bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect Of Immediate Prior-Line Lenalidomide Or Thalidomide Therapy On Response To Pomalidomide In Multiple Myeloma
Authors: Hwa Yi Lisa , Laumann Kristina M , LaPlant Betsy R , Buadi Francis , Dingli David , Dispenzieri Angela , Gertz Morie A , Go Ronald S. , Hayman Suzanne R , Kapoor Prashant , Kumar Shaji K , Leung Nelson , Lin Yi , Lust John A , McCurdy Arleigh , Mikhael Joseph , Roy Vivek , Russell Stephen , Zeldenrust Steven R , Rajkumar S. Vincent , Lacy Martha Q
Published: October 2019

Background

Pomalidomide (POM) is the newest Immunomodulatory agent (IMiDs) that has shown clinical efficacy in relapsed multiple myeloma (MM) refractory to lenalidomide (LEN) and thalidomide (THAL). Studies of MM cell microenvironment suggest that tumor subclones compete for dominance during changes in drug therapy. It was previously observed that a minor clonal population increased in prominence of MM cells after 1 year of treatment with LEN. We examined whether the patients having THAL or LEN as the most recent prior-line therapy affects response to POM.

Methods

We studied 208 patients enrolled in a phase II trial of POM / dexamethasone between November 2007 and March 2012 at Mayo Clinic, Rochester. We reviewed the immediate prior-line therapy before enrollment in our trial. Comparison of treatment duration of POM and response rates were conducted between patients who had either THAL or LEN as latest prior-line therapy, and patients who were treated with non-IMiDs immediate before enrollment in POM trial. Treatment duration of POM was estimated using Kaplan Meier method and the survival curves were compared using log-rank test. We used univariate Cox proportional hazard models to estimate the prognostic impact of different variables.

Results

The median age of patients was 63 (range from 32 to 88). 68 % were males. 80 patients (38.5%) had THAL or LEN immediate prior to POM. Median prior-line therapies were 3. Patients who received THAL or LEN immediately prior to POM had a lower response rate than those who had non-IMiDs as latest therapy (29% vs 44%, p 0.04) and shorter median duration of POM treatment (5.7 months vs 7.3 months, p 0.02). In 190 (91%) patients who were previously treated with LEN and THAL at any time, median time between the end of LEN or THAL treatment and the initiation of POM was higher for POM responders than non-responders (10 months vs 3 months, p 0.0008). Patients responded to POM had a shorter duration of prior LEN or THAL treatment compared to non-responders (9 months vs 15 months, p 0.001).

Conclusion

THAL or LEN as an immediate prior-line therapy to POM is associated with a lower response rate and shorter effective treatment duration. Longer interval between prior immunomodulatory compounds and POM, and shorter exposure to previous immunomodulatory compounds are associated with better POM treatment response.

Disclosures:

Gertz: Celgene: Honoraria. Kumar:Merck: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Company: Research Funding; Novartis: Research Funding; Genzyme: Research Funding. Lacy:Celgene Corporation: Research Funding.
Increased Frequencies Of Myeloid-Derived Suppressor Cells and Regulatory T-Cells Decrease Response To Donor Lymphocyte Infusions, Independent Of Graft Versus Host Disease In Multiple Myeloma
Authors: Franssen Laurens E. , Emmelot Maarten E. , van de Donk Niels W.C.J. , Lokhorst Henk , Mutis Tuna
Published: October 2019

Background

Allogeneic stem cell transplantation (allo-SCT) followed by donor lymphocyte infusions (DLI) can induce durable responses in multiple myeloma (MM) by virtue of the graft versus myeloma (GvM) effect. However, this is only true for a minority of patients and relapse rates after reaching an initial remission remain high. Also, transplant-related mortality and Graft-versus-Host-Disease (GvHD) are still major complications. Towards improving cellular immunotherapy we set out to identify the immune cell subsets that are involved in the GvM effect and GvHD.

Myeloid-Derived Suppressor Cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells, with strong immunosuppressive capacity. In general, they are divided into monocyte-like M-MDSCs en granulocyte-like G-MDSCs. In myeloma, they have been shown to be present in increased frequencies, inhibit T-cell proliferation and promote multiple myeloma cell growth in vitro. Furthermore, regulatory T-cells (T-regs) seem to suppress cellular anti-tumor responses in MM.

Methods

To determine whether MDSCs and T-regs hamper immunotherapy, we investigated MDSCs and T-regs in peripheral blood (PB) (n=43) from post-allo SCT MM patients, prior to their first DLI, and from healthy donors (n=13). All patients had persistent or progressive disease after allo-SCT. DLI dose was 107 T-cells/kg for sibling donors (n=20) and 106 T-cells/kg for matched unrelated donors (n=23).

Results

We observed a significantly higher frequency of M-MDSCs (CD14+ HLA-DR-/low) (mean 5.7 vs. 0.7%, P<0.001) and T-regs (CD4+ CD25+ CD127-/low) (11.4 vs. 5.5%, P<0.01), but not of G-MDSCs (CD11b+ HLA-DR-/low CD14-CD33+) in PB of MM patients versus healthy donors. Interestingly, patients responding to DLI had significantly lower amounts of immunosuppressive G-MDSCs (mean 0.6 vs. 1.2%, P=0.04) and T-regs (7.9 vs. 13.9%, P=0.02) in their PB prior to DLI. There was no difference in M-MDSCs between responding and non-responding patients. This effect on GvM was independent of GvHD, as levels of G-MDSCs, M-MDSCs and T-regs did not differ between GvHD+ and GvHD-groups.

Conclusion

We show that increased frequencies of G-MDSCs and T-regs are associated with resistance to DLI, independent of the occurrence of GvHD. Our data suggest that targeting G-MDSCs as well as T-regs may improve cellular immunotherapy in multiple myeloma.

Disclosures:

van de Donk: Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.
Stem Cell Transplant Versus Chemotherapy in Older Adults with Acute Myelogenous Leukemia In First Remission
Authors: Berman Ellin , Maloy Molly , Devlin Sean M. , Papadopoulos Esperanza B , Jakubowski Ann A.
Published: October 2019

Introduction

Optimal therapy for older adults with acute myelogenous leukemia (AML) who achieve remission following induction chemotherapy has not been determined. Options include consolidation chemotherapy (CC) or stem cell transplant (SCT) if an appropriate donor is identified. In order to determine whether SCT improved overall survival (OS) or whether associated toxicity was increased, we performed a retrospective study comparing SCT with CC in this older age group.

Methods

All adult patients ages 60 to 75 years with AML in 1st remission (CR1) who underwent a SCT at MSKCC between 2001 and 2013 were reviewed and compared to age-matched patients with AML who achieved CR1 and received CC. A landmark analysis at 3 months following CR1 was used to compare OS for the 2 patient groups. Only SCT patients transplanted by landmark time were included in the analysis. Overall survival was compared using Kaplan-Meier methodology.

Results

Sixty-eight patients were identified for the SCT group. Thirty-two patients were identified for the CC group (Table). Stem cell sources included: peripheral blood (n=63), cord blood (n=4) and bone marrow (n=1). Fifty-six patients received a T cell depleted transplant (32 with ClinMACsTM and 24 with IsolexTM ) and 12 received an unmodified product. Conditioning regimens were busulfan/melphalan/ fludarabine (n=54), melphalan/fludarabine (n=4), cyclophosphamide/fludarabine/thiotepa/ TBI (n=4), fludarabine/busulfan (n=3), busulfan/melphalan (n=2) and thiotepa/fludarabine/TBI (n=1). Donors included matched unrelated (n=28), matched related (n=25), mismatched unrelated (n=11) and mismatched cord blood (n=4). For patients in the CC group, induction chemotherapy included cytarabine in combination with either idarubicin (n=21), daunorubicin (n=10), or mitoxantrone plus etoposide (n=1). Forty-four patients received their transplant by the 3 month landmark and 30 patients in the CC group were alive at the landmark and were included in the OS analysis.

Deaths in the SCT group included 4 patients from infection, 1 from treatment-related toxicity, and 1 from relapsed disease. The estimated OS at 2 years in the landmark groups were 64% in the SCT group and 42% in the CC group (p=0.04).

Conclusions

Recognizing the inherent bias when retrospective studies compare SCT and CC, these data support the use of SCT for older patients with AML in CR1 who have an appropriate donor. Despite the older age, there was a statistically significant better OS with low 100 day mortality for those patients who underwent SCT.

Disclosures:

No relevant conflicts of interest to declare.
TRIM28 Is Essential For Erythroblast Differentiation In The Mouse
Authors: Tomonori Hosoya , Clifford Mary , Engel James
Published: October 2019

We previously identified TRIM28 (TIF1β, KAP-1) as a component of the mammalian DRED complex that prior studies had implicated as an embryonic/fetal β-type globin gene repressor. TRIM28 has been previously characterized as a transcriptional co-repressor thst interacts with heterochromatin protein 1 (HP1) and histone H3K9 methyltransferase SETDB1. We tested the contribution of TRIM28 to globin gene regulation and erythropoiesis by employing a mutant mouse that could be inducibly ablated for the Trim28 gene during the adult stage. The Mx1Cre transgene was activated by injecting poly(I:C) 5 times into these compound mutant mice every 48 hours. We hypothesized that TRIM28 might normally contribute to silencing of the embryonic and/or fetal globin genes and therefore that one might anticipate that the mouse εY- and/or βH1-globin genes (the structural homologues of human embryonic ε− and fetal γ-globin genes, respectively) would be de-repressed in the absence of TRIM28. To our surprise, the expression of εY- and βH1-globin mRNAs was not altered after induced genetic loss of Trim28. However, we discovered that its loss results in defective immature erythropoiesis in bone marrow and consequently to normocytic anemia. To address possible mechanisms that might be responsible for this observed defect in erythropoiesis, we analyzed staged, immature erythroid cells isolated from the Trim28 mutant mice by RNA-Seq and qRT-PCR. We observed diminished expression of multiple erythroid transcription factors and heme biosynthetic enzymes in the mutant. We also discovered increased expression of multiple apoptosis-related genes, and experimentally confirmed an increase in early apoptotic cells (Annexin V+ DAPI-) in the Trim28 mutant erythroblasts. We conclude that TRIM28 is vital for immature erythropoiesis in the adult bone marrow.

Disclosures:

No relevant conflicts of interest to declare.
The Development Of The Humanized Darc Transgenic Mouse
Authors: Novitzky-Basso Igor N , Ulvmar Maria H , Bacon Andrea , Chaganti Sridhar , Moss Paul AH , Rot Antal
Published: October 2019

The Duffy Antigen Receptor for Chemokines (DARC) is an atypical chemokine receptor which binds CXC and CC chemokines with no measurable downstream signaling. Erythrocyte DARC serves as a chemokine sink and reservoir. DARC on endothelial cells transports chemokines thus promoting transendothelial leukocyte migration. Two common polymorphisms define the Duffy blood group, FYA and FYB, whilst the third polymorphism which is widely prevalent in West Africa, FYB(ES), abolishes erythrocyte expression of DARC. DARC is a portal of entry for plasmodium vivax suggesting that FYB(ES) was selected due to it conferring resistance to infection. The impact of the FYB(ES) polymorphism extends to a genetic association with the benign ethnic neutropenia. In order to study the mechanisms of this association we have developed a murine transgenic models of the Fy(a-b-) phenotype as encoded by FYB(ES), and Fy(a-b+) phenotype as encoded by FYB. Cloned genes were introduced by pronuclear micro-injection into fertilized oocytes of murine-DARC knockouts. The resulting embryos were transferred into pseudopregnant mice and offspring screened for transgene insertion by PCR genotyping. Gene expression in founder animals was measured by qPCR, demonstrating expression in lung, kidney, liver, spleen, skin and cerebellum. Protein expression was confirmed by immunofluorescence on frozen sections in spleen and skin in a distribution identical to human. Erythrocyte phenotyping by FACS using anti-Fy3 and anti-Fy6 monoclonal antibodies demonstrated the lack of DARC expression in mice with FYB(ES) and confirmed expression on FYB transgenic erythrocytes. We show that murine chemokines bind to human DARC and we plan to use this model to investigate the impact of FYB(ES) on in vivo chemokine functions and equilibria. These models will prove useful in the dissection of the mechanisms of ethnic neutropenia and improve the understanding in the role of DARC in chemokine homeostasis.

Disclosures:

Chaganti: Roche: Membership on an entity’s Board of Directors or advisory committees, Receipt of travel grant Other.
Critical Roles Of The IDH2 Mutation In Development and Maintenance Of Acute Myeloid Leukemia
Authors: Ogawara Yoko , Katsumoto Takuo , Aikawa Yukiko , Shima Yutaka , Kagiyama Yuki , Matsunaga Hironori , Seki Takahiko , Kitabayashi Issay
Published: October 2019

Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system.

The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy.

Disclosures:

No relevant conflicts of interest to declare.
Activation Of TLR4-MyD88 Pathway Impairs HSC Function During Acute Inflammation
Authors: Zhang Huajia , Rodriguez-Rodriguez Sonia , Wang Lin , Mumaw Christen , Zollman Amy , Liu Yunlong , Cardoso Angelo A. , Carlesso Nadia
Published: October 2019

Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. High mortality rates in sepsis (200,000/yr in the USA) are associated with the host’s failure to eradicate pathogens due to the lack of neutrophils, excessive pro-inflammatory cytokines, tissue damage and multiple organ failure. While most studies have focused on the late consequences of sepsis, little is known about the changes occurring in the bone marrow (BM) at early stages of hematopoiesis and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, which closely recapitulate lethal sepsis in burn patients, we have previously reported that HSC undergo a significant expansion in the BM associated with a block of myeloid differentiation. Furthermore, we found that expanded HSC were unable to generate the downstream progenitors (common myeloid progenitors and granulocytes/monocytes progenitors) necessary to produce neutrophils, and had reduced self-renewal. All these effects were TLR4-dependent. TLR4 is activated by bacterial LPS and signal through two major pathways: TRIF-dependent and MyD88-dependent. In this study, we show the different contribution of the TLR4-TRIF and the TLR4-MyD88-dependent pathways to the BM response to Pseudomonas aeruginosa LPS. LPS challenge conducted on TRIF-null and MyD88-null mice demonstrated that TRIF was involved in the expansion of the HSC pool, but did not play a major role in the myelosuppression, whereas MyD88 activation was required for LPS-induced myeloid suppression. Moreover, we found that the impaired engraftment at long-term observed in wild-type LPS-challenged HSC during transplantation was rescued by MyD88 loss of function. Taken together, our results indicate a distinct role of theTLR4-TRIF and -MyD88 pathways in the regulation of the primitive stem/progenitor pool during sepsis and provide insights for a better understanding of the molecular mechanisms leading to neutropenia.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stem Cell-Derived Vesicles Reverse Hematopoietic Radiation Damage
Authors: Wen Sicheng , Goldberg Laura R , Dooner Mark S , Reagan John L , Quesenberry Peter J
Published: October 2019

Extracellular vesicles, including exosomes and microvesicles, have been shown to epigenetically modulate cell fate (Aliotta et al JEV, 2013). We have evaluated the effect of murine or human mesenchymal stem cell (MSC)-derived vesicles on marrow from irradiated mice or on the marrow cell line, FDC-P1, irradiated in vitro. B6.SJL mice were exposed to 0 or 100 cGy whole body irradiation and 7 days later marrow was harvested, lineage depleted and then cultured with or without the addition of murine MSC-derived vesicles for 48 hours. The cells were then engrafted into 200 cGy exposed C57BL/6J mice and engraftment was analyzed at 3, 12, 24 and 36 weeks post-transplant. Vesicle exposure led to a statistically significant increase in engraftment by the irradiated cells at 24 and 36 weeks with the average percent donor engraftment equal to 8.8 ± 4% and 9.6 ± 5% for the vesicle treated group compared to 3.7 ± 2% and 3 ± 2% for the non-vesicle treated groups at 24 weeks and 36 weeks, respectively (Figure 1). In a similar experiment, murine marrow was harvested 24 hours after 100 cGy whole body irradiation, and cultured with and without vesicles for 24 hours. There was a statistically significant increase in donor engraftment by the Lineage negative cells incubated with MSC-derived vesicles at 3 and 6 months (Figure 2). The increase in engraftment potential with vesicle exposure persisted in serial transplantation. These studies indicate that MSC-derived vesicles can reverse radiation damage to marrow when administered either 1 or 7 days after irradiation. The in vivo effect of murine MSC-derived vesicles on post-radiation blood cell count recovery was tested. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-derived vesicles were then injected intravenously 6, 24 and 72 hours after irradiation. WBC recovery compared to non- irradiated mice was 17% in the non-vesicle group and 37% in the vesicle- injected group at 14 days; the values at 28 days were 61 and 89%, respectively, indicating that vesicle injection enhanced leukocyte recovery following radiation damage. Marrow cells from the non-irradiated, the irradiated and the irradiated plus vesicle-treated mice were then analyzed for gene expression. Twelve genes were elevated in the irradiated cells and the elevations were reversed by vesicle exposure while 17 genes were depressed in the irradiated cells and the depressions reversed by vesicle exposure. Studies with the marrow cell line, FDC-P1, showed that apoptosis in irradiated cells was reversed by exposure to MSC-derived vesicles and growth of irradiated cells was restored. Restoration of growth by in vitro exposure to MSC-derived vesicles is shown in Figure 3. In summary, these data indicate that MSC-derived vesicles have the capacity to ameliorate radiation-induced damage to marrow and may represent an important biologic for use in radiation exposures.

Disclosures:

No relevant conflicts of interest to declare.
A Bi-Specific T Cell Engaging Monoclonal Antibody (mAb) Derived From a TCR-Like Mab Specific For WT1/HLA-A0201 (ESK-BiTE) Shows a Potent Activity Against Human AML and Ph+ ALL In Mouse Models
Authors: Pankov Dmitry , Dao Tao , Wang Yiyang , Scott Andrew , Korontsvit Tatyana , Zakhaleva Victoria , Veomett Nicholas , Curcio Michael , Doubrovina Ekaterina , O'Reilly Richard J. , Liu Cheng , Scheinberg David A.
Published: October 2019

The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density.

Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766.

Disclosures:

No relevant conflicts of interest to declare.
Loss Of BTG1 Function Promotes ETV6-RUNX1-Mediated Leukemic Transformation
Authors: Tijchon Esther J.H. , van Emst Liesbeth , Schenau Dorette van Ingen , van der Meer Laurens T. , Williams Owen , Hoogerbrugge Peter M. , Scheijen Blanca , van Leeuwen Frank N.
Published: October 2019

Background

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and is characterized by the acquisition of recurrent genetic aberrations, which include chromosomal translocations, submicroscopic deletions and point mutations. The t(12;21) ETV6-RUNX1 translocation is present in about 25% of all pediatric B-cell precursor ALL (BCP-ALL) cases and represents an early genetic event, which can already be detected in utero. However, ETV6-RUNX1 expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop.

Results

Using extensive genomic profiling of pediatric BCP-ALL we and others have demonstrated that several recurrent gene deletions can be found in ETV6-RUNX1-positive leukemia. These include focal deletions affecting the B-cell translocation gene 1 (BTG1), which is a member of the BTG/Tob family of anti-proliferation genes. BTG1 deletions are present in 9% of all BCP-ALL cases, but appear to be specifically enriched (20%) in ETV6-RUNX1-positive leukemia. BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors, including RUNX1 and ATF4.

To investigate whether loss of BTG1 function cooperates with ETV6-RUNX1 in leukemic transformation we developed an in vitro transformation assay. To this end, primitive fetal liver progenitors (FLPs) were purified as c-Kit+ Ter119- cells from fetal livers of embryonic day 13.5 (E13.5) of C57Bl6/J wild-type and Btg1-/- mice. After transduction with control pMSCV-IRES-GFP (pMIG) and pMIG-ETV6-RUNX1 retrovirus, cells were serially replated in methylcellulose or liquid culture in the presence of cytokines SCF, FLT3L and IL-7. We observed a proliferative growth advantage of ETV6-RUNX1 over control virus, and in BTG1-deficient FLPs as compared to wild-type FLPs. Notably, the proliferative advantage of BTG1-deficient FLPs was even further enhanced by co-expression of ETV6-RUNX1. By immunoprecipitation from FLPs, we could demonstrate that endogenous PRMT1 binds to ETV6-RUNX1, and this interaction is lost in BTG1-deficient FLPs, arguing that BTG1 is required for the interaction between PRMT1 and ETV6-RUNX1.

In summary, our data indicate that loss of BTG1 function promotes leukemic transformation induced by oncogenic fusion protein ETV6-RUNX1, which implies that BTG1 gene deletions can act as a cooperating event in ETV6-RUNX1-driven leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Oral Arsenic Trioxide - Based Maintenance Without Hematopoietic Stem Cell Transplantation At Second Remission In Acute Promyelocytic Leukemia - A Prospective Follow-Up Study Of 65 Patients
Authors: Gill Harinder , Au Wing-Yan , Kwong Yok Lam
Published: October 2019

Approximately 20 percent of patients with acute promyelocytic leukemia (APL) relapse after initial remission. More than 80 percent of these patients achieve a second complete remission (CR2). Autologous hematopoietic stem cell transplantation (HSCT) at CR2 is a widely accepted post-remission strategy. However, only a few prospective trials have examined the optimal post-remission therapy for relapsed APL.

In this study, we prospectively evaluated the outcome of 65 patients with APL in their first relapse (R1) treated with oral arsenic trioxide (As2O3) and chemotherapy-based re-induction followed by oral arsenic trioxide-based maintenance. Sixty-five patients (men=37; women=28) at a median age of 39 (3-76) years were treated with oral As2O3, all-trans retinoic acid (ATRA), and ascorbic acid combined with idarubicin (6mg/m2/day for 5 days) for re-induction. On achieving CR2, 2 cycles of idarubicin consolidation (6mg/m2/day for 2 days each cycle) were given. That was followed by oral As2O3-based maintenance (2 weeks every 2 months) for 2 years.

The median duration of follow-up was 90 (21–378) months. All evaluable patients achieved CR2. Twenty-one patients (32.8%) subsequently had a second relapse (R2). On multivariate analysis, male gender was associated with an increased risk of second relapse (P=0.01). The age, white blood cell (WBC) count, platelet count, peak WBC during reinduction and the occurrence of differentiation syndrome did not impact on the risk of second relapse. The median overall survival (OS) was not reached. The 5-year and 10-year OS were 82.2% and 74.4% respectively. Significantly inferior OS was associated with male gender (P=0.02), prior oral As2O3-based maintenance at CR1 (P=0.01) and central nervous system (CNS) involvement at relapse (P<0.001). The median leukemia-free survival (LFS) after CR2 was not reached. The 2-year and 5-year LFS were 72.4% and 64.1% respectively. Male gender and relapse from prior oral As2O3-based maintenance at CR1 were associated with worse LFS (P=0.01 and P=0.04 respectively).

In conclusion, our study showed that durable remissions and long-term survival were achieved with oral As2O3-based maintenance without the need of autologous HSCT at CR2.

Disclosures:

No relevant conflicts of interest to declare.
Decitabine Is More Cost Effective Than Standard Conventional Induction Therapy In Elderly Acute Myeloid Leukemia Patients
Authors: Batty Nicolas , Wiles Samuel , Kabalan Mathew , Sharma Rohit , Shatzel Joseph , Pang Jonathan , Yi David , Alatovic Iris , Saif Sana , Narasimha Deepika , LaPenna Joseph , Troitino Anthony , Attwood Kristopher , Yin Yong , Wetzler Meir
Published: October 2019

Introduction

Decitabine (Dec) is not approved in the United States (US) for acute myeloid leukemia (AML) because it did not improve overall survival compared with standard conventional induction treatment. We asked what would be the cost effectiveness of Dec versus conventional induction therapy in AML patients (pts) older than 60 years of age.

Methods

The standard conventional induction including cytarabine, and daunorubicin, (AD) (N Engl J Med. 2009 361:1235-48) was compared with Dec (Haematologica. 2012 97:393-401) using a semi-Markov model compiling survival and cost data. Survival probabilities were retrieved from the literature. Data accounted for re-induction therapy with IDA-FLAG (idarubicin, fludarabine, cytarabine and granulocyte colony-stimulating factor) and consolidation therapy with high-dose cytarabine (HiDAC) but not for stem cell transplantation. The assumption-based model considered a maximum of 4 cycles of HiDAC and continuing Dec until loss of benefit. Drug costs were derived from the 2012 US market. Hospital costs accrued were evaluated in a diagnosis-related group (DRG) system. Drug dosage was estimated based on a body surface area of 1.85 m2. The quality of life (QoL) was assumed as 1 for healthy individuals; 0 for death; 0.524 for active disease; 0.91 for AML in remission on AD; 0.71 and 0.524 for AML being actively treated with Dec or AD, and 0.81 for AML in remission treated with Dec or HIDAC. QoL data were based on the literature except for pts on consolidation therapy. The latter was the mean of QoL of AML in remission and AML actively being treated.

Results

Assuming 1,000 pts for each treatment arm in a semi-Markov model over 1 year time horizon, the quality-adjusted life year (QALY) for AD vs. Dec was 0.1754 and 0.5982. The percentage survival for AD and Dec was 45.2% and 50.5%. Their costs were $127,867 and $55,777. The incremental cost-effectiveness ratio (ICER) was -$72,090/0.4228 = -$170,506/year. By sensitivity analysis, Dec was superior to AD to all parameters (Table 1).

Conclusion

Dec is a more cost-effective therapy for pts older than 60 years of age than conventional induction therapy. Given the economic pressures in the US Health System, one should consider approving Dec for newly diagnosed AML pts older than 60 years of age.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis
Authors: Giallongo Cesarina , Parrinello Nunziatina , Tibullo Daniele , La Cava Piera , Romano Alessandra , Chiarenza Annalisa , Stagno Fabio , Vigneri Paolo G. , Palumbo Giuseppe A. , Raimondo Francesco Di
Published: October 2019

Introduction

In some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients.

Methods

MDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation.

Results

CML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05).

Conclusions

MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation Of Bcl2 Family Proteins Induced By JAK2V617F Mutation Contributes To The Abnormal Expansion Of Neoplastic Initiating Cells
Authors: Figueiredo-Pontes Lorena Lobo , Welner Robert S , Mishima Yuta , Yamamoto Mihoko , Mullally Ann , Tenen Daniel G. , Kobayashi Susumu
Published: October 2019

The treatment of myeloproliferative neoplasms (MPN) harboring JAK2V617F mutation remains non-curative with the current strategies despite the development of JAK2 inhibitors, which improve the symptoms of the disease but do not effectively reduce the allele burden of the mutation. In addition, it has been shown that, although JAK2 inhibitors can control the expansion of the myeloerythroid precursors that contribute to the clinical phenotype, they fail to eliminate the most primitive stem cells, which are responsible for the initiation of the disease. The differential expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. We have shown that JAK2 inhibition induces down-regulation of the anti-apoptotic protein Bcl-xL and up-regulation of the pro-apoptotic BH3-only protein, Bim, in human cells harboring the JAK2V617F mutation, suggesting a key role of these Bcl-2 family proteins as potential targets for JAK2-mediated apoptosis. Therefore, we aimed to study the contribution of the Bcl-2 family proteins to MPNs by the use of a conditional JAK2V617F knock-in model, particularly focusing on MPN initiating cells. The heterozygous Jak2V617F expressing animals (Jak2+/VF) develop a lethal MPN characterized by an elevated hematocrit and splenomegaly due to extramedullary hematopoiesis, with a prominent expansion of early (Lin- CD71+ Ter119+) and late (Lin- CD71- Ter119+) erythroid cells in the spleen, and increased pre-megakaryocyte-erythrocyte (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105- CD150+), megakaryocyte (Lin- Sca1- cKIThi CD41+ CD150+) and erythroid (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105+ CD150+) precursors in the bone marrow. HSC numbers (Lin- Sca1- cKIThi CD105+ CD150+) are also increased in Jak2+/VF as compared to the Jak2+/+ controls. Gene expression analysis revealed that Bcl-xL was upregulated in sorted LSKs (Lin- Sca-1+ cKIThi) containing HSCs but not in the more committed progenitors (CMP, GMP, MEP). By contrast, the pro-apoptotic Bim was downregulated in CMPs and MEPs but not at the LSK cells containing HSCs. Expression of Bcl-2 showed no difference between Jak2+/VF and Jak2+/+ mice. These data suggest that signals from JAK2V617F mutation may be cell-stage specific, which may explain why JAK2 inhibitors fail to eliminate MPN initiating cells. Modulating the overexpression of Bcl2 antiapoptotic proteins at the MPN initiating cells level could potentially render these cells sensitive to the JAK2 inhibition.

Disclosures:

No relevant conflicts of interest to declare.
Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown
Authors: Sun Zejin , Enzor Rikki , Rio Paula , Clapp D. Wade , Hanenberg Helmut
Published: October 2019

Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA.

Disclosures:

No relevant conflicts of interest to declare.
Smac Mimetic-Mediated Sensitization Of Childhood ALL Cells For Drug-Induced Apoptosis Independent Of TNFα and NFκB Signaling Pathways
Authors: Schmelz Karin , Weichert Nina , Proba Jutta , Erdmann Marie-Sophie , Hundsdoerfer Patrick
Published: October 2019

Targeting inhibitor of apoptosis proteins (IAPs) using small molecular Smac mimetics (SM) has been shown to offer a novel promising treatment strategy for resistant malignant diseases including childhood acute lymphoblastic leukemia (ALL). The effect of SM alone has been shown to be associated with endogenous TNFα expression, therefore tumor cells can be classified into sensitive or resistant against apoptosis induction by SM alone. In SM sensitive tumor cells the effect of SM has been shown to be mediated mainly by degradation of cellular IAP (cIAP) and activation of TNFα and NFκB signaling pathways but not inhibition of XIAP.

We show here, that sensitivity of ALL cells to SM alone (as well as TNFα expression) is highly variable. Nevertheless even in ALL cells resistant against SM alone, treatment with SM resulted in significant sensitization for drugs used within standard induction therapy for childhood ALL. Sensitization for drug-induced apoptosis by SM was not only mediated by activation of the intrinsic (cleavage of caspase 9) but also extrinsic apoptosis pathway (cleavage of caspase 8). Surprisingly, SM-induced cIAP degradation alone was not sufficient for caspase 8 activation and apoptosis induction. Consistently, SM-mediated sensitization for drug-induced apoptosis was independent of TNFα and NFκB signaling pathways. We demonstrate that caspase 8 activation by combined treatment with SM and cytostatic drugs is blocked by inhibition of caspase 3 and caspase 9 and therefore occurs downstream of intrinsic apoptosis pathway activation.

In conclusion, our data argue for a model comprising inhibition of XIAP-mediated blockade of caspase 3/9 as the central effect of SM in chemo-sensitization of childhood ALL cells resistant against SM-alone.

Disclosures:

No relevant conflicts of interest to declare.
Impact Of Allogeneic Stem Cell Transplantation On The Health and Economic Burden Of Sickle Cell Disease In Children
Authors: Dalal Jignesh , Lee Brian R , Hall Matt , Woods Gerald M , Kalpatthi Ram
Published: October 2019

Background

Hospitalization is the major driver of health care utilization and costs in sickle cell disease (SCD) [Kauf et al. Am J Hematol 2009]. Allogeneic stem cell transplantation (alloSCT) is the only curative therapy available for sickle cell disease. However, it is a highly specialized, resource intense and expensive medical procedure. The objective of our study is to analyze the impact of alloSCT on the hospitalization patterns and resource utilization in children with SCD.

Methods

We used the Pediatric Health Information System (PHIS), a database of clinical and financial data from free-standing children's hospitals in the US. Using ICD-9 procedure and diagnosis codes, patients under the age of 21 who underwent alloSCT at one of the 26 PHIS hospitals from 2000-2011 were identified. We abstracted data on hospitalizations, alloSCT, resource utilization and costs. Pre and post alloSCT hospitalizations were calculated from day 0 (actual alloSCT procedure date) and does not include initial transplant hospitalization. The PHIS database provides an encrypted patient medical record number, thus we were able to follow patients over time. This allowed for a better visualization of the patient's hospitalizations trend over 11 years.

Results

From 2000 to 2011, 186 unique pediatric patients with SCD who underwent alloSCT were identified. The median hospital length of stay (LOS) and the median total hospital charges for the initial BMT hospitalization were 38 days (31, 51) and $143,000 ($109,000, $123,000) respectively. The mean hospitalization rate was significantly higher one year after alloSCT compared to one year before the transplantation (5.8 vs. 3.1 respectively, p=0.01). However, when we expanded the analysis over 2 years, there was no significant difference in the hospitalizations between the pre and post alloSCT groups (4.8 vs. 6.8 respectively, p=0.2). Similarly, the median total hospital charges per patient were significantly higher 1 year post-alloSCT compared to one year pre-alloSCT (Table 1) whereas it was not significant when analyzed 2 years pre/post-alloSCT (Table 2). In addition, both the hospitalization rate and costs decrease consistently for several years post alloSCT suggesting a long term benefit (Figure 1A & 1B).

Conclusions

AlloSCT for SCD increases the hospitalizations and costs in the first post transplantation year. This is consistent with the previous report of increased hospitalizations in the immediate post-transplant period of alloSCT for all pediatric disorders (Shulman et al. Pediatr Blood Cancer 2013). However, over the long term, alloSCT likely improves the quality of life (by decreasing the hospitalization rate) and economic burden of pediatric sickle cell population. Further studies associating various clinical morbidities (pre & post-transplant) and healthcare utilization are needed.

Disclosures:

No relevant conflicts of interest to declare.