bluuebunny/crossref_metadata_2025 huggingface dataset:
MPL Mutation Profile in JAK2 Mutation-Negative Patients with Myeloproliferative Neoplasms.
Authors: Ma Wanlong , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , Uyeji Jennifer , Albitar Maher
Published: October 2019

Abstract 1891

Poster Board I-914

Myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell neoplasms characterized by excess production of various blood cells. Mutations in the thrombopoietin receptor gene (MPL) have been reported in JAK2 V617F-negative patients with MPNs. We evaluated the prevalence of MPL mutations relative to those of JAK2 mutations at V617 or in exons 12 through 15 in a large number of patients with suspected MPNs. A total of 2790 patients with suspected MPNs referred to our institution were first screened for JAK2 V617F and exon 12–15 mutations. Patients with no JAK2 mutations detected were then tested for MPL mutations in exons 10 and 11 by means of a sensitive MPL reverse-transcription-PCR-based assay with direct bidirectional sequencing. All JAK2 and MPL mutation assays were performed on plasma RNA, rather than DNA isolated from blood or bone marrow cells. Of the 2790 patients, 529 (18.96%) had a V617F mutation; 12 (0.43%) had small insertion/deletions in exon 12; and 7 (0.25%) had other JAK2 mutations, including point mutations in exons 13–15 and an exon 14 splice mutation. MPL mutations were identified in 66 of the 2242 (2.94%) JAK2 mutation-negative patients (2.37% of all tested patients). W515L was the dominant MPL mutant detected in 46 patients (70%) including two patients with homozygous W515L mutation. The other W515 variants (W515K, W515R, W515S, W515G, W515A, W515*) comprised 16% (N=11) of the MPL mutations. The remaining MPL mutations (n =9, 14%) were detected at other locations in exon 10 and 11. Two of these were novel exon 10 deletion/insertion mutations and two were unreported exon 11 point mutations. The exon 10 T496-A497ALVI (4AA) homozygous insertion was detected in one patient with a confirmed diagnosis of idiopathic myelofibrosis and the W515-P518 del/ins (KT) mutation was detected in another patient with unspecified MPN. The two novel MPL exon 11 point mutations were D545G and D545N. The S505N mutation (n=2) and V507I (n=1), R514K (n=1), and A519V (n=1) were also detected. Furthermore, three unreported silent polymorphisms/mutations (T496, L543, and D534) were detected. In conclusion, our results demonstrate that for every 100 V617F mutations detected in patients with MPNs, there are 2.3 JAK2 exon 12 mutations, 1.3 JAK2 exon 13–15 mutations, and 12.5 MPL mutations. Thus, MPL mutation detection should be performed on all JAK2 V617F-negative patients with suspected MPNs. In addition, our findings indicate that >20% of MPL mutations would have been missed if only W515L and W515K were analyzed; thus, sequencing of both exon 10 and 11 may be beneficial.

Disclosures:

No relevant conflicts of interest to declare.
The Role of JAK3 mutations in Adult T-Cell Leukemia/Lymphoma.
Authors: Elliott Natalina E , Cleveland Susan , Grann Victor , Janik John E. , Dave Utpal P.
Published: October 2019

Abstract 1940

Poster Board I-963

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive and incurable cancer caused by infection from a retrovirus, Human T-cell Leukemia Virus type-1 (HTLV-1). HTLV-1 infects T cells and induces cell transformation through the expression of viral oncogenes such as tax. In newly infected cells, tax activates the expression of Interleukin-2 and the alpha subunit of its receptor (IL2RA) resulting in autocrine signaling that drives proliferation. In ATLL cells, tax is frequently deleted or silenced by methylation but there is constitutive activation of pathways downstream of IL-2 and related cytokines. These data led us to hypothesize that somatic mutations in this cytokine pathway may play a role in ATLL tumor progression. Encouraged by the recent discovery of activating mutations in JAK3 in diverse hematologic malignancies, we sequenced the exons of JAK3 in a small cohort of ATLL patients and found three missense mutations in the amino terminal FERM domain which were not found in Single Nucleotide Polymorphism databases. IL-2 signaling recapitulated in Human Embryonic Kidney 293T cells showed that these missense mutations caused increased tyrosine phosphorylation of the JAK3 substrate, STAT5A. Furthermore, enforced expression of these mutant JAK3s caused cytokine independent growth of BaF3 cells. The FERM domain of JAK3 is similar to the FERM domain of Focal Adhesion Kinase (FAK), which has a solved crystal structure. Interestingly, the FAK FERM domain contacts and autoinhibits its own kinase domain. Our homology-based model suggests that the JAK3 missense mutations disrupt the secondary structure needed for this intramolecular contact thereby perturbing autoregulation of JAK3 kinase activity. Our findings suggest that JAK3 gain of function may play an important role in ATLL disease progression.

Disclosures:

No relevant conflicts of interest to declare.
G-CSF Priming, Clofarabine and High Dose Cytarabine (GCLAC) for Relapsed or Refractory Acute Myeloid Leukemia (AML).
Authors: Becker Pamela S. , Estey Elihu , Petersdorf Stephen , Storer Barry E , Appelbaum Frederick R
Published: October 2019

Abstract 2068

Poster Board II-45

Background:

Clofarabine is active in both relapsed and newly diagnosed AML. Clofarabine in combination with ara-C at a dose of 1 g/m2 daily for 5 days resulted in 52% complete remission (CR) in newly diagnosed patients age 50 or older. (Faderl et al Blood 108:45, 2006), Addition of low dose cytarabine (ara-C) improved efficacy as compared to clofarabine alone in untreated patients age 60 or older (Faderl et al Blood 112:1638, 2008). As high dose ara-C has considerable efficacy in AML, we combined clofarabine with higher dose ara-C (HiDAc) and granulocyte colony-stimulating factor (G-CSF) priming for patients with relapsed AML or AML that failed to respond to initial therapy. GCLAC is based on the FLAG regimen, substituting clofarabine for fludarabine, given the former's greater anti-AML effect.

Methods:

We initially conducted a phase 1 trial in 19 pts and identified 25 mg/m2 daily days 1-5 as the MTD of clofarabine when combined with ara-C 2 g/m2 daily days 1-5 and G-CSF 5 mcg/kg subcutaneously, beginning 1 day before chemotherapy and continuing daily until neutrophil recovery. A phase 2 expansion was then conducted at the MTD.

Results:

We have treated 38 patients (pts), age range 19-66 years, median 51, 22 at the MTD. Twenty pts received GCLAC as 1st salvage, 15 at the MTD. Seven of the 20 had relapsed after a median 1st CR duration of 6 mos while 13 had not responded to initial 3+7 induction therapy, including 4 who were refractory to > 1 course. The CR rate for all patients or all patients at the MTD was 45% and the CR +CRp rate at the MTD was 64%. These rates are 50% CR (95%CI 27-73%) and 65% CR+CRp among 1st salvage pts (95% CI 41-85%), respectively, and 70% CR + CRp excluding pts who relapsed after allogeneic SCT. Median time to neutrophil recovery (ANC>500) was 21 days (range 13 to 39), and to platelet recovery (platelet count >100,000), 29 days (range 21-42). Four of 22 pts had serious infections and/or asymptomatic grade 3 LFT elevations, a rate not in excess of that seen with other ara-C-containing salvage therapies. Sixty percent of the first salvage patients had poor risk cytogenetics, and 80% of CRs were observed after the first course of therapy. Median overall survival was 7.4 months, and event free survival, 4.3 months. Using a prognostic model that derives expected CR rates with HiDAc or FLAG based on 1st CR duration and number of prior salvage therapies (Blood 1996;88:756), the ratio of observed (with GCLAC) to expected CR was 2.5:1.

Conclusion:

Based on its efficacy, formal comparisons of GCLAC with other salvage regimens are warranted and the combination should also be investigated in untreated pts.

Disclosures:

Becker: Genzyme Oncology: Research Funding. Off Label Use: Clofarabine is approved for relapsed or refractory pediatric ALL .
Robotic High Thoroughput Multiplex PCR Single-Nucleotide Polymorphism Genotyping of Apheresis Platelet Donors.
Authors: Shehata Nadine , Hannach Barbara , Banning Nancy , Freedman John , Denomme Greg
Published: October 2019

Abstract 2106

Poster Board II-83

Background Phenotyping blood donors for human platelet antigens (HPAs) can be limited by the availability of antibodies and methodologies. Robotic high throughput multiplex polymerase chain reaction (PCR) single-nucleotide polymorphism (SNP) technology provides the opportunity to perform mass genotyping for HPAs to screen apheresis donors who are negative for antigens implicated in alloimmune thrombocytopenia. This technology needs to be validated by standard polymerase chain reaction-SSP to establish a repository of platelet donors to allow for timely and adequate platelet support for patients requiring HPA-matched platelets. The objective of this study is to validate the use of high throughput SNP technology. Methods This is a prospective cohort study of 750 regular apheresis donors. Platelet apheresis donors were identified from Canadian Blood Services' Toronto Centre and genotyped for HPA 1-5 and 15 using high throughput SNP (SNPStream®) technology. HPAs were genotyped using both the sense and antisense DNA strands of each polymorphism to ensure maximum specificity. The genotypes identified by SNP were confirmed by standard methods thus all donors found to be negative for antigens implicated in alloimmune thrombocytopenia were retested using manual (sequence specific SSP-PCR at Canadian Blood Services' Platelet Immunology Laboratory and at the Toronto Centre. Results Of the 750 donors that were screened, 130 donors were found to be negative for antigens implicated in alloimmune thrombocytopenia based on SNP technology. The table illustrates genotyping results using SNP technology and SSP-PCR confirmation to date. In some instances, donors were homozygous for two low frequency HPA genotypes: 2 donors were homozygous for both HPA-1b/b and -2b/b, 6 donors for HPA-1b/b and -3b/b, 1 donor for HPA-2b/b and -3b/b, 1 donor for HPA-1b/b and -5b/b, 10 donors for HPA-1b/b and -15b/b, 4 donors for HPA-5b/b and -15b/b and 1 donor for HPA-2b/b and 15-b/b.

HPA System Number of Genotypes Identified by SNP Technology Discrepant results Identified by SSP HPA-1 b/b 15 2 (13%) HPA-2 b/b 8 3 (27%) HPA-3 b/b 25 1 (4%) HPA-5 b/b 1 0 HPA-15 b/b 9 1 (11%)

Conclusions:

Robotic high throughput multiplex PCR SNP is potentially useful for mass genotyping of apheresis platelet donors and can identify both low frequency antigen-negative donors and combinations of these genotypes. This technology needs to be further developed.

Disclosures:

No relevant conflicts of interest to declare.
Tolerance Induced by B-Cell Gene Therapy: Expansion and Increased Activity of Antigen-Specific Tregs.
Authors: Skupsky Jonathan , Su Yan , Scott David W.
Published: October 2019

Abstract 2129

Poster Board II-106

Patients with hemophilia A treated with coagulation co-factor VIII (FVIII) may mount a response against this therapeutic protein, thus forming antibodies that block FVIII function (inhibitors). To treat those currently forming inhibitors (or to prevent inhibitor formation), a B-cell based gene therapy has been used that employs a chimeric antigen-Ig tolerogenic construct comprised of FVIII C2 and A2 domains. We have shown that hemophilic mice receiving B cells transduced with these Ig fusion proteins have diminished Bethesda titers, even after further FVIII treatment. Tolerogenic B-cell therapy requires the presence of CD25+ regulatory T cells (Tregs). We have recently shown that tolerogenic B-cell treatment results in the increase of antigen-specific Treg cells and a selective loss of antigen-specific responder cells. To extend these studies, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using CFSE dilution as a measure of T-cell responsiveness to FVIII, we now show that Tregs from treated mice are more suppressive than those from control treated animals. In order to follow antigen-specific Tregs, we repeated these studies in DO11.10 T-cell receptor transgenic mice that recognize an OVA peptide. Tregs from DO11.10 mice treated with an OVA-Ig construct are also more suppressive than polyclonal Tregs when stimulated with OVA peptide. Although functionally superior, there appear to be no discernible phenotypic differences in these Treg populations. These changes in the lymphocyte population shift the balance away from effector function towards a tolerogenic phenotype and may provide the basis for future therapies in hemophilia A patients with inhibitors. (Supported by NIH RO1 HL061883, NIH T32 HL007698, and AHA Fellowship 0815219E)

Disclosures:

No relevant conflicts of interest to declare.
Effects of Mobilization with Granulocyte Colony Stimulating Factor (G-CSF) and Plerixafor On Lymphocyte Subsets in Autologous Peripheral Blood Stem Cell (PBSC) Apheresis Products.
Authors: Elquza Emad , Spier Catherine , Fiederlein Roger , Salamon David , Yeager Andrew M.
Published: October 2019

Abstract 2145

Poster Board II-122

Introduction:

Plerixafor (AMD3100; PLX), a bicyclam molecule that reversibly inhibits binding of the CXCR4 chemokine receptor to stromal cell derived factor-1, is used clinically in combination with G-CSF for mobilization of hematopoietic stem cells. Although the effects of PLX plus G-CSF on mobilization of CD34+ cells have been well studied, the impact of PLX plus G-CSF on mobilization of lymphocytes and lymphocyte subsets remains unclear. Additionally, comparative effects of PLX on the content and subpopulations of lymphocytes in apheresis products from patients (pts) with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) have not been previously reported.

Patients and Methods:

Thirteen pts (7 NHL, 6 MM) underwent autologous PBSC mobilization with subcutaneous (sq) G-CSF (10 micrograms/kg/day x 4 days) and sq PLX (0.24 mg/kg on the fourth day of G-CSF administration), followed by large-volume apheresis approximately 15 hr after PLX dose. Pts who did not attain target CD34+ collection on first day of apheresis (day 1) received up to 3 additional daily doses of PLX plus G-CSF and up to 3 additional days of apheresis. Aliquots of apheresis products were obtained after each collection for differential WBC count, from which the absolute lymphocyte count (ALC) was calculated, and for flow cytometric quantitation of lymphocyte subsets. Cell doses were expressed per kg of pt weight. A nonparametric (Mann-Whitney) tests was used for all statistical analyses.

Results:

Five pts (1 NHL, 4 MM) collected target CD34+ cell doses (median 7.46 × 106 cells/kg; range, 7.22-22.7 × 106 cells/kg) with 1 apheresis. The remaining 8 pts collected target CD34+ cell doses after a total of 2 (2 NHL, 2MM), 3 (3 NHL) or 4 (1 NHL) aphereses. Doses of CD34+ cells, ALC, CD4+ cells, CD19+ cells and CD56+ cells were significantly higher in day 1 PBSC apheresis products from MM pts than from NHL pts, but neither CD3+ nor CD8+ cell dose was significantly different in the two groups (Table 1). For the pts undergoing 2 or more aphereses, comparison of day 1 and day 2 apheresis products showed significantly decreased doses of ALC, CD3+, CD4+ and CD8+ cells, but no significant differences in doses of CD34+, CD19+ or CD56+ cells (Table 2).

Conclusions:

PLX plus G-CSF effectively mobilizes lymphocytes and lymphocyte subsets as well as CD34+ cells. Pts with MM had significantly higher doses of specific lymphocyte subsets, notably CD4+ and CD56+, than did pts with NHL, potentially reflecting differences in previous treatments (e.g., lenalidomide plus dexamethasone in MM pts compared with ICE or ESHAP in NHL pts). These findings are relevant to the understanding of the effects of infused lymphocyte subsets on outcomes of and immune reconstitution after autologous PBSC transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Treatment with the CXCR4 Antagonist AMD3100 Enhances Antibody Mediated Killing in Disseminated Lymphoma Models.
Authors: Siders William , Hu Yanping , Gale Matthew , Jacques Gary , Fogle Robert , Shields Jacqueline , Garron Carrie , Kaplan Johanne
Published: October 2019

Abstract 2717

Poster Board II-693

Interactions between chemokines and their receptors play an important role in cancer biology and have been shown to influence several processes such as tumor growth and the development of metastatic lesions. Stromal cell derived factor-1 (SDF-1) is a chemokine that binds to the CXCR4 chemokine receptor and stimulates B cell growth. CXCR4 is frequently over-expressed on tumor cells and the SDF-1/CXCR4 axis has been reported to play a role in promoting survival, angiogenesis and metastasis. The role of the stroma, in particular SDF-1/CXCR4 interactions, in providing a “survival niche” for tumor cells is well described for both hematological and solid tumors. We hypothesized that disruption of the protective microenvironment of tumor cells through antagonism of the SDF-1/CXCR4 axis may enhance the sensitivity of the tumor cells to treatment with anti-cancer agents such as monoclonal antibodies. Mozobil (AMD3100) or its structurally related analog, AMD3465, were used in combination with either Campath (anti-CD52) or Rituxan (anti-CD20) in lymphoma xenograft models to determine whether enhanced tumor protection could be observed. Flow cytometry analysis of lymphoma cell lines confirmed the cell surface expression of high levels of CXCR4 as well as the CD20 and CD52 target antigens. Lymphoma cells were seeded by intravenous injection and tumor-bearing mice were treated with multiple injections of the CXCR4 antagonists AMD3465 or AMD3100 alone or in combination with monoclonal antibodies. Treatment with AMD3465 as a single agent was sufficient to increase survival of the animals compared to untreated controls. In addition, the combination of AMD3465 and Campath showed a synergistic effect with a prolongation of survival greater than that obtained with either agent alone (p<0.01). Similarly, combination of AMD3100 and Rituxan also resulted in a significantly prolonged survival compared to either agent alone (p=0.0340). In vivo imaging using a Raji-luciferase cell line also confirmed the impact of treatment on tumor burden. These results suggest that there is a potential role for CXCR4 antagonists in combination with a B-cell targeted therapy in the treatment of B-cell malignancies in the clinic.

Disclosures:

Siders: Genzyme Corporation: Employment. Hu:Genzyme: Employment. Gale:Genzyme: Employment. Jacques:Genzyme: Employment. Fogle:Genzyme: Employment. Shields:Genzyme: Employment. Garron:Genzyme: Employment. Kaplan:Genzyme: Employment.
A Phase II Trial of Rituximab-CHOP Chemotherapy Followed by Yttrium 90 (90Y) Ibritumomab Tiuxetan (90Y-IT) for Previously Untreated Elderly Diffuse Large B-Cell Lymphoma (DLBCL) Patients.
Authors: Zinzani Pier Luigi , Fina Mariapaola , Tani Monica , Stefoni Vittorio , Gandolfi Letizia , Broccoli Alessandro , Pellegrini Cinzia , Derenzini Enrico , Rossi Giuseppe , Angelucci Emanuele , Gaidano Gianluca , Petti Maria Concetta , Martelli Maurizio , Vitolo Umberto , Fanti Stefano , Baccarani Michele
Published: October 2019

Abstract 2720

Poster Board II-696

Introduction:

In 2008 we published a phase II trial abuot the combination of 6 cycles of CHOP plus 90Y-IT for previously untreated elderly patients with DLBCL. The CCR was 95% with OS at 2 years of 95% and PFS at 2 years of 75%. The results of this study support a further evaluation of 90Y-IT in combination of chemotherapy. We conducted a prospective, single-arm, non-randomized, phase II trial with CHOP plus Rituximab followed by 90Y-IT reducing the number of CHOP cycles from 6 to 4 but introducing Rituximab. The rational is to utilize all the therapeutic approaches (chemotherapy, immunotherapy and radioimunotherapy) reducing conventional chemotherapy and probably related toxicity.

Patients and Methods:

Patient elegibility was represented by: patients older than 60 years with biopsy proven, untreated, bidimensionally mesurable stage II, III or IV DLBCL. Expression the CD-20 antigen; WHO performance status of 0 to 2. patients were treated with standard CHOP chemotherapy plus Rituximab every 21 days for 4 cycles. Patients were restaged 4 to 6 weeks after completion of 4 cycles of R-CHOP chemotherapy. Patients achieving CR, PR or SD after chemotherapy were eligible for consolidation with 90Y-IT provided the granulocyte count was greater than 1500/microl, the platelet count exceded 100.000/microl and the bone marrow examination at the completion of chemotherapy demostrated no more than 25% involvement with lymphoma. All patients were to receive a single dose of 90Y-IT 14.8 MBq/kg (0.4 mCi/kg). Fifty-five patients have been enrolled: 26 were male and 29 female; the median age was 70 years (range 60–83); 17 were stage II, 38 were stage III-IV.

Results:

Fifty-one patients had completed the R-CHOP treatment and the overall response rate was 94.1% including 30 (58.8%) of patients in CR and 17 (35.3%) in PR. Treatment was well tolerated; grade 3–4 AEs are comparable with previous experience and the most common grade 3–4 AEs was neutropenia. At this time 47 patients had just received 90Y-IT and 45 are evaluable. In particular, 9/17 (52.9%) patients converted from PR to CR after treatment with 90Y-IT.

Conclusions:

These preliminary data indicate that radioimmunotherapy appears highly effective and feasible as “consolidation” after 4 cycles of immunochemotherapy in elderly DLBCL patients, improving quality of response without any cumulative toxicity.

Disclosures:

Off Label Use: The drug Yttrium 90 (90Y) Ibritumomab Tiuxetan (90Y-IT) (Zevalin) is off-label for Diffuse Large B-Cell Lymphoma (DLBCL).
Combining RAD001 (Everolimus) with Proteasome Inhibitors Bortezomib (Velcade) or MG132 Significantly Enhances Pre-B ALL Cell Death in Vitro.
Authors: Saunders Philip O. , Bradstock Kenneth F , Bendall Linda J.
Published: October 2019

Abstract 2743

Poster Board II-719

Mammalian target of rapamycin (mTOR) inhibitors have shown potential as novel therapeutic agents with efficacy against a wide range of tumors including precursor-B acute lymphoblastic leukemia (pre-B ALL). We have previously reported RAD001 (16μM) induces JNK pathway activation in pre-B ALL cells and that combining RAD001 with DNA damaging agents in vitro significantly enhanced JNK dependent death, via a caspase dependent mechanism. We sought to evaluate agents, which may favor JNK activation and promote JNK dependent cell death in pre-B ALL cells. Bortezomib and MG132 have both been reported to activate the JNK pathway via death receptor activation, with reported efficacy in pre-B ALL. Consistent with the literature we observed enhanced JNK pathway activation in pre-B ALL cells treated with bortezomib. Analysis of annexin V and 7AAD expression by flow cytometry utilizing JNK inhibitor SP600125 (5μM) showed that a significant proportion of pre-B ALL cell death observed with bortezomib was JNK dependent.

Combining RAD001 (4–16μM) with bortezomib in vitro (10–20nM) significantly enhanced cell death in pre-B ALL cell lines and patient cases at 24 hours. This observation was supported by equivalent observations combining MG132 (250–500nM) with RAD001 (8–16μM). The degree of enhanced killing was greater than that achieved combining RAD001 (16μM) with DNA damaging agents. Enhanced killing was also achieved at a significantly lower dose of RAD001 relative to combination therapy with DNA damage. Utilizing JNK inhibitor SP600125 (5μM) we determined that a significant proportion of enhanced killing was JNK dependent.

In conclusion we have identified two novel and clinically available agents which when combined can significantly enhance pre-B ALL cell death. Our observations suggest combining agents which induce JNK activation has the potential to enhance clinical responses in pre-B ALL, particularly for patients with advanced or relapsed disease, for whom treatment with cytotoxic chemotherapy offers little hope of improved survival. In vivo studies will provide further insight into this promising strategy.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Cytogenetic Abnormalities On Outcome Among Patients with Relapsed/Refractory Multiple Myeloma Treated with Bortezomib: Adverse Effect of 1q21 (CKS1B) Amplification.
Authors: Chang Hong , Trieu Young , Qi Connie , Xu Wei , Reece Donna E.
Published: October 2019

Abstract 2826

Poster Board II-802

To investigate whether genetic risk factors affect the outcome of relapsed/refractory myeloma patients undergoing bortezomib therapy, we evaluated the clinical features of 85 patients treated with bortezomib and correlated with the high-risk cytogenetic abnormalities as detected by interphase cytoplasmic fluorescence in situ hybridization (cIg-FISH). Forty (47%) patients had an objective response to bortezomib with median progression free (PFS) and overall survivals (OS) of 9.4 and 12.6 months, respectively. cIg-FISH determination of del(13q), del(17p), del(1p21), t(4;14), and amp. (1q21)(CKS1B) was done on 79, 77, 77, 78, and 80 cases and the frequency of their detection was 38%, 22%, 26%, 18%, and 39%, respectively. There was no statistically significant difference in terms of response or duration of response to bortezomib for patients with or without any of the cytogenetic abnormalities. PFS was also comparable among patients with and without the aforementioned genetic markers. However, patients with 1q21(CKS1B) amplification had significantly shorter OS than those without such abnormality (5.3 vs. 24.6 months, p=0.0005). Multi-variate analysis adjusting for all 5 high-risk genetic factors confirmed that 1q21(CKS1B) amplification is an independent risk factor for OS (p=0.008). Improved therapeutic strategies may be required for this subgroup of patients.

Disclosures:

Reece: Ortho Biotech: Honoraria, Research Funding.
Leukemogenic Transformation by Anterior HOXA-Cluster Genes.
Authors: Buhl Sebastian , Maethner Emanuel , Slany Robert K.
Published: October 2019

Abstract 2962

Poster Board II-938

HOX homeobox genes are important regulators of normal and malignant hematopoiesis and abdominal-type HOXA-cluster genes, in particular HOXA7 to HOXA10, are highly leukemogenic. However, little is known about the transforming abilities of anterior HOXA genes HOXA1 to HOXA6 despite a high prevalence of anterior HOX-expression in acute leukemia. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5 all other HOXA genes caused a block or delay of hematopoietic differentiation in serial replating assays and all HOX genes also cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all other active HOXA genes induced the outgrowth of mixed granulocytic/monocytic cells, HOXA13 preferentially specified the development of monocytes/macrophages. Albeit more weakly than HOXA9 also the anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells and generated permanent myelomonocytic cell lines arrested at various stages of differentiation. HOX-RNA profiling revealed that each of these lines also transcribed HOXA9. However, kinetic studies with inducible HOX-derivatives showed that HOXA9 expression was not under control by anterior HOXA proteins. Cellular differentiation was induced immediately after loss of anterior HOX activity and HOXA9 down regulation was a secondary event during maturation. This proves that anterior HOX proteins are able to transform hematopoetic cells through an individual contribution independent of HOXA9. In summary our results explain how HOXA9 can predominate in hematologic malignancies while simultaneously it is not necessarily required for transformation.

Disclosures:

No relevant conflicts of interest to declare.
Racial Differences in Thrombotic Risk Factors Associated with Adverse Pregnancy Outcome Among Women Obtaining Care in US Thrombosis and Hemostasis Centers.
Authors: Philipp Claire S. , Faiz Ambarina , Shrimanker Sheetal , Beckman Michele G , Bockenstedt Paula L. , James Andra H , Manco-Johnson Marilyn J.
Published: October 2019

Abstract 2982

Poster Board II-958

Pregnancy complications such as recurrent miscarriage, intrauterine growth restriction (IUGR), and preeclampsia are common and have been associated with thrombophilia. However racial differences are poorly described for this population. The objective of this study was to determine racial differences in unexplained adverse pregnancy outcome among women obtaining care in the CDC Thrombosis and Hemostasis Centers Research and Prevention Network. Uniform data were prospectively collected from August 2003 to March 2009 in consenting women with a history of adverse pregnancy outcome obtaining care at Network Centers for pre-pregnancy, pregnancy, or postpartum consultation and/or management. Data from 407 women (mean age 38.5 ±12 yrs), including 326 white women (mean age 39.4 ± 12 yrs), and 60 black women (mean age 33.9 ± 11 yrs) were analyzed. Black women had significantly more second trimester pregnancy losses compared to white women (35% vs 22%, p=0.03) but the proportions of 1st trimester losses, 3rd trimester losses, IUGR, prematurity, abruption, and preeclampsia were not significantly different. The risk of thrombophilia differed by race, with Factor V Leiden mutation more common in white women compared to black women (19% vs 3%, p=0.002). Protein S deficiency was more common in black women compared to white women overall (15% vs 5%, p=0.006) and in the subgroup of non-pregnant women (14% vs 5%, p=0.04). There was no significant difference in the proportion of white and black women with antiphospholipid antibodies. Among 285 women evaluated when non-pregnant, body mass index (BMI) ≥ 25 was more frequent in black women (83%) compared to white women (63%) (p=0.04). Overall, black women had a significantly higher proportion with hypertension (27% vs 11%, p=0.002) and sickle cell disease (7% vs 0,%, p< 0.001). There was no significant racial difference in history of pregnancy associated, provoked, or idiopathic venous thromboembolism (VTE). A family history of thrombophilia (6% vs 0%, p=0.05), VTE (22% vs 7%, p=0.005), myocardial infarction (MI) or stroke (12% vs 0%, p=0.0042) was significantly more common in white women compared to black women (overall 32% vs 8%, p<0.0002). This study demonstrates that comorbidities including hypertension, sickle cell disease and BMI≥25 are more prevalent in black women with adverse pregnancy outcomes whereas white women have a higher prevalence of Factor V Leiden mutation, and positive family history of thrombophilia, VTE, MI, and stroke.

Disclosures:

No relevant conflicts of interest to declare.
A Polymorphism in the XPNPEP2 Gene Is Associated with the Development of Hypotensive Transfusion Reactions without ACE Inhibitors.
Authors: Hui Yiang , Tormey Christopher , Wu Yanyun
Published: October 2019

Abstract 3140

Poster Board III-77

Introduction

Hypotensive transfusion reactions (HTRs) have been associated with the transfusion of different blood products. They are characterized by the rapid onset and disappearance of hypotension upon starting and finishing the transfusion respectively. The use of ACE inhibitors has been associated with HTRs. However, there are reported HTRs in patients not taking ACE inhibitors. An inability to break down bradykinin (BK) generated during the infusion/transfusion is suggested to be responsible for the hypotensive effect. Angiotensin-converting enzyme (ACE) and aminopeptidase P (APP) are primarily responsible for degradation of BK. We hypothesize that the C-2399A single-nucleotide polymorphism in the membrane-bound APP gene XPNPEP2 (SNP rs3747343) may be linked to HTRs. This polymorphism is present upstream of the coding sequence of the APP gene and has already been associated with severe hypotension in an angioedema patient treated with an ACE inhibitor. This suggests that it may have an important regulatory role.

Patients and Methods

With the approval of our IRB, patients' samples were collected, de-identified, and then used for testing. Specimens from 4 patients who presented with classic HTRs while not taking ACE inhibitors were analyzed through sequencing of DNA acquired from pre-transfusion blood samples. The same primers were used for PCR and sequencing (5'-GAGTATTATGTGGGGACCATCC-3' and 5'-ATGCCTCGCAGAGACAAGAG-3'). Samples were sequenced in both sense and antisense directions.

Results

Among these patients, two (50%) were found to possess the C-2399A mutation. One was found to have changes in both reference and complementary strands while the other had a C/A mutation only in one strand.

Conclusions

These results indicate that the C-2399A polymorphism may be a contributing factor in the development of HTRs and that it may promote HTRs in the absence of ACE inhibitors. The hypotensive effect may be due to a structural or regulatory change caused by C-2399A though it also seems that other factors may be involved since not all patients assayed possessed the mutation. Further screening of additional contributing factors responsible for HTRs is necessary to fully comprehend the pathogenesis of this type of transfusion reaction.

Keywords: XPNPEP2, C-2399A, ACE Inhibitors, Hypotensive Transfusion Reactions, aminopeptidase P.

Disclosures

No relevant conflicts of interest to declare.
Epitope Mapping of FIX Inhibitors Identify Contact Residues in the Protease Domain.
Authors: Hoehl Sebastian , Kreuz Wolfhart , Krause Nicole , Stichel Diana , Martinez-Saguer Inmaculada , Kessel Christoph , Klingebiel Thomas , Oldenburg Johannes , Irving James , Königs Christoph
Published: October 2019

Abstract 3172

Poster Board III-112

Hemophilia B is an X-linked bleeding disorder that requires regular substitution of FIX preparations to restore coagulation. A rare but severe complication is the development of FIX inhibitors affecting about 3% of hemophilia B patients. Possible allergic reactions and challenging but often unsuccessful immune tolerance protocols complicate the treatment of affected patients. In the present study, five samples from patients with inhibitors against FIX (including one acquired inhibitor) were screened against random peptide libraries displayed on phage. A biopanning protocol was established to isolate phagotopes specific for FIX inhibitors and thus small peptides that mimic the epitopes of the inhibitors. Binding was confirmed by ELISA and the peptide sequences were determined by translating the DNA sequence of the phage. In total 65 peptide sequences were determined. For all patients except for the patient suffering from acquired FIX inhibitors consensus motifs were identified. These motifs include the amino acids FTK(T), TSL(T) and LRQ. In order to compare the motifs to the surface of FIX for epitope identification a novel structural model of human FIX was generated. Using this model epitopes were identified on the surface of the protease domain of FIX. The residues comprising the conformational epitopes are found in the regions AA290 - 330 and 380 - 390. Small peptides have been identified that mimic the epitopes for FIX inhibitors in hemophilia B patients. These mimotopes mimic conformational epitopes on the surface of the protease domain on FIX. Identifying inhibitor epitopes and isolating molecules that can possibly interfere with inhibitor binding might help to understand and to develop novel approaches to treat this rare but life-threatening complication.

Disclosures

Königs: Octapharma AG: Research Funding; Baxter AG: Research Funding.
Prelinical Evaluation of Lyn Kinase Inhibition for the Treatment of B Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Velcheti Vamsidhar , Li Li , Philips Joseph , Fuloria Jyotsna , Rodwig Francis R , Cole John T. , Samaniego Felipe , Prakash Om
Published: October 2019

Abstract 3447

Poster Board III-335

B-CLL is characterized by the accumulation of mature B lymphocytes incapable of undergoing apoptosis. Although several mechanisms have been implicated in the apoptotic defects in B-CLL cells, the signal transduction pathways underlying these defects remain unresolved.

Lyn kinase is known to be a negative regulator of apoptosis, and is linked to chemo-resistance. Our preliminary study indicated that Lyn activity was 4- to 7-fold higher in primary B cells from six randomly-selected B-CLL patients than in normal B cells from healthy donors. Treatment of the B-CLL cells for 4 h with 10 μM concentration of a Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res. 2004;64:1058) resulted in 40 to 50% inhibition of Lyn kinase activity compared to negligible inhibition with control peptide. Further, treatment of the B-CLL cells for 16 to 20 h with the Lyn inhibitor peptide at 5 μM and 10 μM concentrations, decreased the cell viability by ∼25% and ∼50%, respectively, compared with no peptide or the control peptide. Fludarabine is one of the key chemotherapeutic agents for B-CLL and is known to induce cell death by apoptosis. The combined treatment with Lyn inhibitor peptide (5 μM) and fludarabine (2 μg/ml), decreased cell viability by ∼50% compared to ∼25% decrease with fludarabine or Lyn inhibitor peptide alone. In addition, the combined treatment showed ∼75% increase in caspase-3/7 activity compared to ∼25% increase with the Lyn inhibitor peptide or fludarabine alone. Because, overexpression of antiapoptotic proteins is correlated with apoptotic resistance of B-CLL cells, we examined the effect of Lyn inhibitor peptide on the changes in the expression of antiapoptotic genes, myeloid cell leukemia-1 (Mcl-1), x-linked inhibitor of apoptosis (XIAP), and B-cell leukemia/lymphoma-2 (Bcl-2). Treatment of B-CLL cells with 10 μM Lyn inhibitor peptide for 16 to 20 h resulted in more than 50% decreases in the expression of all the three antiapoptotic genes. Further, the Lyn inhibitor peptide markedly inhibited NF-κB activation (∼60%) and VEGF production (∼80%), both strongly implicated in the apoptotic resistance of B-CLL cells. Collectively, our results suggest that targeting Lyn kinase pathway with a clinically relevant Lyn kinase inhibitor may have a therapeutic potential for B-CLL.

Disclosures

No relevant conflicts of interest to declare.
Apoptosis Gene Expression Profiling in Chronic Lymphocytic Leukemia Using Multiplex Ligation-Dependent Probe Amplification.
Authors: Norberg Maria , Sevov Marie , Mansouri Mahmoud , Tobin Gerard , Merup Mats , Åleskog Anna , Nilsson Kenneth , Rosenquist Richard
Published: October 2019

Abstract 3457

Poster Board III-345

One of the hallmarks of the clinically heterogeneous disease chronic lymphocytic leukemia (CLL) is defective apoptosis, which is considered to contribute not only to cell accumulation but also to disease progression and resistance to therapy. In an effort to further investigate the ‘apoptotic profile’ in CLL, we have utilized the multiplex ligation-dependent probe amplification (MLPA) method to analyze the expression profile of 35 apoptosis-related genes in 67 CLL cases including 49 peripheral blood, 9 lymph node, 7 bone marrow and 2 spleen samples. CLL cells derived from peripheral blood displayed a different overall expression profile when compared to peripheral blood mononuclear cells (PBMC) from five healthy controls, with several genes including the anti-apoptotic genes BCL2A1 and MCL1 and the pro-apoptotic genes BAX and BMF all being significantly differently expressed (>2 fold difference in median expression). Few differences were detected when comparing peripheral blood CLL samples (25/49 IGHV unmutated/IGHV3-21) to lymph node samples (6/9 unmutated/IGHV3-21) or to bone marrow samples (5/7 unmutated/IGHV3-21), with lower relative expression of BIK and BAX in peripheral blood samples. Furthermore, we correlated the expression profiles of all genes included in the assay with IGHV mutation status, IGHV3-21 gene usage and genomic aberrations, but overall few differences were evident between prognostically different subgroups. An exception to this was the BIK gene which showed significantly higher expression in unmutated/IGHV3-21 compared to mutated CLL. Additionally, MCL1 and BNIP3L displayed higher expression in 17p-deleted cases compared to 13q deletion/no aberration cases. High expression of HRK and BAX indicated a correlation with poor overall survival and shorter time to treatment. However, when performing real-time quantitative PCR on an extended patient cohort (n=112) only prediction of time to treatment could be confirmed for HRK and BAX (p=0.0035 and p=0.024, respectively). Finally, in order to validate our MLPA approach, we compared the gene expression levels of four genes in 19 CLL samples using both MLPA and real-time quantitative PCR, which revealed a significant correlation for all genes tested. In summary, MLPA is a quick and cost-effective method for pathway specific gene expression analysis and the present analysis of 67 CLL cases highlights the rather homogenous apoptosis gene expression profiles among different clinical subsets of the disease.

Disclosures

No relevant conflicts of interest to declare.
Effects of Danggui and Its Component Ferulic Acid On Hematopoiesis and Platelet Production.
Authors: Deng Rui Xia , Ye Jie Yu , Liu Chang Charlie , Chan Godfrey ChiFung , Chen Jian Liang , Shen Jian Gang , Yang Mo
Published: October 2019

Abstract 3509

Poster Board III-446

Chinese herbs have been used for the treatment of myelosuppression and thrombocytopenia for centuries and some herb mixtures have been shown to have favorable effects in cancer patients with myelosuppression. Our recent publication has shown that Danggui Buxue Tong, a traditional Chinese medicine decoction containing Radix Angelicae Sinensis (Danggui) and Radix Astragali (Huangqi), promotes hematopoiesis and thrombopoiesis in mouse model (Yang et al, J Ethnopharm, 2009). However, reports about the pharmacological effects of each herb ingredient have been scanty. The root of Danggui is an important ingredient of many commonly used prescriptions in Chinese herbs for promoting blood production. The chemical constituents and the pharmacological action of Danggui have been investigated using modern scientific methods at the beginning of this century. The objective of this study was to compare the effect of Danggui and TPO on hematopoiesis and platelet production in radiation-induced hematocytopenic mouse model. Danggui extracts (10 mg/kg/day) and TPO (10 ug/kg/day) were given by injection daily for 21 days starting from the day after radiotherapy. Peripheral blood platelet, WBC and RBC from Danggui, TPO and water control groups were analyzed at different time-points. On day 21, the mouse was sacrificed and bone marrow cells were harvested for CFU-MK and CFU-GM assays using the plasma clot and methylcellulose methods. The results showed that both Danggui and TPO significantly enhanced the recovery of platelet and WBC count, as well as CFU-MK and CFU-GM formation (n=6). Morphological examination of bone marrow also showed that Danggui treatment significantly increased the recovery of hematopoietic progenitor cells and megakaryocytic cell series. In addition, Danggui also increased the recovery of the body weight and organ weight (liver and spleen) in mouse model. We further analyzed the in vitro effect of Ferulic acid (major component of Danggui) and Astragaloside IV (major component of Huangqi) on hematopoiesis using CFU assay. Ferulic acid plus Astragaloside IV significantly promoted mouse CFU-GEMM formation (n=4). Ferulic acid together with Astragaloside IV also significantly reduced serum free-induced apoptosis in K562 cells by annexin V assay using flow cytometry (n=3). This anti-apoptotic effect can be mediated by reducing the activation of Caspase-3 pathway. In summary, our studies showed that Danggui extracts as well as TPO enhances hematopoiesis and thrombopoiesis in a radiation-induced hematocytopenic mouse model. Ferulic acid, in combination with Astragaloside IV, also has promoting effect on hematopoiesis and anti-apoptotic effect on hematopoietic cells.

Disclosures:

No relevant conflicts of interest to declare.
Transcriptional Silencing of the Wnt-Antagonist Dickkopf-1 (DKK1) by Promoter Methylation Unleashes Aberrant Wnt Signaling In Advanced Multiple Myeloma
Authors: Kocemba Kinga A , Groen Richard W , van Andel Harmen , Mahtouk Karene , Kersten Marie Jose , Spaargaren Marcel , Pals Steven T
Published: October 2019

Abstract 1919

Aberrant activation of the Wnt/β-catenin pathway is implicated in driving the formation of various human cancers. Recent studies indicate that the Wnt pathway plays at least two distinct roles in the pathogenesis of multiple myeloma (MM): i) Aberrant, presumably autocrine, activation of canonical Wnt signaling in MM cells promotes tumor proliferation and metastasis; ii) Overexpression of the Wnt inhibitor Dickkopf1 (DKK1), contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggests the presence of a negative feedback loop in MM, in which DKK1 acts as a potential tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is lost in most MM cell lines and in a subset of patients with advanced MM. This loss is correlated with activation of the Wnt pathway, as demonstrated by increased nuclear accumulation of β-catenin. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

Disclosures:

No relevant conflicts of interest to declare.