bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloma and the Microenvironment: Macrophages Are a Protector of Myeloma Cells Apoptosis Induced by Chemotherapy Drugs.
Authors: Yang Jing , Zheng Yuhuan , Cai Zhen , Qian Jianfei , Hong Sungyoul , Li Haiyan , Matthews Jairo A. , Wang Michael , Yi Qing
Published: October 2019

Abstract 4804

Multiple myeloma is a B-cell malignancy characterized by the proliferation of plasma cells in the bone marrow. It is the second most common hematological malignancy and is still largely incurable. One of the major problems is that myeloma cells develop drug resistance upon interaction with bone marrow stromal cells. To better understand the importance of different stromal cell components in the bone marrow microenvironment, we examined the effects of macrophages on myeloma cell survival and myeloma cell response to chemotherapy. We report here that macrophages, in particular tumor-associated macrophages obtained by culturing macrophages with myeloma cell culture supernatants, are a protector of myeloma cells. Macrophages protected both myeloma cell lines and primary myeloma cells isolated from patients from spontaneous and chemotherapy drug-induced apoptosis via attenuating the activation of caspase-dependent apoptotic signaling. The protective effect was dependent on direct contact between macrophages and myeloma cells. Although tumor-associated macrophages secreted large amounts of IL-6, which is the most important survival factor for myeloma cells, our results showed that IL-6 neutralizing antibodies fail to significantly affect the protective effects of tumor-associated macrophages. The reduced numbers of apoptotic tumor cells in the cocultures were not the result of macrophage-uptake of apoptotic cells, because macrophages with or without the ability to phagocytose apoptotic cells provide similar protection to myeloma cells against chemotherapy-induced apoptosis. These findings are clinically relevant, because we examined bone marrow biopsies of patients by immunochemical analysis and found that CD68+ macrophages are heavily infiltrated in the bone marrow (tumor bed) of patients with myeloma but not control patients. Thus, our results indicate that macrophages are an important component of the bone marrow stromal cells and may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo.

Disclosures:

No relevant conflicts of interest to declare.
LACK of IRON Binding by Pixantrone IS ASSOCIATED with Reduced PRODUCTION of Reactive Oxygen SPECIES and Myoctye Cytotoxicity IN VITRO.
Authors: Hacker Miles , McKennon Marc , Singer Jack W.
Published: October 2019

Abstract 4806

Introduction

Pixantrone (PIX), an aza-anthracenedione, which has successfully completed a phase 3 trial (J Clin Oncol 2009; 27:15s, No. 8523) was designed to enhance clinical efficacy while significantly decreasing cardiotoxicity compared to doxorubicin (DOX) and mitoxantrone (MIT). Multidose administration, in animal models of equitoxic doses of PIX, MIT, and DOX, with or without prior therapy with DOX, resulted in minimal evidence for PIX cardiotoxicity compared with the severe histologic lesions seen with these other agents (Cavaletti et al: Investigational New Drugs 2007; 3:187-95). Both DOX and MIT contain a dihydroquinone structural element known to interact with iron. Additionally, DOX contains an aliphatic ketone which, once metabolized to the corresponding secondary alcohol metabolite doxorubicinol, is implicated in release of free iron and the chronic cardiotoxicity observed with DOX. In contrast, PIX has a nitrogen containing heterocycle which replaces the dihydroquinone, forming an aza-anthracenedione structure. PIX also does not contain an aliphatic ketone and cannot form metabolites analogous to doxorubicinol.

Methods

To validate the proposed mechanisms underlying the observed differences in cardiotoxicity, we used established spectrophotometric techniques to quantify iron:drug interactions that are thought to be mechanistic for chronic doxorubicin cardiotoxicity (Menna et al: Cardiovasc Toxicol 2007; 7:80–85).

Results

Adding increasing amounts of iron to drug solution, we observed that DOX and MIT underwent changes in visible range absorbance patterns, characteristic of drug:iron complex formation, confirming the expected 1:3 Fe(II)-drug ratio for both DOX and MIT. In contrast, no spectrophotometric changes were observed with iron added to PIX, clearly demonstrating that PIX does not bind iron. In vitro studies using H2C9 rat myocardial cells indicate that PIX (ID50 >50 μg/ml) is far less toxic than DOX (ID50= 1 μ/ml). Moreover, PIX does not induce significant reactive oxygen species (ROS) production in the H2C9 cells compared to DOX.

Conclusion

These results demonstrate that PIX does not bind iron and that its inability to bind iron and its reduced propensity to generate ROS may be the mechanism for reduced PIX cardiotoxicity in animal models compared to DOX or MIT.

Disclosures:

McKennon: Cell Therapeutics, Inc: Employment. Singer:Cell Therapeutics, Inc: Employment.
Selective Protein Kinase C β Inhibition Induces Apoptosis and Arrests Cell Cycle in AIDS-Related Non-Hodgkin Lymphoma Cell Lines.
Authors: Saba Nakhle S , Safah Hana F , Levy Laura S
Published: October 2019

Abstract 4807

AIDS-related Non-Hodgkin Lymphoma (AIDS-NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate and resistance to chemotherapy. In the present era of target-specific therapy, PKCβ targeting showed promising results in preclinical studies, as well as phase II clinical trials involving a wide variety of cancers. Studies describing the role of PKCβ in AIDS-NHL are primitive if not lacking, and HIV infection has been considered an exclusion criterion in clinical studies involving PKCβ inhibition in NHL patients. In the present study, we measured the sensitivity of a variety of AIDS-NHL cell lines to PKCβ inhibition. Three cell lines were studied: 2F7 (AIDS-Burkitt's Lymphoma), BCBL-1 (AIDS-Primary Effusion Lymphoma) and UMCL01-101 (AIDS-Diffuse Large B Cell Lymphoma). Cells were tested for PKCβ1 and PKCβ2 expression by immunoblot. Cell viability was measured in the presence of a PKCβ-specific inhibitor at concentrations of 5, 10, 20 and 30 μM for 48 hours in the presence of 10% fetal bovine serum (FBS). The IC50 at 48 hours was determined for each cell line, and cells were cultured in the presence of the correspondent IC50 for 24 and 72 hours. MTS assay was performed to quantify cell viability, and TUNEL assay with propidium iodide staining was used to detect apoptotic induction and effect on cell cycle. Results showed that 2F7 and UMCL01-101 cell lines express PKCβ1 and PKCβ2, while BCBL-1 expresses only PKCβ1 and lacks PKCβ2 expression. 2F7 and BCBL-1 were sensitive to PKCβ inhibitor with IC50 of 14 and 15 μM of inhibitor, respectively. UMCL01-101 showed relative resistance to PKCβ inhibition with an IC50 of 28 μM. Incubation in the presence of the correspondent IC50 induced significant apoptosis in all cell lines starting after 2 hours of treatment; UMCL01-101 cell line was not significantly affected when incubated in the presence of 14 μM. Our results also showed cell cycle inhibition in 2F7 and BCBL-1 starting respectively after 2 and 8 hours of incubation with the correspondent IC50; UMCL01-101 showed no features of cell cycle inhibition when cultured in the presence of high concentration (IC50) or low concentration (14 μM) of the inhibitor. BCBL-1 cell line findings were unexpected in the absence of PKCβ2 expression and implicate PKCβ1 as a regulator in these cells. These results indicate that PKCβ plays an important role in AIDS-related NHL survival, and suggest that PKCβ targeting should be considered in a broader spectrum of NHL. Ongoing studies will detail the mechanism of PKCβ inhibition and uncover the underlying mechanism of resistance in PKCβ expressing UMCL01-101 cells. This mechanism may be considered for exclusion from treatment.

Disclosures:

No relevant conflicts of interest to declare.
L-Gossypol Inhibits NF- κB, Down Regulates Mcl-1 and Induces Apoptosis of Primary Acute Myeloid Leukaemia Cells.
Authors: Pepper Chris J , Osman Hani Y , Hewamana Saman , Walsby Elisabeth J , Burnett Alan K , Knapper Steven
Published: October 2019

Abstract 4813

Standard treatments for acute myeloid leukaemia (AML) result in a median survival of approximately 1 year. There is now a realisation that in order to significantly improve outcomes in this disease more targeted therapies that take account of the specific biology of the tumour cell are required. L-Gossypol is a polyphenolic oil cotton seed extract that has anti-tumour activity against a range of haematological malignancies but has never been evaluated in AML cells. It is known to act as a BH3-mimetic, binding to the BH3 pocket of anti-apoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge of the molecular events that underpin its downstream effects is limited. In this study we analysed the in vitro effects of L-Gossypol in 50 primary AML samples in order to determine its efficacy and mode of action. Apoptosis was induced in all the samples tested in a dose- and time-dependent manner as evidenced by increased Annexin V / propidium iodide labelling and the activation of caspase-9 and caspase-3. The median LD50 value (the concentration of drug required to kill 50% of the cells) was 27.5μM ± 18.3μM. There was no association between LD50 and age, sex, presenting white cell count, FLT3 mutation status or karyotype. Mechanistically, L-gossypol decreased the DNA binding activity of the NF-κB subunit, Rel A, in a concentration-dependent manner; this inhibition was evident after only 4 hours and preceded the induction of apoptosis. Furthermore, treatment with L-Gossypol inhibited the transcription of the NF-κB-regulated genes CFLAR, BCL2, BIRC5 and MCL1 in the same timeframe. Finally, studies of Mcl-1 protein expression showed down regulation in a dose-dependent manner but this was only apparent after 8 hours exposure to L-Gossypol. Taken together, our data demonstrate that L-Gossypol works, at least in part, through the inhibition of NF-κB and our data provides a rationale for clinical investigations of this agent in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.
Authors: Sun Zejin , Li Yan , Li Jingling , Cai Shanbao , Zeng Yi , Lindemann Dirk , Pollok Karen E , Hanenberg Helmut , Clapp D. Wade
Published: October 2019

Abstract 696

Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited.

In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
C-Myc, but Not Akt, Can Substitute for Notch in Lymphomagenesis.
Authors: Chiang Mark Y , Childs M. Eden , Romany Candice , Shestova Olga , Aster Jon , Pear Warren S.
Published: October 2019

Abstract 7

Notch signaling is activated in ∼70% of human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) samples and many human and mouse T-ALL cell lines require Notch signals for growth and survival. To gain insight into the role of Notch during induction of T-ALL, we used a fully penetrant, conditional, transgenic KrasG12D mouse model in which ∼80% of T-ALLs acquire activating Notch1 mutations in the endogenous locus. We crossed mice bearing this transgene with Rosa26-DNMAMLf/f mice, which conditionally express the pan-Notch inhibitor DNMAML. T-ALL developed in these mice despite the expression of DNMAML throughout T-cell development. ∼75% of T-ALL tumors acquired activating Notch1 mutations and suppressed expression of DNMAML, which is consistent with frequent “escape” of Notch from inhibition for efficient T-ALL development. We next compared T-ALL cells that lacked DNMAML expression with T-ALL cells that continued to express DNMAML. T-ALL cells lacking DNMAML expressed the direct Notch target c-Myc at higher levels, proliferated at a higher rate, and contained ∼10-fold higher levels of leukemia-initiating cells. Moreover, DNMAML-positive T-ALLs lost DNMAML after transfer into secondary recipients. These data underscore the strong selective pressure for Notch signals during generation and maintenance of T-ALL. We next sought a mechanistic answer for the strong selective pressure for Notch activation. c-Myc and Akt have both been posited to be critical targets of oncogenic Notch signals. To compare the relative contributions of c-Myc and Akt to lymphomagenesis, we overexpressed c-Myc and activated AKT in the KrasG12D-driven mouse model. T-ALLs induced by KrasG12D and Akt acquired activating Notch1 mutations in ∼70% of tumors, which were sensitive to Notch inhibitors (gamma-secretase inhibitors [GSI]). In contrast, T-ALLs induced by KrasG12D and c-Myc did not acquire Notch1 mutations and were resistant to GSI. We conclude that upregulation of c-Myc is sufficient to substitute for Notch in lymphomagenesis, whereas activation of Akt signaling is not. These data identify c-Myc not AKT as the driving force behind Notch-induced lymphomagenesis. These data emphasize the Notch/c-Myc axis as an attractive, rational, therapeutic target in T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Hodgkin Lymphoma in Adolescents Treated with Adult Protocols: An Analysis From the German Hodgkin Study Group (GHSG).
Authors: Eichenauer Dennis A. , Bredenfeld Henning , Franklin Jeremy , Haverkamp Heinz , Mueller Horst , Eich Hans T. , Mueller Rolf-Peter , Fuchs Michael , Borchmann Peter , Diehl Volker , Engert Andreas
Published: October 2019

Abstract 719

Introduction:

The standard of care for adolescent Hodgkin lymphoma (HL) patients is undefined, particularly the choice between adult and pediatric protocols. We thus analyzed and compared risk factors, outcome and incidence of secondary malignancies in adolescents and young adults treated within study protocols of the German Hodgkin Study Group (GHSG) to evaluate whether adolescents represent a distinct patient group.

Patients and Methods:

3785 patients of all stages treated within the second (HD4-HD6, 1988-1993) and third (HD7-HD9, 1993-1998) trial generation of the GHSG were retrospectively analyzed. 557 patients were adolescents aged 15 to 20 and 3228 patients were young adults aged 21 to 45. Treatment consisted of chemo- and/or radiotherapy.

Results:

The risk factors large mediastinal mass (more than 1/3 of the maximum intrathoracic diameter) and involvement of three or more lymph node areas were more common in adolescents (30.2% vs. 20.9% and 67.7% vs. 58.7%, respectively, p<.001). The incidence of other risk factors did not differ significantly between age groups. With a median follow-up of 81 months for freedom from treatment failure (FFTF) and 85 months for overall survival (OS), FFTF rates in both groups were comparable (p=.305) while adolescents had a superior OS (p=.008). 6-year FFTF estimates were 80.2% and 79.7%, 6-year OS estimates were 93.6% and 90.9% in adolescents and young adults, respectively. Young adults had a higher risk to develop secondary malignancies. Secondary malignancy rates were 1.6% (9 of 557) in adolescents and 3.1% (101 of 3228) in young adults with secondary hematological malignancies (AML/MDS, NHL) representing the majority of cases in both groups (6/9 in adolescents and 55/101 in young adults). Since adolescents differ from young adults in terms of cancer risk in the general population, standardized incidence ratio (SIR) and absolute excess risk (AER) per 100.000 person-years were calculated. The SIR was higher in adolescents (5.56 vs. 4.58) while the AER was higher in young adults (394 vs. 194). However, differences between age groups were not significant for both.

Conclusions:

There are only slight differences between adolescents and young adults regarding risk factors, outcome and incidence of secondary malignancies. Therefore, treatment with adult protocols can be considered effective and safe in adolescent HL patients.

Disclosures:

No relevant conflicts of interest to declare.
Structural Basis for MLL Cxxc Domain Protection against CpG DNA Methylation and the Essential Role of This Function in MLL-AF9 Leukemia.
Authors: Risner Laurie , Cierpicki Tomasz , Grembecka Jolanta , Lukasik Stephen M , Popovic Relja , Shultis David S , Zeleznik-Le Nancy J. , Bushweller John H.
Published: October 2019

Abstract 763

MLL is the target of chromosomal translocations which cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. We present the solution structure of the MLL CXXC domain in complex with DNA, showing for the first time how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. Based on the structure, we designed point mutations which disrupt DNA binding. Introduction of these mutations into MLL-AF9 results in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9 locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also validate this interaction as a potential target for therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
Novel PDGFRA Point Mutations in Hypereosinophilic Syndrome Induce Growth Factor Independence and Leukemia in Vitro and In Vivo.
Authors: Elling Christian , Erben Philipp , Walz Christoph , Frickenhaus Marie , Schemionek Mirle , Stehling Martin , Serve Hubert , Cross Nick C.P. , Berdel Wolfgang , Mueller-Tidow Carsten , Reiter Andreas , Koschmieder Steffen
Published: October 2019

Abstract 767

Considerable progress has been achieved in our understanding of the pathogenesis of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) by identification of constitutively activated tyrosine kinase fusion genes, e.g. FIP1L1-PDGFRA or ETV6-PDGFRB. However, the overall incidence of those fusion genes in HES/CEL is below 15%, and the molecular pathogenesis of the remaining cases remains elusive. We therefore established generic quantitative RT-PCR assays (RQ-PCR) to detect overexpression of 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion gene or point mutation. Patients with known fusion genes involving PDGFRA (n=5, 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 vs. 0.0066 vs. 0.0064, p<0.0001; PDGFRB/ABL: 196 vs. 3.8 vs. 5.85, p<0.0001). In all patients with significantly increased expression levels who were negative for fusion genes, functionally relevant regions of PDGFRA were sequenced. Several novel mutations (R481G, I562M, H570R, M628T, L705P, G729D) as well as a double mutation (H650Q and R748G) were identified. When cloned into 32D cells, M628T, H650Q, and R748G mutants separately induced growth factor-independent proliferation and clonogenic growth, and this was associated with constitutive phosphorylation of downstream targets STAT5, ERK, and AKT. Low doses of imatinib antagonized all of these effects in vitro. M628T and R748G but not H650Q 32D cell mutants induced acute leukemia after injection into congenic C3H/HeJ mice, similar to FIP1L1-PDGFRA. Interestingly, these two mutants showed a significantly higher propensity to invade the lymph nodes than the FIP1L1-PDGFRA fusion. Oral administration of imatinib to injected mice significantly decreased leukemic growth in vivo and significantly prolonged survival of the recipients. In conclusion, we demonstrate that novel point mutations of the PDGFRA gene found in patients with HES/CEL induce growth factor independence and leukemia in vitro and in vivo and suggest that these patients may benefit from treatment with imatinib.

*CE, PE, AR, and SK contributed equally to this work.

Disclosures:

No relevant conflicts of interest to declare.
Impaired Expression of p66Shc, a Novel Regulator of B-Cell Survival, in Chronic Lymphocytic Leukemia.
Authors: Baldari Cosima T. , Capitani Nagaja , Lucherini Orso Maria , Sozzi Elisa , Ferro Micol , Giommoni Nico , Finetti Francesca , De Falco Giulia , Cencini Emanuele , Pelicci Pier Guiseppe , Lauria Francesco , Forconi Francesco
Published: October 2019

Abstract 801

Intrinsic defects in the apoptotic circuitry underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL) and are moreover believed to be responsible for their resistance to chemotherapy. We have recently demonstrated that p66Shc, a member of Shc family of protein adapters, acts as a promoter of apoptosis in T cells. Here we show that p66Shc uncouples the B-cell antigen receptor (BCR) from the Erk and Akt dependent survival pathways, thereby enhancing B-cell apoptosis. Expression of p66Shc was found to be profoundly and consistently impaired in CLL B cells compared to peripheral blood B cells form healthy donors. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, classified on the basis of the mutational status of the IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes previously implicated in the apoptosis defects of CLL B cells revealed a selective alteration in the balance of pro- and anti-apoptotic members of the Bcl-2 family in these patients. Reconstitution experiments in CLL B cells, as well as data obtained on B cells from p66Shc-/- mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family towards the pro-apoptotic members. Collectively, the data identify p66Shc as a novel regulator of B cell apoptosis which attenuates survival signals emanating from the BCR and modulates expression of the Bcl-2 family. They moreover provide evidence that the defect in p66Shc expression identified in CLL B cells may be causally related to the imbalance towards the anti-apoptotic Bcl-2 family members observed in these cells.

Disclosures:

No relevant conflicts of interest to declare.
Polymorphisms in the CYP450 Genes Influences Regimen Related Toxicity and Outcome in Patients with Beta Thalassaemia Major Undergoing HSCT.
Authors: Balasubramanian Poonkuzhali , Desire Salamun , Mathews Vikram , Lakshmi Kavitha M , Velayudhan Shaji R , George Biju , Viswabandya Auro , Chandy Mammen , Srivastava Alok
Published: October 2019

Abstract 869

Polymorphisms in drug metabolizing enzymes are known to contribute to inter-individual differences in the pharmacokinetics (PK) of the two most commonly used drugs for conditioning for hematopoietic stem cell transplantation (HSCT), busulfan (Bu) and cyclophosphamide (Cy) and their metabolites in plasma. We have previously reported the impact of CYP genes on the PK of Cy, [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 99] and the influence of Cy PK on transplant outcome [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 1820]. We have now extended this study to evaluate a total of 19 polymorphisms in 11 genes that are known to be involved in the metabolism of Bu and Cy. 180 of the 276 patients with thalassemia major who underwent HSCT between March 1991 and Dec 2008 and for who genomic DNA was available were included in the study. The following polymorphisms were screened using PCR followed by RFLP and/ or gel electrophoresis: GSTA1*B, GSTM1 and GSTT1 deletion, GSTP1*B, CYP2B6*2, *3, *4, *5 and *6, CYP2C9*2, *3 and *4, CYP2C19*2, *3, CYP3A4*1B, CYP3A5*3, *6 and ALDH1A1*2 and ALDH3A1*2. Polymorphism frequencies were associated with regimen related toxicities, other transplant related complications using Fischer's Exact test and Cox-proportional hazard's model.. Significant associations are shown in the Table. On univariate analysis, CYP2B6*4 variant genotype was associated with incidence of hemorrhagic cystitis (HC); CYP2C9*3 variant genotype was associated with the severity of HC; CYP2C19*3 and 2C9*2 genotypes were associated with overall and even-free survival (OS and EFS) and CYP2C9*2 and CYP2C9*3 genotype was associated with transplant related mortality (TRM). Multivariate analyses performed adjusting for known clinical risk factors still showed these genotypes to be significantly associated with outcome parameters. Variant genotypes of polymorphisms that result in decreased metabolism of Cy are protective against regimen related toxicities while these polymorphisms were risk factors for EFS and OS in the present study. This is the first report on the influence of common GST, CYP and ALDH polymorphisms on outcome of HSCT in patients with thalassaemia major. Screening for these polymorphisms in patients with beta thalassaemia undergoing HSCT can help identify patients at higher risk of complications.Table:EndpointGenotypeRelative risk (95% CI)P- value HCCYP2B6*4 variant0.3 (0.13-0.889)0.028 HC grade 1 vs. HC grade 2-4CYP2C9*3 variant0.2 (0.073-0.962)0.043 TRM2C9*2 variant2.7 (1.08-6.77)0.034 2C9*3 variant2.3 (1.0-5.7)0.049 OS and EFS2C19*3 variant3.3 (1.2-9.3)0.018 2C9*2 variant1.3 (0.93-2.03)0.070

Disclosures:

No relevant conflicts of interest to declare.
Racial Differences in the Presentation and Outcomes of Diffuse Large B-Cell Lymphoma in the United States.
Authors: Malik Neha , Shenoy Pareen J. , Bumpers Kevin , Sinha Rajni , Flowers Christopher R.
Published: October 2019

Abstract 898

Background:

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), and can often be cured with standard chemoimmunotherapy, but there is great heterogeneity in patient outcomes.

Methods:

To examine the baseline characteristics and outcomes in a national cohort of patients(pts) with DLBCL we performed a retrospective cohort analysis of DLBCL cases diagnosed from 1992 to 2005 in 13 Surveillance, Epidemiology and End Results (SEER) registries. International Classification of Diseases for Oncology histology codes 9680, 9684, 9697 were used to identify cases. Cases with known race were grouped into White (W), Black (B), and others (O; Asians or Pacific Islanders, and American Indians or Alaska Natives). Subtype-specific DLBCL incidence rates were calculated for population subgroups according to age, sex, race, registry and time period, expressed as new cases per 100,000 population and age-adjusted to the 2000 US standard population. Incidence rates were compared by sex and by race by computing incidence rate ratios (IRR) and 95% Confidence Intervals (95% CI). Two- and 5-year relative survival rates (RSR) were calculated by actuarial methods.

Results:

Between 1992 and 2005, 34,436 cases of DLBCL were recorded in SEER. Over this period, the incidence of imunoblastic DLBCL decreased from 1.7 to 0.1 cases per 100,000 population while the incidence of DLBCL as a whole remained stable. Incidence rates were higher among W than among B or O pts with DLBCL (IRR W:B 1.52, 95% CI 1.45-1.59, W:O 1.26, 95% CI 1.21-1.31) and among those with immunoblastic DLBCL but not thymic DLBCL. 65% of B pts compared to 37% of W pts presented at age ≤60 years (p<0.001). Among pts with complete staging (n=5,285), 54% of B compared with 47% of W and 41% of O pts presented with stage III/IV disease (W vs. B p=0.05, W vs. O p=0.002). There were no differences in 2-yr RSR by gender. 2-yr RSR rates were 53% for W, 49% for O, and 46% for B pts (W vs. B p=0.02, W vs. O p=0.09), and improved from 1992 to 2001 for all racial groups. 5-yr RSR rates varied by race (46% for W, 47% for O, and 38% for B pts; (W vs. B p=0.02, W vs. O p=0.09) and gender (47% for females and 43% for males; p=0.03) and remained stable from 1992 to 1997.

Conclusions:

These data indicate that B pts present with DLBCL at a younger age, more advanced stage, and have inferior 2-yr and 5-yr RSR. Given the known differences in outcomes for activated B-cell (ABC) and germinal center B-cell DLBCL subtypes, to address these differences in survival it will be necessary for future studies investigating racial disparities in treatment outcomes to ascertain whether there are racial differences in the incidence of ABC DLBCL.

Disclosures:

Flowers: Amos Medical Faculty Development Program grant from the American Society of Hematology/Robert Wood Johnson Foundation: Research Funding.
Cranial Irradiation Does Not Result in Pituitary-Gonadal Axis Dysfunction in Very Long-Term Male Survivors of Childhood Acute Lymphoblastic Leukemia.
Authors: van Casteren Niels J. , Pieters Rob , Dohle Gert , van Baalen Manita , Neggers Sebastian , Van Den Heuvel M.M.
Published: October 2019

Abstract 989

Poster Board I-11

Introduction:

One of the risks of childhood cancer treatment is fertility impairment later in life. In the past a large proportion of children with acute lymphoblastic leukemia (ALL) has received cranial irradiation as part of their treatment. The aim of this study was to evaluate whether cranial irradiation negatively affects pituitary regulated gonadal function in male survivors of childhood ALL.

Patients and Methods:

We examined gonadal function, including Inhibin B, LH, FSH, testosterone, and pituitary axis function by measuring TSH, Free-T4 and IGF-I levels in 89 long-term male survivors of childhood ALL after a median follow-up time of 19 year (range 7-34 years).

Results:

Twenty-nine out of 89 male ALL survivors received cranial irradiation. Inhibin, FSH, LH, Testosterone, testicular volume as well as TSH and Free-T4 levels were not different in the cranial irradiated group as compared to the non-irradiated group (table 1). In contrast, IGF-I levels were significantly lower in the cranial irradiated group. Survivors treated with total body irradiation or testicular irradiation had significantly decreased gonadal function based on hormone levels.

Conclusions:

These data show that, in contrast to the negative influence on the growth hormone axis, cranial radiotherapy as part of ALL treatment does not have a deleterious long-term effect on the hypothalamic–pituitary-gonadal axis or pituitary-thyroid axis.

Disclosures:

No relevant conflicts of interest to declare.
The Influence of Hemophilia on Wound Healing.
Authors: Hoffman Maureane
Published: October 2019

Abstract SCI-21

Hemostasis is considered to be the first phase of wound healing, followed by inflammation, proliferation and remodeling of the reconstituted tissue. However, very little has been written on the overall effect of impaired hemostatic function on wound healing. Our group has studied the effects of hemophilia B and some other coagulation impairments on healing in a mouse skin punch biopsy model. In this model, a defect of the epidermis and dermis must be replaced. Normally a platelet and fibrin clot fills the defect and forms a framework for the repair process. In addition, growth factors and cytokines are released during hemostasis that influence cellular activities necessary for healing. However, hemophilia and other hemostatic defects delay healing and alter the histology of the process. In our punch biopsy model, the epidermal defect is closed in 8 days in wild type mice, and 10 days in hemophilia B (HB) mice. Histologically, the influx of neutrophils and monocytes is delayed and reduced in HB. This finding supports a key role for thrombin and/or fibrin in mediating the inflammatory response. While the ingrowth of fibroblasts and endothelial sprouts (angiogenesis) appears normal in tempo and histology in HB mice, there are greater numbers of new vessels in the granulation tissue iand much greater microvascular bleeding in HB mice. In addition, hematomas are found adjacent to the vessels from which angiogenic vessels sprout. While reconsitution of thrombin generation at the time of wounding restored clot structure and the initial inflammatory influx, it did not prevent later bleeding into the granulation tissue and hematoma formation. In the mouse model, 5 days of hemostatic therapy (replacement or bypassing) were needed to restore normal healing. Thus, we conclude that adequate hemostatic function is required for an extended period of time to assure normal wound healing. Angiogenesis during healing predisposes to bleeding which can lead to a cycle of inflammation, enhanced angiogenesis and recurrent bleeding. A better understanding of the roles played by hemostasis in the subsequent response to injury holds the promise of improving the management of patients with hemophilia, as well as improving tissue recovery after trauma or surgery.

Disclosures

No relevant conflicts of interest to declare.
New Insights into the Biology of Toll-Like Receptors and Inflammasomes.
Authors: Latz Eicke
Published: October 2019

Abstract SCI-23

Innate immunity evolved to recognize microbial infection and to respond to danger signals that appear under disease conditions. The most recently described innate immune receptor family is the Nod-like receptor (NLR) family. The inflammatory cytokines IL-1b and IL-18 are released only upon cleavage by the inflammatory caspase-1. Caspase-1 activity itself is controlled by a cytosolic signaling complex consisting of the NLR member NALP3 and the adapter protein ASC. After activation NALP3, ASC and caspase-1 form a multi-molecular complex termed the NALP3 inflammasome. The NALP3 inflammasome is activated in response to various membrane active bacterial toxins (e.g. nigericin, maitotoxin or gramicidin) or by incubation with crystalline materials (e.g. silica, asbestos, monosodium urate or alum). The mechanisms by which the NALP3 inflammasome is activated by physico-chemical diverse activators are not well understood. We demonstrate that crystals activate the NALP3 inflammasome in a process that requires phagocytosis and crystal uptake leads to lysosomal damage and rupture. Furthermore, sterile lysosomal damage is also sufficient to induce NALP3 activation and inhibition of phagosomal acidification or inhibition or lack of cathepsins impairs NALP3 activation. These results indicate that the NALP3 inflammasome can sense lysosomal damage induced by various means as an endogenous danger signal. Our results demonstrate a novel strategy of immune cells to recognize different classes of stimuli by a common, indirect mechanism. Cytosolic DNA can activate a NALP3 independent yet ASC dependent inflammasome. AIM2, a member of the PYHIN protein family, has a pyrin domain and a HIN200 DNA binding domain. We discovered that AIM2 binds to dsDNA and forms an inflammasome with ASC leading to caspase 1 activation. These pathways are promising new targets for pharmacological inhibitors with broad clinical significance.

Disclosures

No relevant conflicts of interest to declare.
Role of Myeloid and Other Stromal Cell Types in Tumor Angiogenesis.
Authors: Ferrara Napoleone
Published: October 2019

Abstract SCI-32

Role of myeloid and other stromal cell types in tumor angiogenesis Napoleone Ferrara Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080, USA. Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor, involved in both physiological and pathological growth of blood vessels. An anti-VEGF-A monoclonal antibodies and two small molecule VEGF RTK inhibitors have been approved by the FDA for cancer therapy. We have been investigating the mechanisms of refractoriness/resistance to anti-VEGF therapies. Our analysis points to the mobilization and recruitment of CD11b+ Gr1+myeloid cells from the bone marrow in the tumor as key mechanisms for VEGF-independent angiogenesis, at least in mouse models. Tumors that are refractory to anti-VEGF treatment result in increased peripheral mobilization and tumor homing of of CD11b+Gr1+ cells compared to sensitive tumors. Addition of CD11b+Gr1+ cells from resistant tumors was able to sustain the growth of sensitive tumors in the presence of anti-VEGF antibodies. In subsequent studies, we identified the secreted protein Bv8 as a mediator of myeloid cell-dependent angiogenesis. Blocking Bv8 using neutralizing antibodies inhibited the growth of several tumor cell lines transplanted in nude mice and also reduced the angiogenic switch in the RIP-Tag, a transgenic model of insulinoma. The expression of Bv8 by CD11b+ Gr1+myeloid was regulated by hematopoietic growth factor, especially G-CSF. Other studies have implicated tumor-associated fibroblasts as a source of alternative pro-angiogenic factors following VEGF blockade. We identified PDGF-C as one of such mediators. In summary, multiple stromal cell types and signaling pathways may contribute to inherent refractoriness and, potentially, acquired resistance to anti-VEGF therapies in tumors.

Disclosures

Ferrara: Genentech, Inc.: Employment.
Counterpoint: In Some AMLs, Stem Cells are More Mature and Present at High Frequency.
Authors: Cleary Michael L.
Published: October 2019

Abstract SCI-35

Leukemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease and, therefore, are promising targets for therapy. The current paradigm for LSC frequency, maturation and hierarchical organization is primarily based on transplantation studies in xenograft mouse models. To circumvent potential limitations of this experimental approach, investigators have recently employed syngeneic mouse models to study LSCs. In a mouse model of AML initiated by MLL oncogenes, which are associated with the FAB-M4 or M5 subtypes of human AML, LSCs are remarkably frequent, accounting for up to one-quarter of malignant myeloid cells at late-stage disease. Even in this syngeneic setting, however, transplant assays alone markedly underestimate LSC frequency due to poor engraftment efficiency. LSCs are organized in a phenotypic and functional hierarchy, and express myeloid lineage-specific antigens, placing them downstream of the known hematopoietic progenitor compartments. Thus, LSCs in this model are not synonymous with normal upstream progenitors that are targeted for leukemia initiation, but rather constitute myeloid lineage cells that have acquired an aberrant self-renewal program as well as other biologic features of hematopoietic stem cells. Gene expression profiling confirms the downstream myeloid character of LSCs in this model, and further demonstrates the aberrant expression of a stem cell associated transcriptional subprogram. However, LSC maintenance in the self-renewing compartment of AML employs a global transcriptional program more akin to embryonic rather than adult stem cells. Expression of LSC maintenance program genes is enriched in poor prognosis human malignancies, suggesting that the frequency of aberrantly self-renewing progenitor-like cancer stem cells may be linked to prognosis in human cancer. Consistent with this possibility, LSC frequencies in different syngeneic models of Hox-associated AML can vary over three orders of magnitude, depending on the particular initiating oncogene and expression levels of Hox pathway co-regulators, and correlate with leukemia biology. Studies in a human cord blood cell transduction/transplantation model of AML further support the downstream character of MLL LSCs. These findings prompt a revision of the current paradigm that AML leukemia stem cells are always rare and solely located within the most immature bone marrow progenitor compartments. The fact that LSCs can be more analogous to precursors and employ ESC-like genetic programs for their maintenance, may allow for their selective therapeutic targeting that spares HSCs required for hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Role of the Glycoprotein (GP)IbαaIb Cytoplasmic Domain in Regulating the Von Willebrand Factor-Binding Functions of the Platelet GPIb-IX-V Complex.
Authors: López José A. , Munday Adam
Published: October 2019

Abstract SCI-39

The platelet glycoprotein (GP) Ib-IX-V complex mediates the initial attachment of platelets to regions of blood vessel damage by binding von Willebrand factor (VWF) exposed in the matrix at the site of injury. This interaction is especially important in the arterial system and microvasculature, given that it is the preferred platelet adhesive event at high shear stress. The interaction is positively and negatively regulated in several ways, including through exposure of the GPIb-binding site on VWF by shear stress or collagen binding, cleavage of VWF by the metalloprotease ADAMTS13, and in the platelet by the targeting of the GPIb-IX-V complex to lipid rafts, cytoskeletal attachments, and binding of the adaptor protein 14-3-3. Association with 14-3-3 has complex roles in regulating accessibility of GPIbα (the ligand-binding subunit of the complex), as several binding sites have been identified within the cytoplasmic domains for this adaptor molecule, and these are regulated by phosphorylation. To address the role of GPIbα phosphorylation in regulating platelet adhesion to VWF, we mutated 5 cytoplasmic Ser and Thr residues that were predicted by bioinformatics programs to be targets for serine/threonine kinases (Thr547, Ser566, Ser587, Ser590, Ser609) and expressed the mutants either as an isolated cytoplasmic domain or in intact GP Ibα as part of a GP Ib-IX complex expressed in CHO cells. We tested whether the following kinases could phosphorylate a recombinant GP Iba cytoplasmic tail: protein kinase A (PKA), protein kinase C (PKC), calmodulin-dependent kinase II (CaMKII) and Akt. All but Akt phosphorylated the region. We then tested polypeptides with various combinations of mutations to these residues for phosphorylation by PKA, and found that this kinase phosphorylated each of the residues except Ser590. We also examined the effect of the mutations on 14-3-3z association and on cell attachment and rolling on VWF. The findings from these studies were complex, with the results of 14-3-3 association suggesting a hierarchy of 14-3-3 binding. This binding did not correlate with the effect of the mutations on attachment on VWF or the rolling velocity profiles. Nevertheless, mutants could be clustered in terms of these effects, the aggregate of the data suggesting a very complex pattern of GPIb-IX-V regulation by the GP Ibα cytoplasmic tail, which will have to be taken into account when considering the association of GP Ibα with 14-3-3 as an antithrombotic target.

Disclosures

No relevant conflicts of interest to declare.
Manipulating Stem Cells for Therapeutic Expansion.
Authors: Sauvageau Guy
Published: October 2019

Abstract SCI-42

“Self-renewal” is a process central to the expansion of normal and cancerous stem cells and its understanding is critical for future advances in transplantation-based therapies and cancer treatment. Even today, many patients are deprived of the benefit of a successful blood stem cell transplant because the number of allogeneic or autologous stem cells available is insufficient, which results in delayed hematopoietic recovery post-transplant, or exclusion of a transplant-based therapeutic option altogether. The molecular machinery controlling self-renewal of hematopoietic stem cells (HSCs) remains poorly defined with the exception of a few genes such as HOX4 and Bmi1. We and others recently demonstrated the capacity of a recombinant HOXB4 protein (TAT-HOXB4 fusion protein) to stimulate mouse and human HSC self-renewal divisions in culture. Technical difficulties inherent to this recombinant protein have postponed the initiation of clinical trials. In part to overcome this hurdle, we have developed a novel in vitro/in vivo gain-of-function screen and identified several nuclear factors which expand hematopoietic stem cell ex vivo. A significant proportion of these factors display HOXB4-like properties and show non-cell autonomous activity. Initial results suggest that some of these new factors are also active with human cord blood derived HSCs. The generation of novel TAT fusion proteins will open new possibilities in the therapeutic expansion of human HSCs.

Disclosures

No relevant conflicts of interest to declare.