bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary Central Nervous System Lymphoma (PCNSL) in Immunocompetent Patients: An Analysis of Prognostic Factors and Treatment Strategies
Authors: McFarland Daniel C , Gregory Stephanie A. , Li Yan , Deng Youping , Venugopal Parameswaran
Published: October 2019

Abstract 4955

Background:

The incidence of primary CNS lymphoma in immunocompetent patients is increasing for unknown reasons. Survival estimates from academic centers show an improvement in overall survival (OS) while community epidemiological data estimate minimal difference in OS in the last thirty years. Age greater than 60, poor performance status, elevated initial LDH, high CSF protein and involvement of deep regions of the brain have been associated with worse survival. Optimal dosing strategies and delivery of chemotherapy as well as the role of radiation therapy (RT) have yet to be clearly defined given the limitations of treatment related toxicity in patients achieving remissions.

Purpose:

To characterize the PCNSL patient population treated at a single institution by studying prognostic factors and assessing OS according to treatment strategies.

Methods:

45 cases of confirmed CNS Lymphoma diagnosed at Rush University Medical Center between January 2000 and November 2010 were studied. Demographic, clinical and treatment data were collected and reviewed. Hospital records and the Social Security Death Index were used to determine death dates. Survival data were compared based on treatment groups.

Results:

23 of the 45 cases met all inclusion criteria and had no evidence of previous systemic lymphoma or pre-morbid immunocompromised conditions (i.e. HIV). The age range at diagnosis was from 21 to 81 years old with a median age of 68. One patient had ocular involvement. Three patients had malignant cells in the CSF at diagnosis and 4 patients had multifocal tumors. Biopsies revealed one case of a cerebral T-cell lymphoma and 22 were diffuse large B-cell lymphoma (DLBCL). Elevated CSF protein above the mean (96.1 mg/dl) was associated with worse OS (p=0.02). Elevated LDH above the mean (227 U/L) was associated with worse OS (p= 0.039). Age, performance status and location of tumor within the brain (deep vs. superficial) did not correlate with survival patterns. Comparative survival analysis between treatment groups revealed no statistically significant difference (p=0.134). In addition to systemic chemotherapy, 15 patients received intrathecal chemotherapy

Conclusions:

The description of PCNSL in immunocompetent patients outside of the clinical-trial setting is necessary to inform prospective trial designs. This study supports the use of LDH and CSF protein in determining prognosis and treatment strategy. This analysis of conventional treatment is ongoing. Collaboration on gathering descriptive data for PCNSL in immunocompetent patients should be pursued.

Disclosures:

Gregory: Genentech:.
Long-Term Follow-up of Patients with HIV-Related Diffuse Large B-Cell Lymphoma (DLBCL) Treated in the Phase II Study with Rituximab and CHOP (R-CHOP)
Authors: Ribera Josep-Maria , Morgades Mireia , Oriol Albert , González-Barca Eva , Miralles Pilar , Lopez-Guillermo Armando , Gardella Santiago , López Andrés , Abella Eugènia , Xicoy Blanca , Garcia Marta , Feliu Evarist
Published: October 2019

Abstract 4980

Background and objective.

In the highly active antiretroviral treatment (HAART) era R-CHOP based chemotherapy has proven to be feasible and effective in HIV-related DLBCL. However, the available information on long-term follow-up of patients in remission of lymphoma is scarce. In addition, solid tumors constitute an emerging cause of cancer in HIV-infected patients under HAART. The follow-up of patients in complete response (CR) included in a phase II clinical trial of R-CHOP every 21 days conducted by the Spanish PETHEMA, GESIDA, GELTAMO and GELCAB groups (Ribera JM et al. Br J Haematol 2007; 140: 411–419) has been analyzed.

Patients and methods.

Out of 81 patients included in the trial 55 patients were in CR. The following data were recorded in these patients: NHL relapse, opportunistic infections (OI) and other cancers. Cumulative probabilities of OI and second cancers, as well as overall survival (OS) and event-free survival (EFS) were calculated.

Results.

Median follow-up of alive patients was 6.4 yr (range: 4.6–9.5). One patient was lost of follow-up in CR, 8 relapsed, 5 had OI (meningoencephalitis [2], Pneumocystis jiroveci pneumonia [1], varicella pneumonia [1], pneumoccal pneumonia [1], esophageal candidiasis [1], and CMV colitis [1]; 2 patients suffered 2 OI during their evolution) and 5 developed a second cancer (invasive carcinoma of cervix [1], squamous lung cancer [1], lung adenocarcinoma [1], pancreatic adenocarcinoma [1], and metastatic sarcoma of unknown origin [1]). Eight-year cumulative probability for OI was 15% (95%CI: 7%-23%) and for second cancer was 12% (95%CI: 2%-22%). Fourteen patients have died: 5 due to lymphoma relapse, 3 due to OI, 4 due to second cancer and 2 due to other reasons (sudden death and assassinate). Eight-year OS probability for the 55 patients in first CR of the lymphoma was 67% (95%CI: 48%-86%) and EFS probability was 59% (95%CI: 42%-76%).

Conclusions.

HIV-infected patients with DLBCL treated with R-CHOP and HAART followed for long-time have a significant frequency of OI and second cancers, with impact on their survival probability.

Supported in part with grants RD06/0020/1056 from RTICC, Instituto Carlos III, 36606/06 from FIPSE and P-EF/10 from FJC.

Disclosures:

No relevant conflicts of interest to declare.
Population Pharmacokinetics Study of a New Humanized Anti-CD20 Monoclonal Antibody AME-133v (LY2469298) in Patients with Previously Treated Follicular Lymphoma (FL)
Authors: Cronier Damien , Radtke David , Carpenter Susan P. , Wooldridge James
Published: October 2019

Abstract 4982

Background:

AME-133v is a humanized monoclonal antibody that was engineered to have increased affinity to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab in vitro. The safety, pharmacokinetics (PK) and preliminary efficacy of AME-133v were assessed in a Phase 1/2 clinical trial in patients with previously treated follicular lymphoma (FL). The objective of this study is to characterize the pharmacokinetics (PK) of AME-133v in the target patient population.

Methods:

5 dose levels of AME-133v (2, 7.5, 30, 100, and 375 mg/m2) were tested in a total of 67 patients with previously treated CD20+ FL. AME-133v was administered intravenously in four weekly infusions. Blood samples were obtained pre-dose and 1, 3–5 days after infusion 1, pre-dose and post-dose during infusions 2, 3 and 4, and 1, 5 and 9 weeks after infusion 4. The PK database (399 data points) collected from the patients was analyzed by using the nonlinear mixed-effect model (NONMEM) program. A number of covariates including demographic characteristics and the FcγRIIIa receptor genotype were evaluated for their influence on the AME-133v population PK parameters.

Results:

Owing to serum concentrations values falling below the limit of detection, the 2 mg/m2 dose group was not included in the analysis. The basic model selected was a two-compartment pharmacokinetic model with first-order elimination. However, a different typical value had to be determined for CL in each dose group. The typical values of V1, Q and V2 were 2.99 L, 0.94 L/day, and 3.31 L, respectively. The typical value for CL was 0.70, 0.53, 0.26, 0.27 L/day-1 for 7.5, 30, 100 and 375 mg/m2, respectively, which indicates a linearization of the elimination rate of AME-133v at doses of 100 mg/m2 and above. Inter-individual variability was moderate to high with CVs of 45.9, 34.1 and 50.0% on CL, V1 and V2, respectively. The only covariate found to influence the PK of AME-133v was BSA which explained 9.6% of the variability observed on V1. The form of the FcγRIIIa receptor was not found to have a significant effect on the PK of AME-133v.

Conclusions:

The PK of AME-133v were best described by a 2-compartment model. Clearance was found to be dose-dependent with a linearization of the elimination rate at doses of 100 mg/m2 and above. BSA has a statistically significant influence on V1 whereas the FcγRIIIa genotype does not seem to influence the disposition of AME-133v.

Disclosures:

Cronier: Eli Lilly and Comany: Employment, Equity Ownership. Off Label Use: AME-133v is an investigational agent. Radtke:Eli Lilly and Comany: Employment, Equity Ownership. Wooldridge:Eli Lilly and Company: Employment, Equity Ownership.
Pernicious Anemia Mimicking MDS by Del(3p) in Cytogenetics and Monocytic Leukemia in Trephine Biopsy by Highly Expression of CD163 Positive Cells
Authors: Parmentier Stefani , Meinel Jörg , Jakob Christiane , Frömmel Anke , Mohr Brigitte , Ehninger Gerhard , Schaich Markus , Platzbecker Uwe
Published: October 2019

Abstract 5038

Case Report

We report of a 53 year old woman who presented repeatedly with syncopes over a period of 3 months. Laboratory results revealed severe pancytopenia (Hb 4.6 mmol/l (7.4 g/dl), WBC 1.22 Gpt/L, platelet count 13 Gpt/L) and severe hemolysis (haptoglobin < 0.20 g/l, normal range 0.3–2.0 g/l; LDH 33.1 μmol/s*l, normal range 2.25–3.55 μmol/s*l). Routine examination of the bone marrow aspirate showed typical features of megaloblastic erythropoiesis, which could be confirmed by a low serum cobalamin level (53 pg/ml, normal 211–911 pg/ml) and the presence of anti-intrinsic factor antibody (16.84 U/ml). Additionally, atrophic gastritis was seen in biopsies taken of gastric mucosa. The diagnosis of pernicious anemia was suspected and the patient treated with cobalamin. Except for hemoglobin, the peripheral blood counts recovered within one week. Meanwhile, cytogenetics from the bone marrow revealed metaphases with del(3p) and histopathological results were suspicious of an increased number of blast cells with highly expression of CD163 possibly mimicking MDS or (acute) monocytic leukemia. Therefore, bone marrow examination was repeated two weeks after recovery, which still showed dysplastic changes paralleling hematopoietic recovery but no increased number of blast cells. Additionally, the cytogenetic aberration had disappeared.

Discussion

Diagnostic work-up for megaloblastic anemia rarely includes cytogenetic analysis of bone marrow cells. Therefore, the finding of a transient cytogenetic aberration has possibly not reported frequently before in the literature. In our case, the initial finding of del(3p) appears to be due to ineffective hematopoiesis caused by vitamin B12 deficiency which leads to impaired DNA synthesis and genomic instability. This might be an explanation for this cytogenetic abnormality which disappeared after substitution of cobalamin. CD163 is exclusively expressed on monocytes and macrophages and with signs of (slightly) increased blast counts might mimic (acute) monocytic leukemia. However, in pernicious anemia with severe hemolysis as seen in this case it might reflect an acute phase reaction, as CD163 represents a signal-inducing macrophage receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin-hemoglobin complexes.

In conclusion, vitamin B12 deficiency might be associated with cytogenetic abnormalities and thus in addition to the bone marrow morphology feign certain haematological diseases.

Disclosures:

No relevant conflicts of interest to declare.
Anti-CTLA-4 Antibody Is Not Able to Trigger Apoptosis in CTLA-4 Expressing Myeloid Tumor Cells
Authors: van Bijnen Sandra , Rogkoti Maria , Withaar Marian , de Witte Theo , Muus Petra , Dolstra Harry
Published: October 2019

Abstract 5054

Cytoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a co-inhibitory molecule normally expressed on activated effector T cells and a subset of regulatory T cells. However, it has been reported also to be expressed on acute myeloid leukemia (AML) cells, CD34+ hematopoietic progenitor cells after stimulation as well as several solid tumor cell types. Furthermore, CTLA-4 engagement with recombinant CD80 and CD86 ligands has been shown to induce apoptosis in AML cells (Laurent et al. Br J Haematol 2007). In this study, we investigated CTLA-4 expression on different cell populations in bone marrow aspirates of myelodysplastic syndrome (MDS) patients, and explored the possibility of targeting CTLA-4 for apoptosis induction using the CTLA-4 antibody tremelimumab. Flow cytometry analysis showed CTLA-4 surface expression on immature CD34+, CD117+ and CD33+ myeloid cells, as well as CD14+ monocytic cells from MDS patients (n=11). In addition, CTLA-4 was expressed by both CD4+ and CD8+ T cells in bone marrow of both low and high risk MDS patients. In order to address whether anti-CTLA-4 antibody is able to induce apoptosis in myeloid leukemic progenitor cells, we incubated the CTLA-4-expressing myeloid cell lines HL-60 and KG1a in vitro for various time points (24h, 48h, 72h) with 25 microgram/ml tremelimumab. However, anti-CTLA-4 antibody alone as well as cross linking with a secondary antibody was unable to induce apoptosis, while serum-free conditions and irradiation induced high numbers of Annexin V and/or 7AAD positive cells. These data indicate that the anti-CTLA-4 antibody tremelimumab is unable to induce an apoptotic signal into CTLA-4-expressing myeloid tumor cells.

Disclosures:

No relevant conflicts of interest to declare.
Multiple Myeloma Variant with Ascites and Cytoplasmic Crystalline Granules in Plasma Cells
Authors: Rodriguez Cesar , Janckila Anthony , Potenziani Sylvia , Sharma Vivek R.
Published: October 2019

Abstract 5088

CASE REPORT:

CLINICAL HISTORY:

A 62-year-old male patient who presented with anemia and findings of monoclonal gammopathy of IgA type was referred to the Hematology clinic for work-up and management of possible multiple myeloma. At the same time, the patient was being managed by the Gastroenterology service with diuretics and recurrent paracentesis for an ongoing significant ascites of unclear etiology. These interventions had been performed for a year on a biweekly basis.

CLINICAL WORK-UP:

The patient had IgA gammopathy with a monoclonal protein level of 1.2 Gm/dL and findings on skeletal survey consistent with lytic lesions in the pelvic bones.

PROCEDURE:

A bone marrow biopsy obtained from the patient revealed a plasmacytosis of 15% with atypical crystalline cytoplasmic inclusions seen in the majority of plasma cells and some histiocytes as well. The transudative ascitic fluid was analyzed for crystalline deposits, but these could not be conclusively detected even though there were inclusions seen in some macrophages that may represent remnants of the crystals.

TREATMENT:

The patient was diagnosed with IgA myeloma given the above findings and was treated with a combination of Revlemid, Velcade and Dexamethazone with good response in terms of his IgA monoclonal protein as well as in significant improvement in his ascites, which essentially resolved. He no longer required therapeutic paracentesis upon initiation of chemotherapy and was able to get weaned off of diuretics as part of his treatment.

CONCLUSION:

This case represents what we believe to be a rare variant of multiple myeloma that presented with monoclonal gammopathy in the blood as well as concomitant ascites. The bone marrow showed plasma cells containing crystalline inclusions. The precise nature of these inclusions is presently unknown, however it is felt most likely to be precipitated immunoglobulins. We plan to investigate this further. For reasons that are not entirely clear, this particular biology appears to induce a reactive process in the peritoneum causing a development of ascites. The resolution of such ascites with treatment directed toward the myeloma seems to indicate that the two are related. A case similar to this was reported by Doctors Martin, et al in 1987 with a patient presenting with similar type of crystalline inclusions in plasma cells with clinically associated ascites.1 There was no information available on treatment effect and so it was not possible to tie the clinical presentation of the myeloma and ascites together.

Disclosures:

No relevant conflicts of interest to declare.
Sulforaphane Synergistically Enhances the Cytotoxicity of Arsenic Trioxide in Multiple Myeloma Cell Lines Through Generation of ER Stress
Authors: Doudican Nicole A , Mazumder Amitabha , Orlow Seth J
Published: October 2019

Abstract 5140

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic recticulum (ER) that is exquisitely sensitive to perturbations in protein synthesis. Targeting ER stress- related signaling has been clinically validated in the treatment of multiple myeloma (MM) as evidenced by the response to treatment with bortezomib (BTZ). Despite impressive response rates, BTZ carries the potential for serious side effects, and the development of resistance to BTZ is a clinical issue. We therefore sought to identify novel drug combinations that effectively generate ER stress. Here, we report that sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent that has shown clinical activity in MM, in a panel of MM cell lines. As single agents, both 1 μM sulforaphane and 0.5 μM ATO have a modest effect on cellular proliferation in a panel of MM lines. However, when the agents are administered in combination, cellular proliferation is dramatically reduced. For example, in PCNY-1 MM cells, 1 μM sulforaphane has no effect and 0.5μM ATO causes a 29% reduction in proliferation. However, when administered together, the agents enhance growth inhibition to 73%, with a CI of 0.632 indicative of synergy. Four out of 5 MM cell lines tested displayed sulforaphane and ATO synergy. Combination treatment resulted in enhanced apoptotic induction as demonstrated by cleavage of PARP. Enhanced induction of ER stress signaling and activation of the unfolded protein response (UPR) upon combination treatment was demonstrated by enhanced expression of the molecular chaperone HSP90 along with increased phosphorylation of PERK (an ER transmembrane kinase and proximal effector of the UPR) and eIF2 (translational initiation factor). Additionally, increased splicing of XBP1 (a transcription factor of UPR target genes) was apparent upon combination treatment as compared to treatment with either agent alone. Our results show that sulforaphane can synergistically sensitize MM cells to the cytotoxic effects of ATO through promotion of ER stress generating mechanisms. Based upon these promising results, further evaluation of this safe, natural product as an ATO sensitizer in a clinical trial of MM patients is warranted. Additionally, this approach holds the promise as a means to identify and clinically validate natural products effective in the treatment of MM and/or inhibition of progression of asymptomatic MM.

Disclosures:

No relevant conflicts of interest to declare.
DISTINCT CLINICOLABORATORY CHARACTERISTICS of the t(14;19)(q32;q13)-POSITIVE SPLENIC MARGINAL ZONE LYMPHOMA
Authors: Julhakyan Hunan , Magomedova Aminat , Kravchenko Sergey K , Obukhova Tatyana N , Samoylova Rima , Kaplanskaya Irina B
Published: October 2019

Abstract 5200

Background:

Splenic marginal zone lymphoma (SMZL) is a well recognized B-cell neoplasm which is characterized by splenomegaly, bone marrow involvement, immunologically by typical phenotype of marginal zone cells. The most frequent cytogenetic findings are involvement of chromosomes 1, 3, 7(usually deleted in 7q) and 8. The t(14;19)(q32;q13) is a rare cytogenetic abnormality with bcl-3 rearrangement that has been reported in other B-cell lymphomas.

Aims:

To describe the clinical, morphological, immunophenotypic findings in SMZL associated with t(14;19)(q32;q13).

Methods and results:

In Hematological Research Centre, Moscow between January 2001 and May 2011 three cases SMZL with t(14;19)(q32;q13) were identified. All patients were males with age 51, 58, 67 y.o. Lymphoma presented with B-symptoms, high level of lactate dehydrogenase (LDH), hepatosplenomegaly and regional lymphadenopathy (enlarge splenic hilar lymphnodes). The hemoglobin was 92 g/l, 110 g/l, 122 g/l. All patients had normal count of leukocytes with an absolute lymphocytosis (lymphocytes count 72 × 109 g/l, 79 × 109 g/l, 83 × 109/l) and thrombocytopenia. Morphological examination of peripheral blood and bone marrow lymphocytes showed that all lymphocytes are atypical with wide cytoplasm and nuclear indentation. In all cases there was nodular type of bone marrow involvement, composed of majority medium sized cells. Immunophenotypic analysis has shown the expression of mature B-cells antigens (CD19, CD20, CD22, FMC7, sIg) and absence of ÑD10, CD23, CD5, CD43, CyclinD1.

Two patients were treated with CHOP-regimen without any response. They progressed with spleen enlargement and decreased of thrombocytes counts. All 3 patients undergo splenectomy. Weight of spleen was 1800 g, 2083 g, 2850 g. Splenic section generally show massive nodular pattern (involvement of the white and red pulp) associated with diffuse invasion of the sinuses. In all cases discovered high Ki-67. All patients demonstrated progression after splenectomy during 3–6 months that was characterized by increase of leukocytes count (range 45,4 – 101,8 × 109 /l), high level of LDH, appearance of peripheral and visceral lymph nodes. Considering the increase leucocytes, presence of lymphadenopathy in all cases CHOP, FMC regimen were used. All patients died of disease progression and infectious complications. Time of observation was 21, 30, 34 months.

Conclusions:

The t(14;19)(q32;q13)-positive SMZL is distinct variant which is characterized by rapid progression after splenectomy, poor responses to chemotherapy and short survival. So t(14;19)(q32;q13) may be regarded as a poor prognostic factor.

Disclosures:

No relevant conflicts of interest to declare.
Endocrine Late Sequelae in Long-Term Survivors of Childhood Non-Hodgkin Lymphoma
Authors: van Waas Marjolein , Neggers Sebastian J.C.M.M. , te Winkel Mariel Lizet , Beishuizen Auke , Pieters Rob , van den Heuvel-Eibrink Marry M.
Published: October 2019

Abstract 5215

Introduction.

Currently, the 5-year survival for childhood NHL is ∼80%. Because of this high survival rate and since treatment is administered in a growing and developing individual, long-term side effects are an important issue. Studies on endocrine long-term effects in survivors of childhood non-Hodgkin lymphoma (NHL) are scarce and often limited by small sample sizes, or by the fact that data of NHL survivors have been combined with that of survivors of other malignancies. Aim of this study was to investigate the long-term endocrine effects of treatment for childhood NHL.

Methods.

A single centre cohort of 84 survivors (22 females) was included in this retrospective study. Median age was 21 yrs (9–40 yrs) and time after cessation of therapy 12 yrs (4–30 yrs). Height, weight, percentage fat, lean body mass (LBM), bone mineral content (BMC), bone mineral density of total body (BMDTB) and lumbar spine (BMDLS) were measured. Bone mineral apparent density of the lumbar spine (BMADLS) was calculated to correct for bone size. Thyroid stimulating hormone (TSH), free thyroxin (fT4), insulin-like growth factor-1 (IGF-1), Inhibin-B and anti-müllerian hormone (AMH) levels were measured. Results were compared with Dutch controls.

Results.

Height was lower in survivors at follow-up (mean SDS −0.36, P=0.002), but further analysis showed that shorter stature was already present at diagnosis (mean SDS −0.28, P=0.023). BMI, percentage fat, BMC, BMDTB, BMDLS and BMADLS were not different from controls. LBM was lower in survivors (mean SDS −0.47, P=0.008). TSH, fT4 and IGF-1 were normal in all survivors. Three out of 20 adult females had low AMH levels and 55% (23/42) of adult males had low Inhibin-B levels.

Conclusion.

Twelve years after cessation of therapy, survivors of NHL did not develope adiposity, osteopenia or osteoporosis or thyroid disease. Male NHL survivors seem to be at risk for infertility.

Disclosures:

No relevant conflicts of interest to declare.
Relationship Between Intestinal Marginal Zone B-Cell Lymphoma and Enterobacterias Including Camphylobacter Jejuni
Authors: Oh Sung Yong , Kim Won Seog , Kim Seok Jin , Hong Junshik , Lee Soon Il , Lee Jae Hoon , Do Young Rok , Kim Min Kyung , Lee Ho Sup , Suh Cheolwon , Yi Seong Yoon , KIm Jong Hyun , Hong sook Hee
Published: October 2019

Abstract 5224

Background:

Even though one great study had been addressed for identifying relationship Camphylobacter jejuni (C.jejuni) and IPSID, association is not proof for causation; it is usually the first step in proving the microbial etiology of a disease. Intestinal-marginal zone B-cell lymphoma (I-MZL) in Korean result, many points of clinical presentations were quietly different from classical IPSID. Median age was about 60 not a young child, common involving site was ileo-cecal valve not a jejunum, limited segmental involvement was dominant not a diffuse involvement of whole intestine. Therefore, we propose this study for identifying the causative etiology of I-MZL. Thus firstly we planned to demonstrate the enterobacterias including C.jejuni and I-MZL with Korean specimen.

Patients and Methods:

Histologically confirmed 33 patients of intestinal MZL were evaluated for this study. DNA preparing from FFPE will be used Ambion RecoverAllTM Total Nucleic Acid Isolation Kit for FFPE tissue. Universal primers PB (5'TAACACATGCAAGTCGAACG3') and DG74 5'AGGAGGTGATCCAACCGCA3') has been used to amplify bacterial 16S ribosomal DNA (rDNA). Established primers directed at C. jejuni (CCCJ609F 5'AATCTAATGGCTTAACCATTA3' and CCJ1442R 5'GTAACTAGTTTAGTATTCCGG3') will be used to generate an 852-bp 16S rDNA amplification. Cycling conditions for all PCR will be as follows: 7 minutes at 95°C, 35 cycles of denaturation for 30 seconds at 95°C, annealing for 20 seconds at 55°C, and 2 minutes of extension at 72°C and then 10 minutes at 72°C.With the similar concept, Salmonella typi, Vibrio Cholerae, Escherichia coli, Shigella also were evaluated for evaluating the etiology of the I-MZL.

Result:

Between 1999 and October 2009, a total of 24 patients with histological diagnosis of I-MZL from 9 different institutions in Korea were analyzed. The male/female ratio was 14 to 10. The median age was 57 years (range, 15–80). The number of involved site was duodenum-1, jejunum- 2, ileum-7, ileo-cecal valve-3, ascending colon-2, sigmoid colon-1, rectum- 5, and multiple intestine involvement was 3. Four bands were appeared in I-MZL patient. All of two jejunum involved MZL tissue had positive result. But one patient was ileum and the other one was ileo-cecal valve involved MZL. Salmonella typi, Vibrio Cholerae, Escherichia coli, Shigella were all negative in I-MZL tissue

Conclusion:

It was a difficult to identify the relationship several enterobacterias and I-MZL. However C. jejuni could be a possibility of etiologic organism of jejunum involved MZL. Furthermore, extra-jejunum I-MZL has a need for more investigation to define the etiology of I-MZL

Disclosures:

No relevant conflicts of interest to declare.
Prox1 Suppressed Tumor Cell Growth by the Down Regulation of PKCβII Through the DNA Methylation
Authors: Hagiwara Kazumi , Murate Takashi , Miyata Yasuhiko , Hotta Tomomitsu , Nagai Hirokazu
Published: October 2019

Abstract 5227

BACKGROUND:

The homeobox gene Prox1 is a transcription factor related to the Drosophila prospero gene and known to be the master gene which controls the development of lymphatic vasculature. Recent studies have suggested that Prox1 is a strong candidate tumor suppressor gene in several kinds of malignancies. We previously demonstrated that the expression of Prox1 gene was frequently silenced by aberrant DNA methylation in about 60% of diffuse large B cell lymphoma (DLBCL). In this study, we showed that Prox1 regulated Protein Kinase C beta II (PKCβII) expression, which was overexpressed in poor risk DLBCL, and analyzed the inhibitory mechanism of tumor suppression by Prox1.

METHODS and RESULTS:

We established the Prox1 stable transformants of Hela cells which originally lacked Prox1 expression, and examined them in vitro cell proliferation and in vivo tumor formation using nude mice. Our experiments showed that Prox1 had inhibitory effect on both cell proliferation and tumor formation. cDNA microarray analysis indicated that the expression of PKCβII was highly suppressed by Prox1 overexpression. In Prox1-expressing Hela cells, mRNA expression of PKCβII was undetectable and protein was significantly down-regulated compared with Mock cells. The selective PKCβII inhibitor, enzastaurin was less effective on cell proliferation of Prox1-expressing cells compared with Mock cells. These results showed that PKCβII was required for tumor cell proliferation. We focused on the regulatory mechanism of PKCβII expression by Prox1. The 5'-promoter analysis of PKC-β gene revealed that the proximal region between 110bp of the promoter containing 2 Sp1 binding sites was responsible for the endogenous promoter activity, and Prox1 did not affect that. Mithramycin A, a relatively specific Sp1 inhibitor, and Sp family knockdown by siRNA on Hela cells decreased PKCβII expression, suggesting that Sp family was important for PKCβII transcription. Interestingly, 5-aza-deoxycytidine (5-Az) treatment restored PKCβII mRNA expression in Prox1-expressing Hela cells, suggesting that 5'-promoter region of PKCβII was methylated in these cells. Actually, bisulfite sequencing showed that the predicted CpG island of PKC-β gene, especially around these Sp1 binding sites, was highly methylated in Prox1-expressing Hela cells compared with Mock cells and 5-Az relieved DNA methylation.

CONCLUSION:

Our study suggested that the decreased PKCβII expression suppressed tumor cell proliferation and Prox1 down-regulated PKCβII expression by enhancing DNA methylation of PKC-β 5'-promoter around Sp1 binding sites. This is the first to report the inverse relationship between Prox1 and PKCβII expression in cancer cells and the novel regulatory mechanism of PKC-β gene expression by Prox1 transcription factor.

Disclosures:

No relevant conflicts of interest to declare.
The Comparison of Fibrin Monomer (FM) Performance to Other Activation Markers (TAT, PF1.2 and DD)
Authors: Patel Priti , Telesca Mayra , Ku De-Hui
Published: October 2019

Abstract 5259

In this study, we attempted to determine the performance of the soluble fibrin monomer complex (SFMC) test in comparison to known activation markers, such as prothrombin fragment 1.2 (PF1.2), thrombin antithrombin complex (TAT) and d-dimer (DD). We have used the STA(R)-Liatest FM and STA(R)-Liatest D-Di kits to measure the SFMC and d-dimer levels.

A total of 25 patients were included in this study. The data and demographic information are shown in the table below.

ID GENDER AGE FM TAT PF1.2 D-dimer 1 M 66 0.42 2.3 1988 0.05 2 F 31 27.93 >60.0 >12000 2.39 3 F 24 3.41 14.1 1367 0.48 4 F 61 2.22 7.7 335 0.63 5 F 18 4.61 6.9 940 0.22 6 F 38 156.52 43.5 >12000 10.37 7 F 20 0.42 2.2 149 0.66 8 F 21 0.42 2.4 143 0.24 9 M 44 >150 15.4 11043 5.5 10 F 42 3.4 <2.0 134 0.24 11 M 46 0 4.8 271 0.3 12 M 76 90.73 29.4 1712 >20.0 13 F 61 3.41 3.8 484 0.52 14 M 1 0.1 3.8 116 0.33 15 F 33 4.2 37 6346 2.03 16 F 35 3.2 2.6 325 0.09 17 F 23 1.1 4.7 232 0.2 18 M 64 2.3 2.6 189 0.15 19 M 1 1.7 2.9 253 0.49 20 M 64 2.2 33.2 451 2.43 21 M 60 1.6 2.9 126 0.42 22 F 55 2.2 2.6 189 0.97 23 F 65 1.3 2.3 79 0.25 24 F 39 3.6 8.1 222 0.18 25 M 2 4.8 <2.0 109 0.53 Reference range >6.0 ug/mL <4 mcg/L 41–372 pmol/L <0.45 ug/mL

The data indicate that in 16 out 25 patients, the SFMC levels are correlated with TAT and PF1.2. Only 3 out of 25 patients have elevated TAT and PF1.2 with normal SFMC levels. Therefore, our data would tend to indicate that SFMC level could be used as an activation marker to assess an ongoing prothrombotic process. In order to more fully determine the efficacy of the SFMC assay, we will obtain a detailed clinical history for the cases shown above.

Disclosures:

No relevant conflicts of interest to declare.
Src Family Kinases Contribute to GI and Gq-Mediated Platelet Activation
Authors: Xiang Binggang , Zhang Guoying , Li Zhenyu
Published: October 2019

Abstract 5263

The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y12 antagonist AR-C69931MX or P2Y12 knockout but was not affected by the P2Y1 antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi pathway activated via its receptor P2Y12. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of Gi is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y12−/−, but not in Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-dependent SFK phosphorylation is downstream from the Ca2+ signaling. The calcium ionophore, A23187-induced TXA2 synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA2 synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin aIIbb3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca2+/SFKs and PKC represent two parallel signaling pathways mediating Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation.

Disclosures:

No relevant conflicts of interest to declare.
Serum Prohepcidin and Iron Homeostasis in Patients with Breast Cancer
Authors: Grudeva Janet G. , Nenova Ivanka Slavejnova , Terzieva Dora Dimitrova , Spasova Maria Ivanova , Boyadjiev Nikolay Petrov
Published: October 2019

Abstract 5291

Introduction:

Cancer-related anemia (CRA) has multifactorial etiology and complex pathogenesis. It is defined as normochromic, normocytic anemia with reticulocyto-penia and hypoferremia. Hepcidin is recognized as the central factor in causing CRA. Objective: To investigate the changes in the serum levels of prohepcidin (pHp) and markers of iron homeostasis for gathering more data on the pathogenesis of CRA.

Patients and Methods:

The authors analyzed prospectively 46 newly diagnosed women with breast cancer, aged 29–72 years (average 47.5±9.0 SD), who had the same clinical stage, histology and hormonal status. They were diagnosed and treated in the Clinic of Oncology and Hematology - University Hospital for a 2-year period (2009–2010). Serum pHp levels and common markers of iron status including serum iron (sFe), transferrin saturation, soluble tranferrin receptor (sTfR), Zn-protoporphyrin (ZPP), ferritin as well as vitamin B12 and folate were measured before treatment initiation and two months afterwards. Serum pHp was determined by a competitive immunotest. All statistical data was computed by the methods of variational and correlation analyses.

Results and Discussion:

For the two-month follow up interval parameters of full blood count remained without statistically significant deviation. Although the analyzed parameters in the first and second blood samples remained within the reference intervals, the decrease of pHp and the changes of iron-containing substances and ZPP in red blood cells cannot be neglected. However the correlation coefficient (R) between decreased pHp, increased sFe (R=0.314), sTfR (R=0.258), ZPP (R=0.118) and decreased ferritin (R=0.099), were low instead of the expected higher relationship. The obtained results do not support the idea that iron increase and other changes of iron homeostasis in these patients is a direct result of regulatory decrease of pHp. The two-month interval is likely short enough to rule out a potential direct suppressive effect of the specific cytotoxic treatment, and therefore we do not discuss such potential influence on iron homeostasis.

Conclusion:

Iron homeostasis dysregulation is one of CRA components. In its complex pathogenesis several other factors interfere e.g. such related to the disease; therapy related factors; altered erythropoietin production; activation of cytokines (IL-6 is the major promoter of hepcidin production).

Disclosures:

No relevant conflicts of interest to declare.
Impact of Different Immunosuppresive Drugs on the Endothelium. Protective Effect of Defibrotide
Authors: Diaz-Ricart Maribel , Carmona Alba , Palomo Marta , Rovira Montserrat , Escolar Gines , Carreras Enric
Published: October 2019

Abstract 5319

Endothelial dysfunction is a key factor in the development of hematopoietic stem cell transplantation (HSCT) complications due largely to the conditioning treatments. The effect of immunosuppressive therapy on endothelial function has not been explored in detail in this setting. We evaluated the effect of three immunosuppressive drugs: two calcineurin inhibitors (cyclosporine and tacrolimus) and an inhibitor of mTOR (rapamycin). Microvascular endothelial cells (HMEC) were exposed to the three drugs to assess changes in ICAM-1 expression on the cell surface, the reactivity of the extracellular matrix (ECM) generated, and in signaling proteins. Studies were conducted in the absence and presence of defibrotide (DF) to assess its protective effect. Only the exposure of HMEC to cyclosporine (CyA) increased significantly the expression of ICAM-1 (4-fold, p<0.01) and the reactivity of the ECM (surface covered by platelets of 32.1±2.6% vs. 26.5±2.5%, p<0.01). While tacrolimus itself only increased slightly ICAM-1 expression, rapamycin moderately decreased the ECM reactivity. Those changes due to CyA were prevented by DF. CyA induced the activation of AKT and Raf, among others, whose expression was inversely related (4- and 0.5-fold, respectively, with respect to control cells). DF reversed this trend (0.4- and 2.7-fold, respectively, with respect to CyA treated cells). In conclusion, both calcineurin inhibitors showed a proinflammatory effect, though only CyA exhibited significant proinflammatory and prothrombotic effects on the endothelium that correlated with AKT/Raf pathway activation. Interestingly, the protective anti-inflammatory and anti-thrombotic effect of defibrotide seems to occur through the same signaling pathway. In contrast, rapamycin did not exert any significant action on endothelium.

Disclosures:

No relevant conflicts of interest to declare.
Perioperative Heparin Bridging in Patients Receiving Oral Anticoagulation: Meta-Analysis of Bleeding and Thromboembolic Rates
Authors: Yudin Jovana , Siegal Deborah , Lim Wendy , Kaatz Scott , Douketis James , Spyropoulos Alex C
Published: October 2019

Abstract 545

Background:

Periprocedural bridging using unfractionated heparin (UFH) or low molecular weight heparin (LMWH) in patients receiving chronic oral anticoagulation (OAC) is often utilized with the view to reduce the risk of thromboembolic (TE) events. Optimal perioperative anticoagulant methods have not been established. Methods: Systematic review and meta-analysis of published English-language studies from 2001 to 2010 examining bleeding and TE events in patients receiving bridging therapy during temporary OAC interruption for elective procedures. Results: A search of MEDLINE, EMBASE and Cochrane Collaboration databases yielded 32 studies on 6760 bridged patients. Studies were reviewed by 2 independent data collectors (k=0.869). Study quality was generally poor with risk of bias. Thirty-one studies were observational with 1 randomized controlled trial. Low TE risk and/or non-OAC patient groups were used for comparison in 12 observational studies. Major (22/32, 68.8%) and non-major (27/32, 84.4%) procedures were represented. TE events occurred in 67 of 6760 bridged (0.87%; 95% CI 0.40%–1.35%) and 29 of 4897 non-bridged (0.77%; 95% CI 0.24%–1.30%) patients. Using a random effects model, there was no difference in the risk of TE events in bridged versus non-bridged patients (OR 1.02, 95% CI 0.53–1.95). Bridged patients had a significantly increased risk of overall bleeding (OR 5.47, 95% CI 3.89–7.70) and major bleeding (OR 3.43, 95% CI 1.13–10.4) compared to non-bridged patients. There was no difference in TE events (OR 2.44, 95% CI 0.34–17.4) or overall bleeding (OR 2.40 95% CI 0.72–8.05) in patients receiving full versus intermediate/low dose LMWH. Summary: Patients receiving heparin bridging during OAC interruption appear to be at increased risk of bleeding and similar risk of TE events compared to non-bridged patients. Studies of high methodologic quality are needed to develop an optimal anticoagulation strategy and inform clinical decision-making.

Disclosures:

Lim: Leo Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy; Pfizer: Honoraria. Kaatz:Boehringer-Ingelheim: Consultancy, Research Funding, Speakers Bureau; Bristol Myer Squibb: Consultancy, Research Funding; Bayer: Research Funding; National Institute of Health: Research Funding; Canadian Institute of Health Research: Research Funding; Pfizer: Consultancy; Johnson and Johnson: Consultancy; Ortho-McNeil: Consultancy; GlaxoSmithKline: Speakers Bureau; AC Forum: Membership on an entity's Board of Directors or advisory committees; National Certification Board of Anticoagulation Providers: Membership on an entity's Board of Directors or advisory committees; National Blood Clot Alliance: Membership on an entity's Board of Directors or advisory committees.
MARCKS Confers Resistance to Velcade Through Skp2/p27-Mediated Pathway in Multiple Myeloma
Authors: Yang Yijun , Micallef Johann , Chang Hong
Published: October 2019

Abstract 985

Despite advances in clinical care, multiple myeloma (MM) remains an almost universally fatal disease. Velcade, a novel proteasome inhibitor represents a promising new clinical strategy for relapsed and refractory MM. However, only 30–40% of MM patients respond to this treatment. In order to gain insight into the mechanism of velcade resistance, we examined the differentially expressed proteins between 8226S (velcade sensitive) and 8226R5 (velcade resistant) myeloma cell lines using iTRAQ mass spectrometry (MS). The MS based multiple-reaction-monitoring technique (MRM) was then used to independently verify the quantitative differences of the protein expression levels between the two cell lines. We identified several cancer specific proteins to be highly differentially expressed between 8226S and 8226R5 by iTRAQ mass spectrometry. Of particular interest is Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a PKC substrate that has been implicated in conferring drug resistance to ovarian carcinoma cells. Differential expression of MARCKS between sensitive and resistant cells was verified by MRM and then confirmed by RT-PCR and Western blot analysis. MARCKS over-expression was also found in additional MM cell lines as well as patient samples that were resistant to velcade. Knockdown of MARCKS through siRNA strategies enhanced velcade sensitivity by increasing apoptosis in the velcade treated resistant cell line 8226R5. Moreover, inhibition of PKC, the upstream activator of MARCKS by enzastaurin significantly blocked phosphorylation of MARCKS leading to a caspase dependent apoptosis in the 8226R5 cells. In addition, enzastaurin enhanced velcade proteosome inhibitory effect and rendered the 8226R5 cells more sensitive to velcade treatment. Combination of velcade with enzastaurin showed a synergistic cytotoxic response in MM cells compared to either drug used alone. Furthermore, knockdown of MARCKS or inhibition of MARCKS phosphorylation by enzastaurin led to a decrease in Skp2 expression and an increase in p27 and p21, as well as a reduction of cyclin E and CDK2, resulting in G1/S cell cycle arrest and an increase in apoptosis in MM cells. Taken together our results suggest that phosphorylated MARCKS plays an important role in velcade resistance in MM and as such may be used as an important therapeutic target. In addition, MARCKS may be a biomarker to select patients that would most benefit from patient specific treatment.

Disclosures:

No relevant conflicts of interest to declare.
Iron Regulatory Proteins in Systemic Iron Metabolism and Erythropoiesis
Authors: Hentze Matthias W.
Published: October 2019

Abstract SCI-22

Imbalances of iron homeostasis account for some of the most common human diseases. Pathologies can result from both iron deficiency or overload. The hepcidin/ferroportin and the IRE/IRP regulatory systems balance systemic and cellular iron metabolism, respectively, and understanding their points of intersection and crosstalk represents a major challenge in iron biology. I will discuss an emerging picture from studies with different mutant mouse lines according to which the “cellular” IRE/IRP system determines “set points” via its targets (including ferroportin and HIF2α). These are then subject to modulation via hepcidin in response to systemic cues.

Disclosures:

No relevant conflicts of interest to declare.