bluuebunny/crossref_metadata_2025 huggingface dataset:
Rituximab Plus Gemcitabine and Oxaliplatin as Salvage Therapy In Patients with Relapsed/Refractory Mantle-Cell Lymphoma.
Authors: Gironella Mercedes , López, MD Andrés , Pau Abrisqueta , Anny Jaramillo , Noelia Purroy , Bosch Francesc
Published: October 2019

Abstract 2825

Mantle cell lymphoma (MCL) constitutes one of the lymphomas with poorest prognosis at relapse and there are no effective salvage.

The activity shown by the combination Gemcitabine and Oxaliplatin in several types of limfoma, along with its “in vitro” synergistic effect, made this regimen an attractive regimen for salvaging patients with MCL. Against this background, we performed an off-label pilot study in order to assess efficacy and toxicity of the combination R-GemOx in relapsed or refractory patients with MCL.

For this, 27 patients (70% male, median age 70 years) diagnosed with MCL between November 2004 and January 2010 were included in this study. Inclusion criteria were adequate performance status, confirmed diagnosis of MCL and relapse or refractoriness to the previous treatment. The regimen consisted of Rituximab 375 mg/m2 on day 1, Gemcitabine 1000 mg/m2 and Oxaliplatin 100 mg/m2 on day 2, every 14 days, up to 8 cycles. Dose and interval were adjusted according to hematological and extrahematological toxicities. Median number of previous regimens was 1 (range 1 to 3), being EPOCH-R (n=14), R-CHOP (n=6), and RFC (n=3) the most frequently used induction therapies. Twelve patients relapsed after prior CR, 10 progressed after achieving a PR, whereas 5 were refractory to therapy. At inclusion, 85% of patients were in advanced (III/IV) clinical stage, 40% had bone marrow infiltration, 28% gastrointestinal involvement, and 17% cavum infiltration. Median number of cycles administered was 8 (range, 3 to 8). Doses were reduced in 9 cycles and delayed in 15 cycles. Neutropenia grade 3–4 was observed in 9 cycles and thrombocytopenia grade 3–4 in 6. Hepatotoxicity grade 1–2 in 5 pts and grade 3 in 1, sensitive neurotoxicity grade 1–2 in 12 pts, and renal impairment grade 2 in 1 patient. After completion of the treatment, 21 pts (77%) were considered in CR/uCR, 1 (4%) achieved a PR, 1 a SD, whereas 4 PD. Six patients subsequently received a stem-cell transplantation (4 allogeneic, 2 autologous). Thirteen pts are still alive and out of progression. Ten pts have died (8 due to progression and 2 due to acute GVHD). With a median follow-up of 23 months (range: 3–57), PFS and OS at 2 years are 41% and 58%, respectively. The R-GemOx combination showed a significant activity in relapsed or refractory pts with MCL with a very acceptable toxicity profile. These results prompted us to conduct a multicenter phase II clinical trial that is now ongoing.

Disclosures:

López: Roche Farma: Research Funding, Travel Support. Bosch:Roche Farma: Research Funding.
Lenalidomide In Patients with Relapsed or Refractory Hodgkin Lymphoma.
Authors: Böll Boris , Fuchs Michael , Reiners Katrin S. , Engert Andreas , Borchmann Peter
Published: October 2019

Abstract 2828

The immunomodulatory compound lenalidomide induces apoptosis in tumor cells, exerts antiangiogenic effects and activates immune effector cells. Apoptosis resistance, increased neoangiogenesis and impaired immunity critically contribute to Hodgkin‘s Lymphoma (HL). However, the activity of lenalidomide in HL is unknown so far.

42 patients with multiple relapsed or refractory HL were treated with 25 mg oral lenalidomide for 21 days of a 28-day cycle in a named patient program in 18 different german hospitals. Participants gave written informed consent, had no curative treatment options, an ECOG performance status ≤ 2, and normal organ functions including peripheral blood counts within the normal range. Patients were staged and restaged with computed tomography (CT) of the initially involved sites; the use of PET was optional. Response was defined according to the “Cheson-criteria” from 1999 and evaluated after two to three cycles and every other cycle thereafter. Treatment was continued until disease progression or in case of intolerable toxicity occurred. Concomitant anti-thrombotic prophylaxis with 100 mg daily oral acetylsalicylic acid was mandatory. Patients with a history of thrombosis or thrombembolic events received low molecular heparin.

To date, in the 24 patients who are eligible for analysis, treatment was well tolerated. No hematological or other toxicities above CTC grade-2 were reported; adverse events included diarrhea, constipation, neuropathy, and mild dyspnea. At restaging, eight of the analyzed patients had stable disease and twelve patients achieved clinical responses (11× PR, 1× CR) as measured by CT-scan. However, five of the heavily pretreated patients had progressive disease. Results on the remaining patients are currently being analyzed and will be presented at the ASH meeting.

In conclusion, these first clinical observations indicate that lenalidomide is well tolerated and effective in patients with relapsed or refractory HL. Consequently, international clinical trials evaluating the role of lenalidomide in HL are in preparation and a phase I/II trial evaluating lenalidomide with AVD (Adriamycine, Vinblastine and Dacarbacine) in elderly HL patients is currently recruiting.

Disclosures:

Off Label Use: Lenalidomide approved for Multiple Myeloma and MDS (5q−).
The Histone Deacetylase Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction
Authors: Jona Adam , Khaskhely Noor , Buglio Daniela , Shafer Jessica A. , Derenzini Enrico , Bollard Catherine M , Illes Arpad , Andreef Michael , Younes Anas
Published: October 2019

Abstract 2850

Introduction:

Based on recent favorable in vitro and in vivo activity of several HDACi (histone deacetylase inhibitors) in HL (Hodgkin lymphoma), we investigated the in vitro activity of SNDX-275, an oral, class 1 isoform of selective HDACi in HL-derived cell lines.

Materials and methods:

Proliferation and cell death were examined by MTS assay, Annexin-V/PI and FACS analysis. For combination studies, cells were incubated with SNDX-275 (0.1-2 μM) and either ABT-737 (0.01-0.2 μM), Obatoclax (0.1-2 μM), Gemcitabine (1-20 nM) or Bortezomib (1-20 nM) for 72 hours. Gene and protein expression were measured by RT-PCR, Western blot, and immunohistochemistry. A multiplex assay was used to determine 30 cytokines and chemokines.

Results:

SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased histone-3 acetylation, up-regulated p21 expression, and activated the intrinsic apoptosis pathway by down-regulating the XIAP (X-linked inhibitor of apoptosis protein), which was associated with activation of Caspase 9 and 3. Similarly to other HDACis, SNDX-275 decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, while level of Mcl-1 and pro-apoptotic Bax remained the same level. Combination studies demonstrated that SNDX-275 had more synergistic effect when combined with Bcl-2 inhibitors ABT-737 or Obatoclax and less when combined with Gemcitabine or Bortezomib. Dysregulated cytokine/chemokine production has been shown previously to contribute to HL pathology, including immune tolerance of the cancer cells. Hence, we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Increased IL12 p40-70, IP10, and RANTES, and decreased IL13, IL4 and TARC levels were found, thus favoring Th1-type cytokines/chemokines. Recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of CTAs (cancer testis antigens), leading to a favorable immune response. SNDX-275 was able to induce CTA expression of SSX2 and NY-ESO only in one cell line whereas MAGE-A4 was induced in both HL cell lines.

Conclusion:

Our studies demonstrate that SNDX-275 has a dual effect on apoptotic and immunomodulatory pathways in HL, which can be enhanced by the addition of agents targeting cell survival pathways. Phase II studies with SNDX-275 in HL are ongoing, future clinical studies should investigate combinations with SNDX-275.

Disclosures:

No relevant conflicts of interest to declare.
CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment
Authors: Chandra Abhinav B. , Burton Jack , Stein Rhona , Chen Susan , Mishra Nidhi , Shah Taral , Enwemnwa Nneamaka N , Goldenberg David
Published: October 2019

Abstract 2888

Background:

CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines.

Methods:

CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed.

Results:

In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab.

Conclusions:

CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful.

Supported in part by NIH grant PO1-CA103985 (DMG).

Disclosures:

No relevant conflicts of interest to declare.
Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma
Authors: Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Rieken Malte , Pastore Alessandro , Hartmann Elena , Rosenwald Andreas , Hiddemann Wolfgang , Dreyling Martin H.
Published: October 2019

Abstract 2893

Background:

The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis.

Methods:

Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment.

Results:

Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways.

Conclusion:

In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL.

Disclosures:

Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.
Myeloma Cells Induce Osteoblast Suppression through Sclerostin Secretion
Authors: Colucci Silvia , Brunetti Giacomina , Oranger Angela , Mori Giorgio , Sardone Francesca , Liso Vincenzo , Curci Paola , Miccolis Rosanna M , Rinaldi Erminia , Specchia Giorgina , Passeri Giovanni , Zallone Alberta , Rizzi Rita , Grano Maria
Published: October 2019

Abstract 2961

Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Thalidomide with Melphalan and Prednisone on Health-Related Quality of Life (HRQoL) in Elderly Patients with Newly Diagnosed Multiple Myeloma: A Prospective Analysis In a Randomized Trial
Authors: Verelst S. , Termorshuizen F. , Groot C. Uyl-de , Schaafsma M. R , Ammerlaan R. , Wittebol S. , Sinnige H. , Zweegman S. , Kooy M. van Marwijk , van der Griend R. , Lokhorst H. , Sonneveld P. , Wijermans P.
Published: October 2019

Abstract 2972

Thalidomide (T) with melphalan/prednisone (MPT) was defined as a standard treatment in elderly patients with multiple myeloma (MM) based on 5 randomized trials. Treatment with MPT not only showed improved response rate, significantly better time to response as well as quality of response but also a significant improvement of event free survival (EFS), progression free survival (PFS) and overall survival (OS). In one of these trials, HOVON 49, a prospective Health related – Quality of life (HRQoL) was initiated in order to asses the impact of T on QoL. (Wijermans et al, J Clin Oncol 28:3160-6). Patients aged >65 years with newly diagnosed symptomatic MM were randomized to receive 8 cycles of MP or MPT, followed by T maintenance in MPT arm. 284 patients were included in this HRQoL side study (MP: n=149, MPT: n=135). HRQoL was assessed with the EORTC core QoL Questionnaire (QLQ-C30) and the myeloma-specific module (QLQ-MY24) at baseline and at pre-determined intervals during treatment. Treatment-related toxicity WHO grade 2–4 occurred in 60% of MP and 87% of MP-T treated patients. Most frequently found were neutropenia-related infections, neurotoxicity (mostly T induced peripheral neuropathy) and myelotoxicity.

The QLQ-C30 subscales Physical Function (p=0.044) and Constipation (p<.001) showed a treatment related improvement during induction in favour of the MP arm. During T maintenance, the scores for the QLQ-MY24- Paraesthesia became significantly higher in the MPT arm (p<.001). The QLQ-C30 subscales Pain (p=0.12), Insomnia (p=0.068), Appetite loss (p=0.074) and the QLQ-MY24- item Sick (p=0.086) scored marginally better during T maintenance. The overall QoL scale QLQ-C30-HR-QOL showed a significant time trend towards more favourable mean values during protocol treatment but did not reveal differences between MP and MPT. For the QLQ-C30 subscales Emotional function and Future perspectives, difference in favour of the MPT arm from the start of treatment was observed (p=0.018and p=0.045 respectively) with no significant ‘time × arm' interaction, indicating a persistent better patient perspective with MPT treatment. Since the first questionnaire was filled out prior to treatment but after randomization we hypothesise that the awareness of being treated with a medication that holds out a prospect of better treatment outcome may in itself be associated with improved feeling of well-being and hope for the future.

This prospective study shows that the higher frequency of adverse effects associated with MPT does not translate into a negative effect on HRQoL and that MPT holds a better patient perspective.

Disclosures:

No relevant conflicts of interest to declare.
Prohibitin Serves as a Novel Target Mitochondrial Protein of Phenyl-Chloroethyl Ureas Involved In Their Antiproliferative Effect on Multiple Myeloma Cells
Authors: Kim Hye-Ran , Eom Ha-Young , Kim Dong Min , Kim Il-Chul , Kumar Manian Rajesh , Lee Sun-Woo , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun
Published: October 2019

Abstract 3002

Background:

Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function. The diverse function of PHB, together with the emerging evidence that its function can be modulated specifically in certain diseases, implies that PHB would be a potential target for new therapeutics.

Materials and Methods:

We analyzed mitochondrial proteins and develop new anti-proliferative agents targeting multiple myeloma (MM) cells. Mitochondria were isolated from primary leukemia and MM cell lines (RPMI8226, ARH77, U266 and IM9) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein was identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology).

Results:

Out of 38 remarkable deregulated mitochondrial proteins in MM cell lines, prohibitin (PHB) (gi4505773) was highly expressed in primary MM and leukemia cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary bone marrow cells and sections. Potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized independently from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in MM and leukemia cells. Moreover, notable morphological transformation of MM cells was observed when treated with 10 – 100 umol of CCEU and ICEU for 24 hours. The half of maximal inhibitory concentration (IC50) was 25umol of most MM cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- MM cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in MM cell lines.

Conclusions:

By analyzing mitochondrial protein in leukemia and MM cell, we discovered a new molecular marker, PHB, characteristically overexpressed in leukemia and MM cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in MM cells.

Disclosures:

No relevant conflicts of interest to declare.
HGAL Directly Interacts with Both Myosin and Actin and Increases the Binding of Myosin to Actin
Authors: Lu Xiaoqing , Katarzyna Kazmierczak , Jiang Xiaoyu , Watt James , Moore Jeffrey R , Szczesna-Cordary Danuta , Lossos Izidore S
Published: October 2019

Abstract 3097

HGAL is a recently identified germinal center (GC)–specific gene whose expression by tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and decreasing lymphoma cell migration and dissemination. The latter effect may contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas. Actin filaments, usually in association with myosin, are responsible for many types of cell movements. The interaction between myosin and actin is responsible for muscle contraction, migration of nonmuscle cells and also plays a role in cell division. The movement of cells appears to be driven directly by actin polymerization and by actin-myosin interactions. In our previous work (Lu et al, Blood 2007), we demonstrated by co-immunoprecipitation and co-localization studies that HGAL interacts with myosin II and actin. However, the mechanistic consequences of this interaction are unknown. Because the interaction of actin with myosin is the key factor in cell motility and HGAL negatively regulates lymphocyte migration, we hypothesized that HGAL may decelerate cell migration by affecting the binding of myosin to actin. To confirm the direct and not interdependent interaction between myosin-HGAL and actin-HGAL, we performed in vitro co-sedimentation experiments with recombinant HGAL protein and purified actin and myosin, respectively. These studies demonstrated direct and independent binding of HGAL to both myosin and actin. We next demonstrated that the N terminal portion of the HGAL protein (aa 1–118) can bind the head region of myosin II (S1) that contains the actin and ATP binding sites, and to the rod part of the myosin molecule that is responsible for filament formation. While no effect of the HGAL on the actomyosin ATPase activity was observed, it reduced the rate of actin polymerization from G-actin to F-actin. Further binding studies using pyrene-labeled F-actin showed that HGAL increases the binding of myosin to F-actin. Taken together, our results indicate that HGAL is a cellular regulator of actin assembly that can also regulate the actin-myosin interaction. This in turn may explain the role of HGAL as a favorable prognosis marker for patients with diffuse large B-cell and classical Hodgkin lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Quantification of EBV DNA In Whole Blood In Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma
Authors: Beltran Brady , Salas Renzo , Chunga Ausberto , Sotomayor Eduardo M. , Castillo Jorge J
Published: October 2019

Abstract 3102

Introduction:

Epstein-Barr virus (EBV) is a well-known oncogenic virus associated with the development of several lymphoproliferative disorders. The quantification of EBV viral load in plasma and peripheral blood mononuclear cells has been reported as a useful biomarker for EBV-associated non-Hodgkin lymphoma (NHL). The role of EBV in diffuse large B-cell lymphoma (DLBCL) is unclear at this time. METHODS: In the present prospective study, using real-time quantitative polymerase chain reaction (RQ-PCR), whole blood specimens from patients with a new diagnosis of DLBCL were used to quantitatively determine EBV viral load. Consecutive patients who presented from January 1, 2009 to December 30, 2009 were selected for this study. The pathological samples from the patients showing elevated EBV DNA levels were then investigated for the presence of EBV-encoded RNA (EBER) using a chromogenic in situ hybridization (CISH) technique. Clinical data from these patients was collected and analyzed, and will be presented using descriptive statistics. Overall survival estimates were obtained using the Kaplan-Meier method.

Results:

Forty six patients with a new diagnosis of DLBCL were included in our study; from which five cases showed elevated levels of EBV DNA by RQ-PCR (10.8%). These five patients (100%) were positive for EBER expression within the tumoral cells by EBER CISH. One patient was evaluated at the end of treatment and blood EBV DNA level was undetectable. All EBV DNA-positive cases were male with a median age of 69 years (range 25–73 years); only one case was younger than 50 years. The median EBV DNA viral load was 59 copies/uL (range 0.225–43200 copies/uL). Three patients (60%) presented with advanced clinical stage (III or IV) and three patients (60%) had a primary extranodal site of involvement. Four patients (80%) received CHOP-based chemotherapy regimens. The three patients with advanced stage have deceased with a survival of <3 months. The two patients with early stage experienced a complete response with immunochemotherapy and are alive 6 and 9 months after diagnosis, respectively. The median overall survival is 4 months.

Conclusions:

These results, although preliminary, suggest that whole blood EBV DNA quantification might be useful at detecting patients with EBV-positive DLBCL. Further studies are needed to investigate the potential diagnostic and/or prognostic value of EBV DNA in DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia
Authors: Tao Rong-Hua , Berkova Zuzana , Wise Jillian F , Urszula Daniluk , Bai Li , Ao Xue , Samaniego Felipe
Published: October 2019

Abstract 3148

Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML).

PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis.

Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice.

These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells.

Disclosures:

No relevant conflicts of interest to declare.
Glucocorticoids Inhibit Cell Death Induced by Oncogenic Stress After Imatinib Withdrawal In TKI Resistant p190Bcr/Abl Overexpressing Cells
Authors: Dengler Michael A. , Staiger Annette M. , Hofmann Ute , Gutekunst Matthias , van der Kuip Heiko , Aulitzky Walter E.
Published: October 2019

Abstract 3150

Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl.

In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession.

Disclosures:

No relevant conflicts of interest to declare.
A Predictive Risk Score for Cancer-Associated Thrombosis: Role of Screening In A Prospective Study
Authors: Khorana Alok A. , Herman Kimberly , Rubens Deborah , Francis Charles W.
Published: October 2019

Abstract 3173

Background:

We evaluated the utility of screening for VTE using a previously developed clinical risk score (Khorana et al, Blood 2008) in a prospective cohort of cancer patients initiating outpatient chemotherapy but not receiving thromboprophylaxis.

Methods:

Cancer patients initiating a new chemotherapy regimen and deemed high-risk based on a predictive risk model (score ≥3) were enrolled on an ongoing prospective cohort study with informed consent. Patients were evaluated with baseline and Q4 (± 1) week serial ultrasonography for upto 16 weeks; additionally, computed tomography scans for restaging were also evaluated for VTE.

Results:

Of 30 patients enrolled on study, 8 (27%) developed a VTE. This included 5 patients with DVT alone (17%), 1 patient with PE alone (3%) and 2 (7%) with both. Twenty-seven patients underwent a baseline ultrasound. Of these, 3 asymptomatic DVTs were identified (11%). Subsequent ultrasounds were performed in 18 patients at week 4 (0 DVT), 17 patients at week 8 (0 DVT) and 15 patients at week 12 (1 DVT, 7%). An additional two patients developed symptomatic DVT between weeks 1 and 4. Restaging CT scans identified an asymptomatic PE in 1 patient at week 6 and asymptomatic PE in 1 patient at week 9 with subsequent symptomatic DVT at week 10.

Conclusions:

In a prospective observational study, 27% of cancer outpatients deemed high-risk using a clinical risk score developed VTE, a rate much higher than observed even in hospitalized acutely ill patients. Thus, this study confirms the validity of a previously described risk score. The role of thromboprophylaxis in this population is currently being tested. The value of screening ultrasonography should be considered in high-risk patients based on this risk score.

Disclosures:

No relevant conflicts of interest to declare.
Concurrent BCL2 and MYC Translocations In a Prospective Cohort of Diffuse Large B-Cell Lymphomas
Authors: Pedersen Mette Ølgod , Gang Anne Ortved , Poulsen Tim Svenstrup , Knudsen Helle , Lauritzen Anne F. , Talman MajLis M , Nielsen Signe Ledou , Norgaard Peter H
Published: October 2019

Abstract 319

Background:

Concurrent chromosomal translocations involving the BCL2 and MYC protooncogenes, so-called double-hit, is found both in diffuse large B-cell lymphoma (DLBCL) and in a newly defined B-cell lymphoma category with features overlapping between DLBCL and Burkitt lymphoma (BCLU, 2008 WHO classification). Few studies have been published on series of double-hit B-cell lymphomas, and to our knowledge only retrospective series, reporting an average frequency of around 5%. Therefore some authors suspected that the frequency of double-hit translocations was underestimated. In addition to unanimous reports of highly aggressive clinical behaviour; poor outcome and resistance to chemotherapy, double-hit lymphomas had GCB immunophenotype (Hans), varying morphology from classical Burkitt like to DLBCL-NOS and mostly high proliferation rates (Ki-67).

Purpose:

We conducted a prospective study of DLBCL to evaluate the frequency of double-hit BCL2/MYC translocations in DLBCL. Secondly, we wanted to analyse if any pathologic and/or clinical features correlated with the presence of a double-hit in order to evaluate the need for routine genetic analysis of large B-cell lymphomas in prognostic stratification.

Materials and methods:

All patients diagnosed with DLBCL or BCLU at the Haematopathology Section at Copenhagen University Hospital in Herlev were prospectively collected throughout 2009. Tumours were classified according to their morphology (2008 WHO classification), immunohistochemistry (CD10, BCL6, MUM1, BCL2, Ki-67) and FISH (BCL2, BCL6 and MYC translocations). Clinical data were collected from patient files.

Result:

Double-hit BCL2/MYC translocations were detected in 9 of 93 cases (10%); 7 DLBCL, 1 BCLU, 1 unclassified. All double-hit DLBCL were GCB immunophenotype and showed varying morphology. Ki-67 index ranged from 15–95%. Interestingly, 2 double-hit cases were late relapses (primary double-hit). Characteristic clinical parameters included a high International Prognostic Index (IPI) score, a high stage and an extranodal presentation.

Discussion and conclusion:

In this prospective cohort of DLBCL patients the incidence of BCL2/MYC double-hit was 10%. This suggests that double-hit is more frequent in DLBCL than previously estimated. Double-hit lymphomas had GCB-immunophenotype but could not be identified by morphology or proliferation rate. Therefore if these patients should be identified due to an inferior prognosis (which we could not significantly establish due to limited observation time) routine FISH analysis for double-hit translocations should be performed in all patients with BCLU or DLBCL of GCB-subtype.

Disclosures:

No relevant conflicts of interest to declare.
A novel P2Y12-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors
Authors: Xiang Binggang , Zhang Guoying , Liu Junling , Morris Andrew J. , Smyth Susan S. , Gartner T. Kent , Li Zhenyu
Published: October 2019

Abstract 3191

The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

Disclosures:

No relevant conflicts of interest to declare.
Platelet 12-Lipoxygenase Is Required for Dense Granule Secretion and Platelet Aggregation: Role of 12-hLO In Platelet Hemostasis and Thrombosis
Authors: Apopa Patrick , Patel Megha , Boutaud Olivier , Holinstat Michael
Published: October 2019

Abstract 3203

Platelet activation plays a central role in regulating hemostasis. Uncontrolled activation of circulating platelets can result in the formation of occlusive thrombi and stroke. Following activation, metabolism of arachidonic acid by 12-lipoxygenase (12-hLO) may play a significant role in regulating the degree and stability of platelet reactivity. Using specific inhibitors for 12-hLO which do not interact with other lipoxygenases or enzymes in the COX-1 pathway, we were able for the first time to asses the involvement of 12-hLO in platelet reactivity. In order to assess the role of 12-hLO in platelet activation and thrombosis, dense granule secretion, platelet aggregation, alpha granule secretion, and platelet adhesion and clot formation under flow were measured. Inhibiting 12-hLO results in a complete inhibition of dense granule secretion with only a partial attenuation of alpha granule secretion indicating a novel regulatory scheme for modulating positive autocrine reinforcement of platelet reactivity and clot formation. Addition of the 12-hLO metabolite, 12-HETE (as low as 250 nM), resulted in a significant (25%) increase in PAR1-mediated dense granule secretion compare to agonist alone indicating that 12-HETE may be the crucial metabolite formed by 12-hLO metabolism of arachidonic acid. Importantly, platelet aggregation and adhesion are also significantly attenuated in the absence of 12-hLO. In fact, collagen-mediated platelet aggregation was shifted over 25 fold to the right in the absence of 12-hLO. These studies support the role of 12-hLO in hemostasis and may be a good target for anti-platelet therapy.

Disclosures:

No relevant conflicts of interest to declare.
Diabetic Platelets Are Not Resistant to Aspirin Inhibition
Authors: Schwartz Kenneth A. , Schwartz Dianne , Gossain Ved , Myneni Anjana , Nelson Peggy , Defranco Anthony
Published: October 2019

Abstract 3324

A daily aspirin in patients with myocardial infarctions (MI) effectively reduces the risk of occlusive vascular disease by 25%. This protective effect is reduced or absent in diabetics possibly because aspirin induced inhibition of platelet function is diminished. We tested the hypothesis that the amount of aspirin induced inhibition of platelets in diabetics is decreased.

Methods:

Each subject was evaluated at 2 time points: off-aspirin and 2-hours after observed ingestion of a 325mg aspirin tablet. Platelets were stimulated in a light aggregometer with platelet prostaglandin agonist (PPA). The slope of the PPA aggregation curve decreases with aspirin inhibition and is a sensitive measure of the amount of aspirin induced platelet inhibition (Schwartz J. Lab Clin Med 2002;139:227). Light aggregometry with arachidonic acid was used to verify that the patients had or had not taken aspirin. Maximal aspirin induced decrease in platelet function was defined as the difference between the PPA aggregation curve slopes at the 2 time points, off-aspirin and 2-hours post aspirin ingestion. 187 adult subjects (62% male 38% female) divided into four groups were studied, MI patients without diabetes n=98, diabetics with an MI n=41, diabetics without an MI n=9, and normal subjects n=39.

Results:

Comparisons of the PPA slopes of the 4 groups showed that diabetics had higher slopes than non-diabetics. Platelets from diabetics with and without an MI had significantly (p<0.05) increased off aspirin platelet reactivity (PPA slope) compared to normals. At the 2 hr time point patients with diabetes and an MI had a significantly higher PPA slope than the MI without diabetes group. Maximum aspirin induced decrease in platelet function was negatively correlated with age (r=-0.32, p=0.002). When controlled for age, there was no difference in the maximum decrease in platelet function among the 4 groups.

Conclusions:

1. Platelets from diabetic patients with and without MI have increased platelet reactivity to PPA. 2. The maximum decrease in platelet function observed in diabetic patients with and without an MI was similar to that observed in MI patients without diabetes and in normals. This suggests that the decreased protection from future vascular events observed with aspirin in diabetic patients is not because of an intrinsic resistance to aspirin inhibition of platelets.

Disclosures:

No relevant conflicts of interest to declare.