 2.51 (n=80), 1.83 (n=67), 1.22 (n=45), and 0.97 × 106 CD34+ cells/kg (n=28), respectively. There were no statistically significant differences between mean CD34+ cells collected on days 1–4 between the two groups (all p-values >0.05). We also asked whether the decrease in mean CD34+ cell yield observed as collection progressed was statistically significant. This phenomenon was determined significant (by one-way ANOVA) for both the chemotherapy and no-chemotherapy groups tested separately (p-values 0.02 and 0.003, respectively).

Conclusions:

Chemotherapy plus GF and plerixafor does not improve CD34+ cell collection in poor mobilizers compared to GF+plerixafor alone. Additionally, mean number of CD34+ cells collected decreases each day, regardless of which of the two mobilization regimens is used.

Disclosures:

Pesek: Genzyme: Membership on an entity’s Board of Directors or advisory committees. Cottler-Fox:Genzyme: Membership on an entity’s Board of Directors or advisory committees.
Recovery of Viable MSCs Isolated From Fresh Umbilical Cord Tissue, Measured After Cryopreservation, Is on Average 8-Fold Higher when Compared to Recovery of Viable MSCs Isolated From Previously Cryopreserved Umbilical Cord Tissue
Authors: Briddell Robert , Litkenhaus Frank , Foertsch Gail , Fuhrmann Angela , Foster Karen , Falcon Girard Kate , Fiscus Bruce , Boehm Ann , Brown Margaret , Pettit Mystie , Rigas Bridges Asimena , Nichols Karen , Fodor William , Kraus Morey
Published: October 2019

Abstract 4398

Background.

The human umbilical cord (hUC) is comprised of a gelatinous connective tissue that contains mesenchymal-like stem cells (TC-MSCs), proteoglycans and collagen. Isolation and cryopreservation of TC-MSCs for potential therapeutic use is an active area of research in the stem cell banking industry. We have compared two methods of cryopreservation to determine the optimal method for storing TC-MSCs: (1) cryopreservation of isolated TC-MSCs from fresh hUC tissue; and, (2) cryopreservation of intact hUC tissue followed by TC-MSC isolation.

Methods.

hUCs were aseptically obtained, and parallel hUC segments from the same hUC were processed by one of the two methods. Method 1: TC-MSCs were isolated from partially dissected fresh hUC segments by overnight collagenese digestion (2.5mg/mL-g), filtration and centrifugation. Isolated TC-MSCs from this process were cryopreserved in a cryopreservation solution (11% DMSO, 1% Dextran40, 4% HSA in 0.9% NaCl), frozen and stored at −80°C for < 30 days. Cells were rapidly thawed at 37°C, washed and resuspended in D-PBS for flow cytometric analysis. Method 2: hUC tissue was cryopreserved in the cryopreservation solution as in Method 1, frozen and stored at −80°C for < 30 days. Frozen hUC tissue was rapidly thawed at 37°C and processed as described above to isolate the TC-MSCs. TC-MSCs recovered from each procedure were enumerated and phenotyped by flow cytometry.

Results.

The recovery of viable CD45−/CD90+ TC-MSCs (after thawing) from fresh hUC tissue (Method 1), on average resulted in 5.04×105 cells/gram of tissue (n=15), whereas the yield of CD45−/CD90+ TC-MSCs from cryopreserved/thawed hUC tissue (Method 2) resulted in 8.20×104 cells/gram of tissue (n=15). Cell viabilities from both processes were comparable.

Conclusions.

Significantly higher (p<0.001) viable cell recoveries (8.4-fold average, 6.8-fold median, range 2.1–18.1) were obtained when TC-MSCs were harvested prior to cryopreservation.

Disclosures:

Briddell: PerkinElmer Inc.: Employment. Litkenhaus:PerkinElmer Inc.: Employment. Foertsch:PerkinElmer Inc.: Employment. Fuhrmann:PerkinElmer Inc.: Employment. Foster:PerkinElmer Inc.: Employment. Falcon Girard:PerkinElmer Inc.: Employment. Fiscus:PerkinElmer Inc.: Employment. Boehm:PerkinElmer Inc.: Employment. Brown:PerkinElmer Inc.: Employment. Pettit:PerkinElmer Inc.: Employment. Rigas Bridges:PerkinElmer Inc.: Employment. Nichols:PerkinElmer Inc.: Employment. Fodor:PerkinElmer Inc.: Consultancy. Kraus:PerkinElmer Inc.: Employment.
Molecular Analysis of the t(2;8)/MYC-IGK Translocation in High-Grade Lymphoma/Leukemia by Long-Distance Inverse PCR
Authors: Kroenlein Hannes , Schwartz Stefan , Reinhardt Richard , Molkentin Mara , Goekbuget Nicola , Hoelzer Dieter , Thiel Eckhard , Burmeister Thomas
Published: October 2019

Abstract 4407

Background:

Burkitt lymphoma and a subset of diffuse large B-cell lymphomas are characterized by chromosomal alterations affecting the MYC oncogene on 8q24. In most cases MYC is found juxtaposed to the immunoglobulin heavy chain (IGH) gene locus. Translocations to the immunoglobulin kappa (IGK) gene locus on 2p11 are observed in around 10% of cases. Little data exist on the molecular mechanisms leading to this aberration. The chromosomal breakpoints on chromosome 8 have been found dispersed over a large area 3’ of MYC. Currently, molecular cytogenetics (FISH) and cytogenetics are the methods of choice to detect the t(2;8) translocation and until now there exists no PCR method to reliably detect it.

Objectives:

In order to obtain a better understanding of this chromosomal translocation we developed a long-distance inverse (LDI) PCR method for the identification of chromosomal translocations affecting the IGK locus. The LDI PCR method takes advantage of the fact that the chromosomal breaks occur on chromosome 2 in the vicinity of the IGK joining segments. Using this method we investigated a number of cytogenetically mostly uncharacterized high-grade lymphoma samples.

Results:

A MYC-IGK juxtaposition was identified in 7 patients and three t(2;8)-positive cell lines. The chromosomal breakpoints were molecularly characterized and analyzed. The linear distance of the breakpoints on chromosome 8 to MYC ranged from some 100 bp to more than 0.5 MB. The breakpoints appeared to be clustered in distinctive regions, however, the number is still to small to draw definitive conclusions. The reciprocal translocated allele could be characterized in the majority of cases and putative break mechanisms are proposed. No larger sequence homologies were detected at the break sites. A minority of breaks were located near putative recombination signal sequences (RSS).

Conclusions:

This study represents the largest series of t(2;8)-positive cases analyzed so far and the LDI PCR developed therein is the first universally applicable PCR-based method to detect this aberration. This LDI PCR should furthermore in general be useful for the molecular analysis of chromosomal translocations affecting the IGK locus. These aberrations have been detected in various lymphoma entities but are currently largely unexplored.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Characterization of Novel Chromosomal Translocations Involved with TCR Locus in T-ALL
Authors: Zheng Haitao , Chen Shaohua , Yang Lijian , Li Bo , Przybylski Grzegorz K , Schmidt Christian A , Li Yangqiu
Published: October 2019

Abstract 4409

Chromosomal translocations occur frequently in hematological malignancies, which might involve in cell transformation and initiation of malignancy. In T-cell malignancies, the chromosomal aberrations are more complex due to the TCR gene rearrangements during T-cell differentiation, which provide different breakpoints for the gene recombination. The chromosomal translocations that occur in the cases of T-cell acute lymphocatic leukemia (T-ALL) frequently involve the juxtaposition of enhancer elements and strong promoter from T-cell receptor (TCR) genes on chromosome 14 (TCRA and TCRD) and chromosome 7 (TCRB and TCRG) with transcription factors genes. Using a novel rapid approach of fine-tiling array comparative genomic hybridization (fine-tiling aCGH) with the array coving the TCR&brkbar;Á&brkbar;Ä, TCR&brkbar;Ã and TCR&brkbar;Â loci, chromosomal alterations were analyzed and the breakpoints and their position in the chromosomes in genomic DNA were identified from patients with T-ALL.

Combining with ligation-mediated PCR (LM-PCR) and sequencing techniques, we clarified at the molecular level novel chromosomal translocation t(X;14)(q11.2;q11), inv(14)(q11;q11), t(1;14)(p32;q11), inv(14)(q11;q32) and t(7;14)(q34;q32) in 4 cases with T-ALL. which caused a V&brkbar;Á37 segment fused to ZFRP1 gene locus, J&brkbar;Á61 segment fused to RPSAP4 gene locus, V&brkbar;Â20 segment rearranged to Ig&brkbar;Ê gene segment and so on. The biological function of the novel fusion genes is needed further research.

Disclosures:

Zheng: the Fundamental Research Funds for the Central Universities (No. 21610603): Research Funding; National Natural Science Foundation of China (no. 30871091): Research Funding. Li:National Natural Science Foundation of China (no. 30871091): Research Funding; Fundamental Research Funds for the Central Universities (No. 21610603): Research Funding.
IL10-592 CC Genotype In the Donor Is Associated with a Significant Decrease of Relapse Rate and a Significant Increase of Event-Free Survival and Overall Survival After Allogeneic Hematopoietic Stem Cell Transplantation In Children with Hematological Malignancies
Authors: Gruhn Bernd , Voigt Janine , Pfaffendorf-Regler Nadine , Wolff Ilona , Zintl Felix , Beck James F.
Published: October 2019

Abstract 4459

IL10 is a pivotal immunomodulatory cytokine and is usually regarded as a suppressor of the immune responses. However, IL10 has been shown to have some immunostimulatory effects. The IL10-592 CC genotype is associated wit higher production of IL10. Because IL10 may promote the development of alloimmunity we hypothesized that the IL10-592 CC genotype in the donor reduces the risk of relapse after hematopoietic stem cell transplantation (HSCT). A cohort of 211 children (median age, 12 years) with acute lymphoblastic leukemia (n=100), acute myeloid leukemia (n=62), myelodysplastic syndrome (n=30) or chronic myeloid leukemia (n=19) who underwent allogeneic bone marrow (n=153) or peripheral blood stem cell transplantation (n=58; T-cell depleted: n=26) in a single center and/or their respective donors was genotyped of IL10 gene for rs1800872 using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 48% of transplants and HLA-identical related in 42% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 69% of transplants and cyclosporine A alone in 17% of transplants. Cell samples from the donor were available in 174 cases and from the patient in 197 cases. The IL10-592 CC genotype was present in 82 of the 174 donors (47.1%) and in 104 of the 197 patients (52.8%). Interestingly, we found a significantly reduced incidence of relapse in patients who were transplanted from a donor with the IL10-592 CC genotype (15.9% versus 30.4%; p=0.016). In addition, we observed a significant increase of event-free survival (52.4% versus 33.7%; p=0.019) and a significant increase of overall survival (54.9% versus 37.0%; p=0.040) if the IL10-592 CC genotype was present in the donor. The occurrence of the IL10-592 CC genotype, in either donors or recipients, had no significant impact on treatment related mortality, acute and chronic graft-versus host disease. In conclusion, IL10-592 CC genotype in the donor is associated with a significant decrease of relapse rate and a significant increase of event-free survival and overall survival after HSCT in children with hematological malignancies. This is the first study to describe an association of IL10 gene polymorphism with relapse rate after HSCT. Selecting a donor with the IL10-592 CC genotype could be a useful therapeutic strategy for improving the final outcome after allogeneic HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Impact of KIR B/X Genotype and Missing KIR Ligands in HLA-Matched Unrelated Donor HSCT
Authors: Li Weiyang , He Jun , Bao Xiaojing , Zhou Lina , Depei Wu , Sun Aining
Published: October 2019

Abstract 4488

Objective

To study the prognostic impact of missing ligands for inhibitory killer immunoglobulin-like receptor(KIR) and donor KIR B/X genotype in HLA-matched hematopoietic stem cell transplantation(HSCT) using unrelated donor.

Methods

HLA genotype of 51 patients (ALL 22 cases,AML 13 cases,CML 14 cases,MDS 1 case and HAL 1 case) and their matched unrelated donors was determined by polymerase chain reaction sequence oligonucleotide probes(PCR- SSOP) and sequence specific primers (PCR-SSP).The KIR genotype was determined by PCR-SSP.

Results

Patients were divided into two groups. Group A consisted of 16 patients with donor KIR B/X genotype and missing HLA class I ligands for donor inhibitory KIR, and group B consisted of 35 patients without KIR B/X or missing KIR ligands.The 3-year overall survival(OS) rate for group A was 93.8%, and the OS rate for group B was 61.7% (P=0.058).The 3-year continuous complete remission(CCR) rate for the two groups was 83.3% and 57.2%,respectively(P=0.098).There was no significant difference in neutrophil and platelet recovery,≥ III° acute GVHD(6.3% vs 20.0%,P>0.05) and extensive chronic GVHD (25.0% vs 17.1%,P>0.05) between the two groups.

Conclusions

In HLA-matched unrelated donor HSCT,donor KIR B/X genotype and missing KIR ligands may be associated with decreased severely acute GVHD,improved CCR and OS.

Disclosures:

No relevant conflicts of interest to declare.
CD56-Positive Acute Myeloid Leukemia Has Inferior Outcome After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Tan Tran-Der , Wu Mau-Ching , Chiou Lun-Wei
Published: October 2019

Abstract 4493

Background:

CD56-positive AML patients have more extramedullary infiltrate and inferior outcomes when treated with intensive chemotherapy. We evaluate the outcomes between CD56 positive and negative AML patients after allogeneic hematopoietic stem cell transplantation.

Methods:

Forty-one AML patients underwent hematopoietic stem cell transplantation at our institute between August 2002 and September 2010. Two of them received autologous transplantation and the other 39 were allotransplant. We analyze the survival and relapse rates when transplanted in complete remission between CD56 positive and negative patients.

Results:

Among these 41 patients, 13 were CD56 positive (M/F 7/6 and median age 34.9 years) with 7 of them (53.8%) had extramedullary involvement; 28 were CD56 negative (M/F 14/14) and median age 42.7 years) with 2 of them (7.1%) had extramedullary involvement. Two-year and five-year overall survivals for all patients were 48% and 40%, respectively. For CD56 positive and negative patients, 2-year overall survivals were 22% and 54%, respectively. There were 30 patients undergoing transplant in complete remission with 8 CD56 positive and 22 CD56 negative, and the relapse rates after transplant were 75.0% and 31.8%, respectively, (p = 0.049). High risk cytogenetics were found in 5 out of 13 CD56 positive patients and 5 out of 17 available cytogenetics CD56 negative patients, which is of no statistically different.

Conclusion:

Even following the most intensive treatment hamatopoietic stem cell transplantation, CD56-positive AML patients still have more relapse rate, and poor survival which is independent of cytogenetic risk. This indicated CD56 positivity is truly a poor prognostic factor and needs to consider allogeneic hematopoietic stem cell transplantation early on remission achieved and earlier immunosuppressants withdrawal is mandatory for those who allotransplant.

Disclosures:

No relevant conflicts of interest to declare.
Outcomes of Hematopoietic Stem Cell Transplantation for Childhood Acute Lymphoblastic Leukemia with t(4;11)
Authors: Sekiya Yuko , Narita Atsushi , Yoshida Nao , Matsumoto Kimikazu , Kato Koji
Published: October 2019

Abstract 4502

Introduction:

As the prognosis of childhood acute lymphoblastic leukemia (ALL) with t(4;11) is generally poor, hematopoietic stem cell transplantation(HSCT) at 1st complete remission is recommended in most of Japanese ALL protocol. We analyzed outcomes of HSCT for childhood ALL with t(4;11) at our department, and assessed the outcomes with age and preconditioning at HSCT.

Subjects:

We retrospectively analyzed 15 patients who underwent HSCT between 1991 and 2011. The onset of ALL was less than one year old (infant ALL) in 9 patients and over one year old in 6 patients. The disease status at HSCT was 1st complete remission (CR1) in 10 patients, 2nd complete remission (CR2) in 2 patients, and more advanced stages in 3 patients. 13 patients underwent allogeneic HSCT and 2 patients underwent autologous HSCT at median age of 19 months. In allogeneic HSCT, the number of bone marrow transplantation from HLA matched sibling was 3 and 4 from unrelated donors, and that of cord blood transplantation was 6. Nine patients including 7 infant ALL underwent conventional stem cell transplantation (CST), 6 patients including one infant ALL underwent reduced intensity stem cell transplantation (RIST). Preconditioning of RIST was mostly consisted of fludarabine and melphalan with 3Gy of total body irradiation (TBI).

Results:

Of all 15 patients, 8 patients are alive in CR. Three out of 9 infant ALL and 5 out of 6 ALL patients, whose onset was above 1 year old, are alive in CR. Seven of 10 ALL patients who underwent HSCT in 1st CR are alive, on the other hand, only one of 5 patients in 2nd CR or non CR is alive. Five patients who underwent RIST over 1 year old are alive in CR.

Discussion:

HSCT for childhood ALL with t(4;11) in 1st CR could achieve better outcome than those in 2nd CR or non CR. The transplant outcomes with RIST was better than CST and it could be applied for ALL patients with t(4;11) whose onset is over one year old.

Disclosures:

No relevant conflicts of interest to declare.
KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire
Authors: Lee Dean A. , Senyukov Vladimir V , Trembley Jerome R
Published: October 2019

NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy.

We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks.

First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation.

Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition.

We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained.

These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
An Unexpected Role For αIIbβ3 In Immune Complex Disorders
Authors: Zhi Huiying , Wu Nannan , Dai Jing , Fang Juan , Liu Pu , Liu Junling , Hu Hu , Zhu Jieqing , Newman Debra K. , Newman Peter J. , Gao Cunji
Published: October 2019

IgG immune complexes contribute to the etiology and pathogenesis of a number of autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus (SLE), rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Human platelets express on their surface an Fc receptor, termed FcγRIIa or CD32a, that when exposed to immune complexes initiates a potent signal transduction cascade that results in platelet activation and granule secretion. Patients with immune complex-related disorders are known to be highly susceptible to thrombotic events, and microthrombi have been observed to colocalize histologically in patients with immune complex disorders. To better understand the contribution of platelet adhesion receptors and signaling molecules to IgG immune complex-mediated thrombotic complications, we incubated platelets in microtiter wells that had been precoated with immobilized IgG. Platelets quickly formed filopodia, and then adopted a fully-spread morphology over a 30 minute period of time. Cytosolic proteins known to be involved in platelet spreading, including FcγRIIa, Src, Syk, and pp125FAK also became rapidly tyrosine phosphorylated. Because the integrin αIIbβ3 employs each of these signaling molecules in platelet spreading on immobilized fibrinogen, we next evaluated its role in platelet spreading on immobilized IgG. Interestingly, Fibans - small molecule antagonists of αIIbβ3-fibrinogen interactions – also blocked (1) platelet spreading on immobilized IgG, (2) the associated phosphorylation events, and (3) platelet thrombus formation over immobilized IgG under conditions of flow. Human platelets from Glanzmann thrombasthenic individuals, or murine integrin β3-deficient platelets expressing a human FcγRIIa transgene, also failed to spread on immobilized IgG and form thrombi on immobilized IgG under conditions of flow. FcγRIIa-transgenic mice lacking the Src-family kinase Lyn – thought to be responsible for phosphorylating the ITAM tyrosines of FcγRIIa – also failed to spread or form thrombi over immobilized IgG. Finally, Chinese hamster ovary cells transfected with αIIbβ3 and FcγRIIa failed to spread on immobilized IgG unless small amounts of fibrinogen were added to the IgG preparation before plating. Taken together, our data suggest a complex functional interplay between FcγRIIa and αIIbβ3 in immune complex-mediated thrombotic disorders in which platelets encounter immobilized IgG and become activated to secrete α-granule fibrinogen. Secreted fibrinogen, in turn, then becomes a substrate for αIIbβ3-mediated platelet spreading and subsequent thrombus formation.

Disclosures:

No relevant conflicts of interest to declare.
Role Of B Cell Tolerance In PF4/Heparin Antibody Production
Authors: Zheng Yongwei , Wang Alexander W , Yu Mei , Padmanabhan Anand , Tourdot Benjamin E , Newman Debra K. , White Gilbert C. , Aster Richard H. , Wen Renren , Wang Demin
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis.

Disclosures:

White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.
EVI1, A Potential Regulator Of CEBPA; A Contributory Mechanism In Leukemogenesis?
Authors: Wilson Michael , Zhang Yi , Cui Xiaohui , Wuertzer Charles A. , Perkins Archibald S.
Published: October 2019

Ecotropic Viral Integration Site 1 (EVI1) is a zinc finger oncoprotein implicated in a subtype of acute myeloid leukemia associated with poor prognosis. Leukemic cells overexpressing EVI1 display a block in myeloid differentiation and resistance to apoptosis, both of which are reversed with EVI1 shRNA knockdown. Previous mutagenesis studies have shown that DNA binding via the first set of zinc fingers (ZF1) is critical for leukemic transformation by EVI1. The mediators between EVI1 DNA-binding and leukemogenesis are mostly unknown. Using chromatin immunoprecipitation and sequencing (ChIP-Seq) in myeloid leukemic cells, we have recently identified an evolutionarily conserved EVI1 binding site 35 kilobases downstream of Cebpa, encoding the CCAAT enhancer-binding protein alpha, a master regulator of early granulopoiesis. In order to obtain a better understanding of the potential regulatory role of this region in leukemogenesis, we used genome database analysis to see what other transcription factors or epigenetic marks are present. ChIP-Seq data deposited in the UCSC databank indicates the presence of CTCF, GATA2 and CEBPβ, as well as TAL1, p300, and GATA1 in leukemic cells, the latter three of which are known to bind together in a complex. This data suggests that the Cebpa +35 region serves an important role in regulating hematopoietic development and Cebpa transcription. In order to characterize the binding of EVI1 ZF1 to the Cebpa +35 site, we performed methylation interference assays and oligonucleotide mutagenesis assays. EVI1 ZF1 binds within this +35kb control region to the sequence TGACAGTGACGG with 133% affinity relative to the canonical motif. In an effort to assess any biological effects linked to EVI1 binding at the Cebpa +35 motif, we conducted EVI1 overexpression studies in the EML hematopoietic progenitor cell line. While control EML cells exhibited marked upregulation of Cebpa upon induction, EVI1-transduced cells displayed a significant suppression of Cebpa transcript. To determine if this suppression requires DNA binding, we tested a mutant of EVI1 that is defective in DNA binding (EVI1R205N); this was unable to suppress Cebpa induction. To test whether EVI1 can bind to the +35 KB site, we performed ChIP-PCR, which indeed showed EVI1 occupancy at the +35 KB site. In conclusion, we have shown evidence that EVI1 suppresses Cebpa transcription in early hematopoietic progenitor cells, and that the EVI1 binding site at Cebpa +35 serves an important function in myeloid maturation. This regulatory node represents a possible therapeutic target for the treatment of acute myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Pre-Clinical Development Of a Novel Chimerical Antigen Receptor Targeting High-Risk Pediatric ALL Over-Expressing Tslpr
Authors: Qin Haying , Monica Cho , Haso Waleed , Zhang Ling , Barrett David M. , Orentas Rimas J , Grupp Stephan A. , Fry Terry J
Published: October 2019

Background

While current upfront therapy will cure over 80% of patients, B-cell precursor acute lymphoblastic leukemia ALL (BCP-ALL) remains a leading cause of cancer-related death in children, and outcomes for those patients who relapse have not been changed substantially over recent decades. Immunotherapy with a chimeric antigen receptor (CAR) targeting CD19 has resulted in impressive clinical responses, but alternative CAR targets are needed as not all patients respond and CD19 negative relapses have been observed. Thymic stromal lymphoprotein receptor (TSLPR) is coded by the CRLF2 gene. CRLF2 genetic alterations occur in 5-16% of pediatric and adult BCP-ALL and result in TSLPR over-expression that is associated with poor prognosis. Furthermore, emerging data suggests that TSLPR overexpression is functionally important for leukemic blasts. Thus, TSLPR is an ideal immunotherapeutic target. We constructed TSLPR-specific CARs and tested their therapeutic efficacy in vitro and in vivo.

Methods

Hybridomas producing TSLPR-specific antibodies were used to clone variable regions of the heavy and light chains for constructing TSLPR-specific scFv. The scFv was linked with the CD8α hinge and transmembrane domain, 4-1BB intracellular domain and CD3ζ chain, and then cloned into a 3rd generation lentiviral vector. For optimization of the CAR configuration, an IgG heavy chain constant domain (CH2CH3) spacer was fused to scFv to generate a “long” TSLPR CAR. Human T cells were transduced with CAR vectors and the transduced cells were evaluated in vitro and in vivo against TSLPR-overexpressing ALL, including a TSLPR-transduced REH cell line (REH-TSLPR) and patient-derived xenografts naturally overexpressing TSLPR.

Results

Both long and short TSLPR CARs could be efficiently transduced (approximately 60-80%) into human T cells using a lentiviral system. T cells expressing both long and short TSLPR CARs produced high levels of cytolytic cytokines upon co-culture with of TSLPR-overexpressing ALL. However, only the short TSLPR CAR demonstrated effective killing against TSLPR-expression ALL cells in vitro. Short TSLPR-expressing T cells completely eradicated leukemia in NSG xenografts mice bearing REH-TSLPR (figure 1) as well as TSLPR-expressing patient xenografts. The long TSLPR CAR did not show any activity in vivo.

Conclusion

TSLPR-targeted CAR T cells is promising therapeutic approach in high risk BCP-ALL and represents the first example of an oncogene-specific cellular therapy in BCP-ALL.

Disclosures:

Grupp: Novatis: Research Funding.
Preclinical Testing Of a PI3K Inhibitor In T Lineage Leukemia: Target Validation and Notch1/Myc Down-Regulation In Drug Resistant Clones
Authors: Dail Monique , Wong Jason , Lawrence Jessica , O'Connor Daniel , Nakitandwe Joy , Chen Shann-Ching , Akagi Keiko , Lee Leslie , Aster Jon C. , Pear Warren S. , Sampath Deepak , Downing James R. , Shannon Kevin
Published: October 2019

Mutations that deregulate Notch1 and Ras/PI3K/Akt signaling are common in T-ALL and often coexist. Thus, inhibiting these pathways alone and in combination has been advocated as a rational therapeutic strategy. GDC-0941 is a pan-PI3K inhibitor that is advancing in clinical development that we evaluated for efficacy and mechanisms of drug resistance in T-ALL cell lines and primary murine T-ALLs. Cell lines are uniformly sensitive to GDC-0941; however gradual dose escalation results in resistant lines. By contrast, primary leukemia cells display variable in vivo sensitivity to treatment with GDC-0941 +/- a MEK inhibitor. Importantly, mice invariably relapse with drug resistant clones. A majority of GDC-0941-resistant T-ALL cell lines and primary leukemias unexpectedly showed reduced levels of activated Notch1 protein, down-regulate many Notch1 target genes including Myc, and display cross-resistance to gamma secretase inhibitors (GSIs). In multiple cases, Notch1 mutations that were present in drug-sensitive parental leukemias were absent at relapse. Importantly, resistant clones that emerge both in vitro and in vivo up-regulate PI3K signaling indicating an “on pathway” mechanism of resistance. Consistent with these data, inhibition of Notch1 signaling promotes GDC-0941 resistance and enhances PI3K signaling, whereas expression of activated Notch1 increases the GDC-0941 sensitivity of mouse and human T-ALL cell lines. Thus, oncogenic Notch1 mutations that promote clonal outgrowth during malignant transformation unexpectedly undergo negative selection during treatment with GDC-0941. Together, these in vivo studies: (1) validate PI3K as an important therapeutic target in T-ALL; (2) demonstrate that active Notch1 and elevated Myc expression are dispensable for T-ALL growth in vivo; and, (3) indicate that therapeutic PI3K inhibition selects for the outgrowth of leukemia cells with reduced Notch1 signaling. Importantly, our data also raise the possibility that simultaneously administering Notch1 and PI3K inhibitors will accelerate drug resistance in T-ALL, and support alternative approaches for deploying combination regimens against these dominant oncogenic “driver” pathways.

Disclosures:

Lee: Genentech: Employment. Aster:Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria. Sampath:Genentech: Employment, Equity Ownership.
Vgamma9Vdelta2 T Cells-Based Immunotherapy Targeting BTN3 Molecules in Acute Myeloid Leukemia
Authors: Olive Daniel , Benyamine Audrey , Le Roy Aude , Castellano Rémy , Gertner-Dardenne Julie , Vey Norbert
Published: October 2019

As they can kill Acute Myeloid Leukemia (AML) blasts in vitro and in vivo, Vg9Vd2T cells are key players in the design of new strategies of immunotherapy. AminoBisphonates (NBP) can enhance their activation in vitro and in vivo. Their combination with low-dose IL2 has shown promising results in 2 patients with AML who underwent partial remission. NBP treatment of blasts inhibits the Mevalonate pathway. The subsequent accumulation of Isopentenyl Diphosphate sensitize AML blasts to Vg9Vd2T cells killing but some AML cell lines blasts are resistant to this TCR mediated-lysis. Butyrophilin 3 A1 (BTN3A1) has been shown to be involved in IPP recognition and Vg9Vd2 T cells activation. Agonist monoclonal antibodies (mAb) recognizing the 3 isoforms of BTN3, can trigger BTN3 on tumor cell lines and sensitize them to Vg9Vd2 T cells lysis.

We show that primary AML blasts from patient at diagnosis are heterogeneously killed by allogenic-IL-2-NBP-expanded Vg9Vd2 T. Some are resistant to this lysis and/or poorly sensitized by NBP. BTN3 molecules are highly expressed by blasts of AML cell lines and primary AML samples. We show that treatment of primary AML blasts with agonist anti-BTN3 mAb can overcome the resistance to Vg9Vd2 cells lysis in vitro. We assess this effect in vivo, showing that the addition of agonist anti-BTN3 mAb to Vg9Vd2 cells infusion decreased the tumor burden and increased the survival of NOG mice xenografted with luciferase-transduced U937 cell line. We confirm this effect in a model of mice xenografted with primary AML blasts, showing that treatment with anti-BTN3 mAb added to Vg9Vd2 cells infusion can decrease the number of blastic cells in the spleen, bone marrow and the blood, without requiring additional cytokine infusion.

This drastic effect on sensitization of primary AML blasts to Vg9Vd2T cells killing could be of great interest especially in cases of refractory or relapsing AML.

Disclosures

No relevant conflicts of interest to declare.
a Randomized, Controlled, Prospective Study for Efficacy of Micafungin Versus Itraconazole As Empirical Antifungal Therapy for Febrile Neutropenic Patients with Acute Leukemia
Authors: Jeong Seong Hyun , Kim Dae Young , Jang Jun Ho , Mun Yeung-Chul , Kim Sung-Hyun , Choi Chul Won , Kim Jin Seok , Park Joon Seong
Published: October 2019

Background: Micafungin, one of the powerful ecchinocandins, needs to be clarified its efficacy and safety of empirical therapy on febrile neutropenic patients compared with other anti-fungal drugs such as azole compounds.

Methods: A randomized, multi-center, prospective study (age-, sex-, diagnosis-, risk-controlled) was conducted for a comparison of efficacy and safety between micafungin and intravenous itraconazole as an empirical therapy on febrile neutropenic patients with acute leukemia. Anti-fungal drugs (study drug, micafungin; control drug, itraconazole) were administrated for sustained high fever following appropriate empirical antibiotic therapy (°Ã 3 days of combination antibiotics). The overall success rate [if the patient meets all of the following criteria: (1) no breakthrough fungal infection; (2) survived for 7 days past the end of the therapy; (3) no premature discontinuation; (4) disappearance of fever during neutropenia; (5) successfully treated base-line fungal infection] and side effects were evaluated. And duration of fever and neutropenic fever were estimated and compared between two groups.

Results: A total of 134 patients with acute leukemia were enrolled in the study (86 patients with AML, 41 patients with ALL, 7 patients with secondary leukemia). The overall success rate of micafungin and itraconazole was 63.6% and 53.2%, respectively (p = NS) (Figure 2). However, duration of fever and duration of neutropenic fever were significantly shorter in micafungin group (p = 0.0007, p = 0.0118, respectively) and survival benefit was marginal (OS, median months, 10.93 vs. 4.77, p = NS) (Figure 1). Grade III-IV adverse events including hyperbilirubinemia (2 vs. 7), elevation of transaminase level (2 vs. 4), electrolytes imbalance (1 vs. 2), atrial fibrillation (1 vs. 0), and anaphylaxis (1 vs. 0) occurred at 7 and 13 patients in micafungin (10.4%) and itraconazole (18.8%) group, respectively. All adverse events were manageable however, 4 patients died of progression of infection in each group.

Conclusion: This randomized, controlled, prospective study documented that micafungin, when compared with itraconazole, showed significantly superior effect on shortening the duration of fever and neutropenic fever, resulting in marginal survival benefit on acute leukemia patients who are suffering from life-threatening neutropenic period under anti-leukemic therapy.

Disclosures

No relevant conflicts of interest to declare.
Preclinical Efficacy of MEK Inhibition in Nras Mutant Acute Myeloid Leukemia
Authors: Burgess Michael R. , Hwang Eugene , Firestone Ari J , Huang Tannie , Xu Jin , Zuber Johannes , Bohin Natacha , Wen Tiffany , Kogan Scott C. , Haigis Kevin M. , Lowe Scott W. , Shannon Kevin , Li Qing
Published: October 2019

Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations.

Disclosures

No relevant conflicts of interest to declare.
Genome-Wide Mapping of Binding Sites and Networks of Potential Target Genes of the Fusion Oncogene ETV6/RUNX1 in Acute Lymphoblastic Leukemia in Childhood
Authors: Kaulfuss Katja , Heiden Thomas , Hecht Jochen , Seeger Karl
Published: October 2019

Acute lymphoblastic leukemia (ALL) in childhood, a clinically and biologically heterogeneous disease, represents the most common malignant disease in childhood. Approximately 20-25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most common reciprocal chromosomal translocation in childhood ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, into the fusion oncogene ETV6/RUNX1 (E/R; synonym TEL/AML1).

Recent studies in various animal models have strengthened the view that E/R positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy.

Regarding the molecular mechanism by which the chimeric fusion protein E/R causes gene expression changes, it is assumed that E/R binds with the runt homology domain of RUNX1 (RHD, DNA-binding domain) to RUNX1 target sequences of gene promoters and recruits corepressors and histone deacetylases through its ETV6 portion, leading to chromatin condensation and transcriptional repression. Thus, E/R appears to act mainly as an epigenetic repressor of genes that are normally activated by RUNX1. However, the precise mechanism of cellular transformation and the identity of E/R target genes are largely unknown.

Therefore, we used chromatin immunoprecipitation (ChIP), followed by next generation sequencing (ChIP-Seq) to identify E/R target genes in the E/R positive BCP-ALL cell lines REH and UoC-B6 as well as in primary patient material from children with relapsed E/R positive ALL. We were able to detect a core gene set of 335 candidate target genes common to all samples analyzed. Those genes could be assigned to 15 significantly overrepresented KEGG pathways (e.g. cell cycle, pathways in cancer, hematopoietic cell lineage and B cell receptor signaling pathway).

The results show, besides target genes already reported in the literature such as EPOR, MPO and IGLL1, numerous not previously described candidate E/R target genes, such as LEF1, E2F2, FLT3, FGFR1 and RUNX1 that are potentially important in the pathogenesis of E/R positive ALL and may lead to new treatment options.

Disclosures

No relevant conflicts of interest to declare.