bluuebunny/crossref_metadata_2025 huggingface dataset:
ALDH1 Expression in Diffuse Large B-Cell Lymphoma: A Study of 88 Cases.
Authors: Olson Ryan K. , Li Li , Sheehan C. E , Homan Suzanne M. , Hayner-Buchan Alida , Nazeer Tipu
Published: October 2019

Abstract 5019

Introduction

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous category and tremendous effort has been made to identify sub-groups that are prognostically significant and to guide therapeutic decisions. Aldehyde dehydrogenase (ALDH) a family of ubiquitous enzymes throughout mammalian tissues catalyzes the oxidation of aldehydes to their corresponding carboxylic acids. Isoform 1 (ALDH1) plays a role in the biosynthesis of retinoic acid from Vitamin A. Retinoic acid is a regulator of cellular proliferation, differentiation and survival in normal human tissues. High expression of ALDH1 has been demonstrated in the putative cancer stem cells in malignancies of breast, lung, colon and other organs. Its expression has been associated with increased replicative potential, the ability to differentiate along multiple development lines, high tumorigenic potential and increased metastatic potential. However, more recently ALDH1 expression has been shown to correlate with favorable outcome in ovarian cancer. It has not been studied in DLBCL. The aim of the current study is to evaluate the expression of ALDH1 in DLBCL.

Methods & Materials

Formalin-fixed, paraffin embedded sections from 88 DLBCLs, were immunostained by automated methods (Ventana Medical Systems, Inc, Tucson, AZ) using monoclonal mouse anti-human ALDH antibody (Clone: 44/ALDH; Immunogen: Human ALDH1, aa 7 – 128; BD Transduction Laboratories). Cytoplasmic immunoreactivity was semi quantitatively assessed in all cases. Scoring was based on staining intensity (weak, moderate, intense) and percentage of positive cells (focal <= 25%, regional 26-50%, diffuse >50%).

Results

were correlated with clinicopathologic variables.

Results

High expression of ALDH1 was observed in 26/88 (30%) cases of DLBCL. The staining intensity and distribution pattern varied somewhat by outcome data. Remission status was known in 23 patients. 18 patients achieved complete remission. Interestingly, all 7 patients with high expression of ALDH1 achieved complete remission (p= 0.095). Recurrence information was known in 87 patients. High expression of ALDH1 was seen in 26 cases. Only one of these patients recurred (p=0.19). No association was seen with stage, HIV status, survival or age.

Conclusion

We have demonstrated that ALDH1 is expressed in DLBCL. Moreover, cases with high expression of ALDH1 trend towards complete remission and lack of recurrence. Further studies of ALDH1 expression in DLBCL appear warranted, potentially defining prognostically relevant sub-groups.

Disclosures

No relevant conflicts of interest to declare.
Primary Cutaneous Follicle Center Lymphoma in An HTLV-1 Carrier.
Authors: Phillips Adrienne A. , Oshikanlu Zion , Nuovo Gerard J , Bhagat Govind , Savage David
Published: October 2019

Abstract 5032

Background

Human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus associated with adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm of mature CD4+ T-lymphocytes which always shows monoclonal integration of HTLV-1 provirus DNA. HTLV is not associated with B-cell non-Hodgkin's lymphoma (B-NHL); however rare cases of B-NHL have been reported in HTLV-1 carriers, leading some to hypothesize a role for HTLV-1 in B-cell lymphogenesis. We report a case of a Jamaican male HTLV-1 carrier with primary cutaneous follicle center lymphoma of B-cell origin and explore the link between HTLV-1 and B-NHL.

Case Report

A 55 year old Jamaican male was seen in our hematology clinic for evaluation of multiple scalp masses progressive over six months. The lesions were painful, pruritic, but not associated with other constitutional symptoms. He had been in good health prior to the onset of these symptoms, and denied toxic habits or exposures. Physical examination revealed multiple firm, non-tender scalp masses, the largest measuring 3.0 × 2.0 cm; the overlying skin was nodular and erythematous but without breakdown or discharge. His complete blood count, basic metabolic profile, hepatic function and lactate dehydrogenase were all within normal limits. On excisional biopsy, the dermis showed a dense lymphoid infiltrate of medium to large sized cells, with a nodular and trabecular pattern of growth from the superficial dermis to the aponeurosis. Numerous large centroblast-like cells were present. Immunohistochemical stains demonstrated the following phenotype: CD45+, CD20+, CD79a+, BCL2+/-, BCL6+/-, CD10+, and MUM-/+. A stain for CD21 highlighted compact and fused/contiguous follicular dendritic cell meshworks in association with the neoplastic B-cell infiltrate. Staining with Ki-67 highlighted a variable but high proliferation rate (80%). A moderate infiltrate of small-sized T-cells (CD3+, CD5+) was also present. These features were diagnostic of a primary cutaneous follicle center lymphoma. Evaluation with the MYC breakapart probe showed no evidence of rearrangement; RT in-situ PCR for HTLV-1 showed a signal in many of the infiltrating T-cells. He was seropositive for positive HTLV-1 and negative for HIV and EBV infection. Bone marrow biopsy and aspiration and additional staging CTs were unremarkable. The patient received 4 cycles of R-CHOP chemotherapy and has been in a complete remission for 2 years.

Discussion

We report a case of a Jamaican male HTLV-1 carrier with cutaneous lymphoma of B-cell origin with detection of HTLV-1 proviral DNA in the neoplastic cells by RT in-situ PCR. Our findings lead us to speculate that HTLV-1 infected T-cells and/or an immunodeficient state in certain HTLV-1 carriers might allow for the development of B-cell lymphoma. Further research on the pathogenesis of HTLV-1 in B cell lymphomagenesis needs to be explored.

Disclosures

No relevant conflicts of interest to declare.
Venous Thromboembolism in the Elderly: Epidemiological Data Overview Based On the 2149-Patients Prospective OPTIMEV Cohort.
Authors: Pernod Gilles , Labarere Jose , Sevestre Marie Antoinette , Bosson Jean Luc
Published: October 2019

Abstract 5063

VTE is a multifactorial disease, but one of the strongest risk factor is age. Despite this high incidence, there is surprisingly little known about VTE in this population. So, based on the large OPTIMEV cohort study on VTE, the aim of this study was to focus on epidemiological information regarding VTE among old population.

OPTIMEV was a multicentre prospective cohort study of patients referred for clinically suspected VTE throughout France. VTE presentations were categorized using objective tests including ultrasonography, lung scan and helical CT scan. Subjects without a confirmed diagnosis of VTE were used as controls.

A total of 8256 patients were included in the OPTIMEV study, among which the aged study population (> 75y) was 2149. The median age of the old group was 81 years (76 - 101 years). Among this cohort, a total of 655 patients (30.5%) were positive cases of VTE, including 203 isolated distal DVT (31%), and 452 (69%) proximal DVT +/- PE. Diagnosis of VTE was ruled out in 1494 patients (69.5%). To identify potential specific risk factors in older patients, we compared VTE positive aged patients to younger. For this purpose, we tested by logistic regression the interaction between age (more or less than 75) and risk factors. Personal or familial history of VTE, travel, cardiac or respiratory insufficiency were not specific of the difference in incidence of VTE in older compared to younger patients. Only male gender (OR 1.4, 95% CI 1.1 – 1.9), bed rest (OR 1.4, 95% CI 1.1 – 1.9), lower limb immobilization (OR 1.1, 95% CI 0.5 – 2.5 and surgery <45d (OR 1.2, 95% CI 0.9 – 1.7) were found to be significantly different risk factors for VTE according to age.

Our epidemiological data, based on a large cohort, showed few really specific risk factors in this old population, compared to younger.

Disclosures

No relevant conflicts of interest to declare.
Transient Iron Resistance in Severe Iron Deficiency Anemia.
Authors: Milani Cannon , Colvin Gerald A. , Mazur Eric , Berz David
Published: October 2019

Abstract 5104

Background

Iron deficiency is the primary cause of anemia in the United States. Occasionally the anemia is severe enough to require transfusion of blood products. Anecdotal case reports described a state of transient iron intolerance in patients with profound, prolonged iron deficiency. We are presenting a study, evaluating in a prospective fashion how the iron parameters of patients with severe iron deficiency respond to blood transfusion and compare this to the trajectory of patients with other causes of anemia.

Methods

We included nineteen consecutive patients admitted to the medical floors with the diagnosis of anemia. Six of them had severe iron deficiency. The remaining part of the cohort carried the diagnosis of refractory anemia or anemia of chronic disease. We evaluated serum iron level, total iron binding capacity (TIBC), transferrin, and ferritin levels immediately prior to the transfusion of two units PRBCs and then six, twelve, twenty four, and forty eight hours after the completion of the transfusion, and correlated them to the pre- and post-transfusional IL-1, IL-2, IL-6, IL-10, IL-18, MIB-1β, MCP-1, erythropoietin, and hepcidin values of severely iron deficient patients and patients with other causes of their anemia.

Results

Patients with severe iron deficiency anemia showed a transient peak of the serum iron levels immediately after completion of blood transfusion. This normalized after 24 hours. In the not severely iron deficient population, no peak in the serum iron levels was observed. The remaining iron parameters, namely TIBC, transferrin, and ferritin remained unchanged. The transient elevation of the serum iron levels correlated with changes in the level of different cytokines.

Conclusion

Severely iron deficient patients show a transient intolerance to externally, parenterally delivered iron, that normalizes within 24 hours.

Disclosures

No relevant conflicts of interest to declare.
Efficacy of Ursodeoxycholic Acid with Low-Dose Heparin and Prostaglandin E1 in the Prevention of Hepatic Veno-Occlusive Disease After Allogenic Hematopoietic Stem Cell Transplantation in Children with β-Thalassemia Major.
Authors: Wu Xuedong , Liu Huaying , He Yuelin , Feng Xiaoqin , Chen Geyu , Su Qingxia , Meng Fanyi , Li Chunfu
Published: October 2019

Abstract 5109

Objective

This study was to explore more efficient methods of prevention of VOD in patients with thalassemia. We explore the efficacy of VOD prophylaxis with ursodeoxycholic acid, lipo-prostaglandin E1 and low-dose unfractionated heparin in allogeneic heamatopoietic stem cell transplantation(HSCT) for patients with thalassemia. Methods:Fifty-eight children with β-thalassemia major received allo-SCT for 61 times. For the prevention of VOD, all patients received low-dose heparin(100-150IU/kg · d) and also received PGE1(1-1.5 · d) by continuous intravenous infusion from the beginning of conditioning treatment to the 20th day after allo-SCT. Patients who received ursodeoxycholic acid (12mg/kg.d,oral,from the beginning of conditioning treatment to the 90th day after allo-SCT)were used as the study group (n=33).Patients who did not received ursodeoxycholic acid were used as the control group (n=28).Results:VOD occurred in 3 patients(8.8%) in the study group. Ten patients in the control group had VOD(37.0%).There were no obvious drug adverse effects in the two groups.

Conclusions

Ursodeoxycholic acid with low-dose heparin and PGE1(study group) is more effective than low-dose heparin and PGE1 only (control group) for the prevention of VOD after allo-SCT for children with β-thalassemia major.

Disclosures

No relevant conflicts of interest to declare.
The Preparative Method and Clinical Application of Autologous memory T Lymphocyte.
Authors: LI D. , Ouyang Jian , LI CP , Chen Jh , Zhou M. , Zhou Rong-Fu
Published: October 2019

Abstract 5123

Background

The term autolymphocyte therapy refers to an adoptive cellular transfer strategy based on the ex vivo activation of memory T cells. The assumption is that because patients with metastatic cancer have been previously exposed to tumor antigens, memory T cells exist which can be collected and activated ex vivo. The result in autologous cytokines that can be used to activate other T cells. The method is simpler than it sounds and basically consisted of 2 cell collections using a cell separator and 2 cell cultures. The supernatant from the first culture, which contains the desired cytokines, is used to activate the second cohort of T cells.

Objective

To establish a preparative method of autologous memory T lymphocyte and use this T lymphocyte to treat hematologic malignancies and solid tumor.

Method

14 patients of hematologic malignancies and solid tumor had autolymphocyte therapy in 4 hospitals since 2002(Plasmacytoma1,NHL1, colon cancer3,HCC3,NSCLC1, nasopharyngeal carcinoma3, breast cancer1, Ovarian cancer1). These patients, including 11 male and 3 female, with a mean age of 55 (range, 25-71), were finally confirmed by pathology and histology autologouslymphokine, anti-CD3 antibody, IL-2. Cimetidine and Indomethacin induced peripheral blood mononuclear cell (PBMC) ex vivo to memory T lymphocyte by two steps. We tested memory T lymphocyte by flow cytometry before and after culture, we observed side effect during infusion.

Result

After culturing peripheral blood lymphocyte for 9 days, the number of cell and CD4+/CD45RO+T cell increased. The data demonstrated: PBMC before culture was 1.07-67.2×109(median was 2.16×109),the cell after culture was 2.0-6.5×109(median was 3.2×109),CD4+/CD45RO+T cell rate was 27.59-86%(median was 77.76%) before culture and 82.09-99.55%(median was 89.49%) after culture. The absolute number of CD4+/CD45RO+T cell increased. Among 14 patients after infusion, one patient who had no medicine before infusion had fever of 39°C, 4 patients had low-grade fever and the rest 9 patients had no side effect. Follow-up: 5 patients abroad had no follow-up. In 1 to 10 months after therapy, all patients were alive. 4 patients were in partial remission and 5 patients in stable stage.

Conclusion

Autologous memory T lymphocyte is a safe and an effective way to treat advanced-stage hematologic malignancies and solid tumor. The mechanism and long-tem effect need more research and observe.

Disclosures

No relevant conflicts of interest to declare.
Factor VIII-Fc Fusion Protein Shows Extended Half-Life and Hemostatic Activity in Hemophilia A Dogs.
Authors: Dumont Jennifer A , Kamphaus George D , Fraley Cara , Ashworth Tamera , Franck Helen , Merricks Elizabeth P. , Nichols Timothy C. , Bitonti Alan J
Published: October 2019

Abstract 545

A recombinant B-domain-deleted factor VIII-Fc (rFVIIIFc) fusion protein was created as an approach to extend the half-life of FVIII. The pharmacokinetics and pharmacodynamics of rFVIIIFc were evaluated in the Chapel Hill colony of hemophilia A dogs. These dogs have a severe hemophilic phenotype comparable to the severe form of human disease with F.VIII < 1%. A single intravenous dose (125 IU/kg) was administered to four dogs and immediately corrected the clotting to normal as measured by whole blood clotting time (WBCT) and aPTT. The WBCT remained below 20 min, the time consistent with FVIII:C > 1%, through approximately 96 h. The range of WBCT in our normal dogs is 8 to 12 min. The concentration of rFVIIIFc in the plasma was measured by ELISA and the terminal half-life was 15.7 ± 1.7 hr. Similar results were obtained when rFVIIIFc was measured using a FVIII-specific chromogenic activity assay (half-life was 15.4 ± 0.3 hr). The concentration vs. time curves were similar using both methods. The activity data were converted to ng/mL using the specific activity of the test article that was used to dose the animals, and these data correlated well with the ELISA data, thus demonstrating that the protein that was measured by ELISA was fully active.

Two of the dogs also received a single dose of recombinant B-domain deleted FVIII (rBDD-FVIII, ReFacto®), 114 IU/kg for one dog and 120 IU/kg rBDD-FVIII for the other, and then received rFVIIIFc (125 IU/kg) 72 hr later in a cross over design. Clotting was corrected to normal immediately after dosing with both rBDD-FVIII and rFVIIIFc (determined by WBCT and clotting activity measured using an aPTT assay). However, the WBCT normalization after rFVIIIFc lasted for approximately twice as long compared to rBDD-FVIII and the half-lives determined from the ELISA data for FVIIIFc (15.7 ± 1.7 hr) were twice those determined for rBDD-FVIII (7.0 hr and 6.7 hr). No adverse clinical signs were detected with any of the infusions. Therefore construction of an Fc fusion of FVIII produces a molecule with a defined mechanism of action that has an increased half life and the potential to provide prolonged protection from bleeding.

Disclosures:

Dumont: Biogen Idec (Syntonix Subsidiary): Employment. Kamphaus:Biogen Idec (Syntonix Subsidiary): Employment. Fraley:Biogen Idec/Syntonix Subsidiary: Employment. Ashworth:Biogen Idec (Syntonix Subsidiary): Employment. Bitonti:Biogen Idec/Syntonix Subsidiary: Employment.
Transferrin Receptor 2 Protein Is Expressed in Mitochondrial-Associated Lysosomes During Human Erythropoiesis.
Authors: Noh Seung-Jae , Wu Yun-Ping , Byrnes Colleen , Lee Y. Terry , Miller Jeffery L.
Published: October 2019

Abstract 628

Regulated expression of transferrin receptor 1 (TFR1) provides for the major mechanism of iron binding and absorption by endocytosis in erythroblasts. In contrast, the expression of transferrin receptor 2 (TFR2) in erythroid cells is not well understood. Fourteen-day cultures of human CD34+ cells were utilized to explore TFR2 gene and protein expression during erythropoiesis. Transcriptome analyses revealed an expression of the TFR2 gene on culture days 7 and 14. Protein analyses were performed with a TFR2 antibody that demonstrated a doublet band at 105 Kd in K562 and HepG2 cells, but no band in Jurkat cells. Western analyses of the primary erythroblasts demonstrated maximum TFR2 levels on culture days 6-8 coinciding with the onset of high-level glycophorin A expression and just prior to hemoglobin accumulation in the cells. Cellular localization of TFR2 was assessed by dual-staining confocal microscopy. More than 15 cells were analyzed for the calculation of correlation coefficients from 4-6 separate pictures per each sample. Unlike TFR1, expression of TFR2 on the plasma membrane was low or absent at all stages of erythroblast maturation. Expression of both proteins was detected in the endosomes throughout the cytoplasm on culture days 2-6. After culture day 6, TFR1 and TFR2 localization became divergent, with TFR2 localized almost exclusively to a single perinuclear compartment near the Golgi stack in the region of the centrosome. Co-localization experiments indicated that TFR2 was most highly co-localized with LAMP1, a lysosome marker. DMT1, the divalent metal transporter was also colocalized with the TFR2-lysosomes. The TFR2-lysosomes were additionally surrounded by and closely associated with mitochondria. Live cell imaging revealed dynamic interactions between the lysosomes and surrounding mitochondria. Pulse-chase experiments using Alexafluor 594-labeled transferrin (Tf) showed that endocytosed Tf was initially co-localized with TFR1. However, Tf was transported to the TFR2 localized region after a chase period of 10 minutes. In summary, TFR2 protein expression is highly regulated during erythropoiesis and localized to centrosomal lysosomes. These studies further suggest that iron regulation in erythroblasts includes trafficking to TFR2 and DMT1 containing lysosomes, which are dynamically associated with mitochondria.

Disclosures:

No relevant conflicts of interest to declare.
Evaluating the Response to Imatinib In Philadelphia-Positive Chronic Myeloid Leukemia (Ph+ CML): The Value of Major Molecular Response (MMolR) at 12 Months
Authors: Baccarani Michele , Rosti Gianantonio , Martinelli Giovanni , Castagnetti Fausto , Palandri Francesca , Gugliotta Gabriele , Amabile Marilina , Testoni Nicoletta
Published: October 2019

Abstract 668

The cytogenetic response is a confirmed early surrogate marker of the outcome of Ph+ CML patients treated with imatinib (Baccarani et al, JCO 2009;27:6041-51). Many reports and reviews highlight the value of MMolR, defined as a BCR–ABL value equal/less than 0.1% on the International Scale (Hughes et al, Blood 2006;108:20-37). The European LeukemiaNet recommendations use the BCR-ABL level for the definition of optimal response at 12 months (achievement of MMolR) and suboptimal response at 18 months (less than MMolR), but not for the definition of failure (Baccarani et al, JCO 2009; 27:6041-51). As the prognostic value of achieving a MMolR may increase with the introduction of 2nd generation tyrosine kinase inhibitors (TKIs) in the frontline treatment of Ph+ CML, we have reviewed and compared the cytogenetic and molecular data of 4 company–sponsored studies and 4 independent investigator–sponsored studies, for a total number of 2466 patients treated frontline with imatinib (Table 1). The CCgR rates at 12 months ranged between 49% and 88% (median 66%). The MMolR rates at 12 months ranged over a wider range, between 15% and 65% (median 33%). The ratio CCgR/MMolR ranged between 1.31 and 1.90 (median 1.60) in 5 out of 8 studies, while in the other 3 studies (Hammersmith, ENESTnd, and DASISION) the ratio was much higher, ranging between 2.35 and 4.00. Comparing Hammersmith, ENESTnd and DASISION studies to the other 5 studies (IRIS, TOPS, ELN, GIMEMA, and TIDEL), the CCgR rates were similar (median 64% vs. 67%, range 45–73% vs. 58–88%), while the MMolR rates were lower (median 22% vs. 40%, range 15–31% vs. 26–65%). These data cannot be used to argue that a lab, a dose of imatinib, or a study, is “better or worse” than the others, but higlight the consistency of CCgR worldwide and warns from overemphasizing the MMolR rate at 12 months for response evaluation and for treatment adaptation or modification.

Molecular studies sere performed: IRIS at 3 labs (Hammersmith, Adelaide; Seattle), Hammersmith at 1 lab, TIDEL at 1 lab (Adelaide), ELN at Bologna and many other labs, TOPS at 3 labs (Naples, Adelaide and Seattle), GIMEMA at 1 lab (Bologna), ENESTnd and DASISION at 1 lab (Portland).

Disclosures:

Baccarani: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Research Funding; Wyeth: Consultancy, Research Funding. Rosti:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Gugliotta:Novartis: Honoraria.
Small Leucine Rich Proteoglycans as Novel Tumor Markers In Chronic Lymphocytic Leukemia
Authors: Mikaelsson Eva , Jeddi-Tehrani Mahmood , Ã–Sterborg Anders , Shokri Fazel , Rabbani Hodjattallah , Mellstedt Håkan
Published: October 2019

Abstract 694

Background:

Small leucine rich proteoglycans (SLRPs) are a family of glycosylated proteins normally expressed in the extracelluar matrix (ECM) of collagen rich tissues. The biological role of the SLRPs is multifactoral, but mostly these proteins are secreted and bind to membrane receptors or ECM proteins affecting cell proliferation and cell migration. Some SLRPs (eg. Decorin, Lumican) have been reported to be expressed in cancer, but the expression as well as the glycosylation pattern differ. Microarray studies have revealed that the SLRP family member fibromodulin (FMOD) gene is overexpressed in chronic lymphocytic leukemia (CLL). We later reported the specific expression of FMOD in CLL and mantle cell lymphoma (MCL). FMOD is located on chromosome 1q32 adjacent to two other members of the SLRP family, proline/arginine-rich end leucine-rich repeat protein (PRELP) and opticin (OPTC).

Aims:

To analyse 1) the expression of PRELP and OPTC in CLL and other hematological malignancies. 2) the glycosylation pattern of PRELP and OPTC, and cellular localization of OPTC in CLL cells.

Methods:

PRELP: Gene expression was tested by RT-PCR and realtime-PCR. Protein expression was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the PRELP protein. OPTC: Cell fractionation was performed using four different methods. Protein expression (molecular weight and cellular location) was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the OPTC protein.

Results:

PRELP was expressed at the gene level (RT-PCR) in all CLL patients tested (n=30) and in 3/5 patients with mantle cell lymphoma, but not in normal leukocytes (n=10) or in other hematological malignancies (7 different types, n=35). The PRELP protein was detected in all CLL samples tested but not in leukocytes of healthy donors (western blot). Molecular analysis of the CLL PRELP protein revealed a unique unglycosylated 38 kDa core protein, with an intact signal peptide. This protein was not detected in serum that, in combination with the uncleaved signal peptide, suggests cellular retention. A “normal” OPTC protein (50 kDa) was detected in cell lysates of both CLL tumor cells (n=30) and normal leukocytes (n=10). However, the CLL cells also expressed a 37 kDa OPTC that was not detected in healthy controls. This 37 kDa OPTC was detected in all CLL samples and molecular analysis revealed a unique unglycosylated core protein that was located in the cell nucleus and endoplasmatic reticulum of the CLL cells.

Conclusion:

The unique expression of the SLRPs PRELP and OPTC in CLL (in addition to the previously reported FMOD) is unexpected and merits further studies since the specific expression of three closely related SLRP genes may indicate a role in the pathobiology of the disease.

Disclosures:

No relevant conflicts of interest to declare.
A Predictive Model for the Development of High-Responding Inhibitor In Children with Severe and Moderate Hemophilia A- Results of a Multicenter Cohort Study
Authors: Holzhauer Susanne , Bidlingmaier Christoph , Ettingshausen Carmen Escuriola , Halimeh Susan , Iorio Alfonso , Kenet Gili , Pautard Brigitte , Nowak-Gottl Ulrike
Published: October 2019

Abstract 710

Background:

Pediatric cohort studies in previously untreated [PUPs] children with hemophilia A [HA] identified potential risk factors for clinical meaningful inhibitor development [IH], among them HA severity < 1%, high risk gene mutations [HRS], high dose FVIII replacement therapy [HD] and use of recombinant FVIII concentrates [rFVIII]. The aim of the present study was to develop a simple score to rate inhibitor development in HA children.

Patients and methods:

184 consecutively ascertained PUPs [enrollment between 1982 and 2008] with HA [severity < 5%] followed over a period of 200 exposure days were randomly selected into two groups [G1; G2] of 92 patients each. Three variables [HRS: 2 points], [HD: median single dose > 30 IU/kgbw within 8 to 12 weeks after therapy start] and [rFVIII] 1 point each, were incorporated into a risk assessment model, which was i) evaluated in patient group G1 [pilot cohort: patients not included in the CANAL study] and ii) validated in the second group [G2: confirmation cohort]. The risk discrimination cut-off was calculated using two independent statistical methods [ROC-analysis: MedCalc 11.0.0; CART regression tree: CART 6.0]. In addition, model specific cumulative IH-free survival [Cox regression analysis: hazard ratio (HR) and 95% confidence interval (CI)], negative predictive values [NPV], sensitivity and specificity were calculated. In Cox regression analysis adjustment was performed for i) age at first symptomatic bleed [proxy for early start of prophylaxis], ii) severity of factor VIII-deficiency, and iii) for treatment periods [1982-2008: divided into 3 to 5 year intervals].

Results:

The risk discrimination cut-off in the pilot cohort G1 was calculated as “2”. Model sensitivity was 78.8 % and specificity was 49.8% respectively. In the confirmation cohort G2 seventeen children with HA developed a high responding IH [18.5%]: 16 out of 52 in children with a risk score > 2 [30.8%] and one out of 40 with a risk score < 2 [2.5%: p<0.001]. Compared to children with a risk score < 2 the cumulative IH-free survival in HA patients with a risk score > 2 was significantly reduced with an adjusted HR [CI] of 11.2 [1.5-83.5]. The corresponding NPV to rule out a high responding IH development in the confirmation cohort was 92% [80.5-98.5].

Conclusion:

The model is able to rule out the development of high responding IH in white children with HA < 5% with good discriminative ability. Thus the model can be instrumental to individualize treatment strategies in HA patients. The score needs to be validated in other populations to test its generalizability.

Disclosures:

No relevant conflicts of interest to declare.
Rational Design and Characterization of Platelet Factor 4 Antagonists
Authors: Sachais Bruce S , Rux Ann H , Hinds Jillian L , Rux John J
Published: October 2019

Abstract 724

Background:

Heparin-induced thrombocytopenia and thrombosis (HITT) is a serious complication of heparin therapy, placing patients at increased risk for thrombosis and death. The chemokine platelet factor 4 (PF4) is central to the pathogenesis of HITT. Tetrameric PF4 forms ultralarge complexes (ULC) with heparin, which, when bound to specific antibodies, lead to thrombocytopenia and thrombosis. We have previously shown that the tetrameric form of PF4 is required for ULC formation. Since a large number of hospitalized patients are exposed to heparin, HITT is a major iatrogenic cause of morbidity and mortality in this patient population. There are currently no specific therapies for the treatment of HITT.

Objectives:

Identify small molecule antagonists of PF4 tetramerization to serve as reagents for the mechanistic study of HITT as well as lead compounds for development of novel HITT therapies.

Approach:

Using the crystal structure of PF4, we identified a potential antagonist binding site near residues glutamic acid 28 (E28) and lysine 50 (K50). These residues form salt bridges between two PF4 dimers which are required for tetramer formation. We then performed an in silico screen of 1.1×10^6 lead-like small molecules using the program DOCK to identify compounds likely to bind to this site. A subset of these compounds were obtained and tested for their ability to inhibit PF4 tetramerization and PF4:heparin ULC formation in vitro.

Results:

In silico screening identified 109 compounds with DOCK scores (lower scores predict higher binding affinity) which were greater than 10 standard deviations below the mean (see Figure). We have obtained 12 of these compounds for in vitro testing. The ability of antagonists to inhibit PF4 tetramerization was measured as previously described by quantitation of silver stained SDS-PAGE gels of cross-linked PF4 in the absence and presence of putative antagonists. 5 of the compounds showed inhibition of tetramerization in a dose dependent manner, with 2 of these compounds (PA31 and PA35) demonstrating micromolar potency (IC50 of tetramerization = 500uM and 600uM respectively). The ability of these antagonists to inhibit ULC formation between PF4 and heparin was measured by both gel filtration and ELISA as previously described. PA31 and PA35 both inhibited ULC formation with potencies similar to that for inhibition of tetramerization.

Conclusions:

We have identified two small molecule PF4 antagonists. These compounds will be useful in future mechanistic studies of HITT. They also may serve as lead drug discovery compounds for chemical modification to create PF4 antagonists with increased affinity and potency. Such compounds may be tested as novel therapeutics for the treatment and/or prevention of HITT.

Disclosures:

Rux: In sillico Molecular, LLC: Employment, Owner.
Assessment of Residual Bulky Tumor Using FDG-PET In Patients with Advanced-Stage Hodgkin Lymphoma After Completion of Chemotherapy: Final Report of the GHSG HD15 Trial
Authors: Engert Andreas , Kobe Carsten , Markova Jana , Haverkamp Heinz , Borchmann Peter , Hitz Felicitas , Zijlstra Josee , Eich Hans , Mueller Rolf-Peter , Dietlein Markus , Diehl Volker
Published: October 2019

Abstract 764

Introduction

The role of additional radiotherapy after chemotherapy for advanced-stage Hodgkin lymphoma is unclear. The German Hodgkin Study Group (GHSG) thus performed the HD15 trial in which advanced-stage Hodgkin lymphoma patients having residual disease after 6–8 cycles of BEACOPP were evaluated by 18F-fluorodesoxyglucose positron emission tomography (PET) following chemotherapy.

Methods

Entry criteria for the PET question in HD15 were partial remission (PR) after the end of chemotherapy with at least one involved nodal site measuring more than 2.5 cm in diameter by computed tomography (CT). Exclusion criteria included diabetes, elevated blood sugar levels and skeletal involvement with risk of instability. Calculations were restricted to those cases with either progressive disease (PD) or relapse within 12 months after PET or at least 12 months of follow-up. A total of 2,137 patients with de novo HL were included in HD15 of whom 728 had a tumor bulk ≥ 2.5 cm after BEACOPP chemotherapy and were qualified for the PET question. An expert panel performed the assessment of response and PET. Only PET-positive patients were scheduled for radiotherapy of residual disease. The negative prognostic value (NPV) of PET was defined as the proportion of PET-negative patients without progression, relapse or radiotherapy despite being PET-negative within 12 months.

Results

The full analysis set included 728 patients of whom 699 had at least 12 months of follow-up. Median age was 30 years, 57% were males and 66% had NS histology. Of the 728 qualified patients with residual disease ≥ 2.5 cm after BEACOPP, 74.2% were PET-negative and 25.8% PET-positive. In the PET-negative group, a total of 28 patients relapsed or had radiotherapy despite being PET-negative (8 patients including 1 relapsing patient) resulting in a negative prognostic value of 94.6% (95% CI 92.7% to 96.6%). With a median follow-up of 38 months, the time-to-progression after PET at 3 years was 92.1% for PET-negative patients counting radiotherapy as failure and 86.1% for PET-positive patients (95%-CI for difference -11.9% to -0.1%). Overall, only 11% of patients had additional radiotherapy as compared to 71% after BEACOPPescalated in our prior HD9 trial. In addition, there was no difference in PFS or overall survival as compared to our earlier trials in advanced-stage HL.

Discussion

The NPV of PET of 0.95 suggests that indeed only patients with residual disease after chemotherapy who are PET-positive need additional radiotherapy. PET-negative patients at least after BEACOPP can be spared from additional radiotherapy.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Pro-Survival Function of Cyclin-D1 Underlies Its Oncogenic Role and Potential as a Therapeutic Target In Mantle Cell Lymphoma
Authors: Beltran Elena , Fresquet Vicente , Martinez-Useros Javier , Richter-Larrea Jose A. , Sagardoy Ainara , Sesma Izaskun , Almada Luciana L. , Montes-Moreno Santiago , Gesk Stefan , Siebert Reiner , Calasanz MaJose , Prosper Felipe , Piris Miguel A. , Fernandez-Zapico Martin E. , Martinez-Climent Jose A
Published: October 2019

Abstract 769

Despite the many and diverse therapeutic approaches used to treat patients with mantle cell lymphoma (MCL), it remains an incurable disease. Recently, attention has turned into novel therapies targeting MCL-specific oncogenic pathways important for the growth and maintenance of the transformed phenotype. The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 over-expression is the hallmark of MCL. Constitute cyclin-D1 activation in B-lymphocytes maintains retinoblastoma protein in a phosphorylated state and promotes cell cycling, thus initiating the tumorigenesis process. Cyclin-D1 has been postulated as a putative target for therapeutic intervention, however its evaluation has been hampered by the incomplete understanding of the mechanism underlying this cyclin oncogenic function and by the lack of valid MCL models. To investigate these issues, we developed a combined cellular-genomics screening whereby responses to known cytotoxic compounds targeting cancer-related molecular pathways were correlated with genomic, gene expression and proteomic profiles of human MCL cells. Results showed that cyclin-D1 silencing had minimal antitumoral effects but significantly increased the therapeutic efficacy of several compounds, especially the BH3 mimetics that inhibited anti-apoptotic protein BCL-2. To further evaluate this finding we generated a MCL mouse model by transducing a tetracycline-regulatable cyclin-D1-expressing vector in murine pro-B cells, which allowed modulating cyclin-D1 expression levels. These mice generated lymphomas recapitulating most of the cellular, histopathological and molecular features of human MCL. Similar to the previous in vitro findings, cyclin-D1 inhibition in this model did not induce lymphoma regression, but sensitized cells to apoptosis. Analysis of the mechanisms underlying this therapeutic synergy identified a novel role for cyclin-D1 as a pro-survival molecule. Specifically, cyclin-D1 sequestrated the pro-apoptotic effector protein BAX in MCL cells, thereby favoring BCL2 anti-apoptotic function. Accordingly, therapeutic cyclin-D1 inactivation released BAX, thus sensitizing cells to apoptosis and inducing lymphoma regression. Interestingly, pharmacological blockade in vivo of cyclin-D1 with Roscovitine synergistically cooperated with the BH3 mimetic ABT-737 to effectively inhibit MCL tumor growth. In summary, our study reveals a novel role for cyclin-D1 in deregulating apoptosis in MCL cells and highlights the potential benefit of cyclin-D1 targeting, thus providing the rationale for the clinical evaluation of drugs targeting cell proliferation and survival pathways in MCL.

Disclosures:

Siebert: Abbott: Honoraria.
Up-Regulation of Anti-Inflammatory, STAT3-Activating Hepatocyte Growth Factor and Interleukin-11 In Polycythemia Vera Is Independent of JAK2V617F and Contributes to the Growth of Clonal Erythroblasts
Authors: Boissinot Marjorie , Cleyrat Cedric , Vilaine Mathias , Jacques Yannick , Corre Isabelle , Hermouet Sylvie
Published: October 2019

Abstract 796

Context:

The V617F activating mutation of JAK2, JAK2V617F, characterizes Polycythemia vera (PV). However, expression of JAK2V617F does not always result in clone expansion, implying that additional events are required for the growth of JAK2-mutated progenitors and development of the PV phenotype. In the present study, we provide evidence that anti-inflammatory cytokines are required for the growth of JAK2V617F-mutated erythroid progenitors.

Methods:

We searched for cytokines over-expressed in PV using cytokine antibody arrays and ELISAs for analyses of serum and bone marrow (BM) plasma, and quantitative RT-PCRs for analyses of cells purified from PV patients and controls. Transient transfection of JAK2V617F and invalidation of JAK2V617F with siRNA were used to study the effect of JAK2V617F on cytokine expression.

Results:

We found that PV patients over-expressed anti-inflammatory hepatocyte growth factor (HGF) and interleukin-11 (IL-11), BM stromal cells (BMSC) and erythroblasts being the main producers. HGF induced the production of IL-11 and both cytokines activate the STAT3 pathway. We used neutralizing antibodies specific for IL-11 or c-MET, the HGF receptor, to block autocrine/paracrine cytokine stimulation of erythroblasts in in vitro cultures. The growth of JAK2V617F-mutated PV erythroblasts and HEL cells was inhibited by at least 40%, indicating that JAK2V617F-mutated cells depend on HGF and IL-11 for their growth. Consistent with activation of HGF/IL-11 pathways, mRNA levels of gp130 (the receptor chain responsible for signalling common to cytokines of the IL-11/IL-6 family) and STAT3 were significantly elevated in PV erythroblasts. Moreover, mRNA levels of c-MET, HGF and IL-11 were correlated, a logical finding as c-MET, HGF and IL-11 act in cascade. No correlation was found with JAK2V617F mRNA levels. To the opposite, we provide evidence that the up-regulation of HGF and IL-11 in PV is not a consequence of JAK2V617F: transient expression of JAK2V617F in BaF-3/Epo-R cells and invalidation of JAK2V617F in HEL cells using anti-JAK2 siRNA had no effect on HGF and IL-11 expression.

Conclusion:

Anti-inflammatory, STAT3-activating HGF and IL-11 are up-regulated in PV independently of JAK2V617F. Both cytokines contribute to the proliferation of JAK2V617F-mutated erythroblasts, suggesting that drugs blocking the HGF/IL-11 pathways, such as c-MET antagonists, could be of interest as an additional therapeutic option in PV.

Marjorie Boissinot and Cedric Cleyrat contibuted equally.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome
Authors: Ito Keisuke , Sportoletti Paolo , Clohessy John G , Silvia Grisendi , Pandolfi Pier Paolo
Published: October 2019

Abstract 95

Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia.

NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality.

To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53.

Disclosures:

No relevant conflicts of interest to declare.
Outpatient Treatment In Patients with Acute Pulmonary Embolism: The Hestia Study
Authors: Zondag Wendy , Mos Inge , Creemers Dina , Hoogerbrugge Lidia , Dekkers Olaf , Dolsma Anneke , Eijsvogel Michiel , Faber Laura , Hofstee Herman MA , Hovens Marcel , Jonkers Ge , Kralingen Klaas , Kruip Marieke , Vlasveld Tom , Vreede Marielle , Huisman Menno
Published: October 2019

Abstract LBA-1

Introduction:

Patients with pulmonary embolism (PE) are initially treated in the hospital with low molecular weight heparin (LMWH). The most recent guideline of the American College of Chest Physicians on Antithrombotic therapy 2008 reports some small studies on outpatient treatment in patients with pulmonary embolism, which suggest outpatient treatment in selected patients with PE is potentially effective and safe but firm recommendations for clinical practice are lacking. Clinicians urgently need reliable, easy-to-use selection criteria for selection of patients with pulmonary embolism eligible for outpatient treatment.

Objective:

To evaluate the efficacy and safety of outpatient treatment according to predefined criteria (Hestia criteria) in patients with acute PE.

Patients and Methods:

Open-label, single-arm, multicenter clinical trial of patients with objectively proven acute pulmonary embolism, conducted in twelve hospitals in the Netherlands from 2008 to 2010. Follow-up was completed in September 2010. Patients with acute PE were triaged with the predefined Hestia criteria for eligibility for outpatient treatment starting with therapeutic weight adjusted doses of LMWH (Nadroparin), followed by vitamin K antagonists. All patients eligible for outpatient treatment according to the Hestia criteria, were sent home either immediately or within 24 hours after PE was objectively diagnosed.

Outcome:

Outpatient treatment was evaluated with respect to recurrent venous thromboembolism (VTE), including PE or deep venous thrombosis (DVT), major haemorrhage and total mortality during initial LMWH treatment and 3 months follow up. We considered outpatient treatment to be effective if the upper limit of the 95% confidence interval of the incidence of recurrent VTE would not exceed 7%.

Results:

Of 297 included patients, who all completed follow-up, 6 patients (2.0%; 95% confidence interval [CI], 0.8–4.3) had recurrent VTE (5 PE (1.7%), 1 DVT (0.3 %)). Three patients (1.0%, 95% CI 0.2–2.9) died during three months follow-up, but none as a result of fatal PE. One patient died of fatal intracerebral haemorrhage, the other two patients died of progressive malignancy. In addition to the patient with intracranial bleeding, one other patient had a major bleeding event (0.7 %, 95% CI 0.08%-2.4%).

Conclusion:

Outpatient anticoagulant treatment is effective and safe for patients with pulmonary embolism who have been selected with the Hestia criteria. (Dutch Trial Register NTR1319).

Disclosures:

Huisman: GSK: Research Funding; Actelion: Research Funding; Bayer: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Pfizer: Speakers Bureau.
Micromanipulating Hematological Malignancies and Other Cancers
Authors: Lieberman Judy
Published: October 2019

Abstract SCI-33

MicroRNAs regulate the response of the cell to environmental changes and developmental cues. microRNA expression is dysregulated in cancer – microRNA expression is generally reduced in cancer cells compared to normal tissue and individual microRNAs, termed oncomirs, that act as tumor suppressor genes or oncogenes are frequently aberrantly expressed in cancer and their expression can be linked to prognosis and response to therapy. Because microRNAs regulate cancer cell differentiation, proliferation, survival, and metastasis, manipulating microRNA function, either by mimicking or inhibiting miRNAs implicated in cancer, could provide a powerful therapeutic strategy to interfere with key pathways for cancer progression. This talk will explore some of the opportunities and obstacles to harnessing microRNA biology for cancer therapy.

Disclosures:

No relevant conflicts of interest to declare.