bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potentiation of Bortezomib-Induced Killing of Lymphoma Cells by Inhibition of Autophagy and Prevention of I-κBα Degradation
Authors: Jia Li , Gopinathan Ganga , Sukumar Johanna T , Gribben John G. 
Published: October 2019

Abstract
Abstract 116
Previous studies have shown that germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) are resistant to proteasome inhibitors such as bortezomib. The mechanism by which GCB-DLBCL cells escape from proteasome inhibitor-induced apoptosis is unclear. To investigate this further, we examined the proteasomal pathway, expression of anti-apoptotic proteins and autophagy. Using bortezomib or MG-262, we show that DLBCL cells have differential sensitivity to proteasome inhibitor-mediated cell death, even though the effects on proteasome inhibition were similar. DLBCL cells that either over-express anti-apoptotic proteins such as Bcl-2, or lack pro-apoptotic proteins including Bax/Bak, are more resistant to proteasome inhibitor-induced reduction of mitochondrial membrane potential and activation of caspase-3. Treatment with bortezomib induced autophagy in both sensitive and resistant DLBCL cells, as demonstrated by an accumulation and aggregation of the autophagy marker protein LC3-II. Bortezomib induced accumulation of ubiquitinated proteins and a decrease in the adaptor protein p62, indicating activation of autophagic degradation. Fluorescent microscopy revealed that bortezomib induced p62 recruits both ubiquitinated proteins and LC3-II, suggesting that p62 leads ubiquitinated protein to autophagosomes. Treatment with bortezomib thereby promotes I-kBα degradation, demonstrating that the route of I-κBα degradation is not via the ubiquitin-proteasome degradation system. Bortezomib-induced I-kBα degradation was detected in both DLBCL cell lines and primary DLBCL and follicular lymphoma samples. Chloroquine (CQ), an inhibitor of autophagy, significantly increased bortezomib-induced accumulation of p62 and ubiquitinated proteins, including I-κBα, Bax and p53. CQ alone induced a dose-dependent increase in I-kBα protein levels, indicating that I-κBα protein can be degraded by autophagy even in the absence of proteasome inhibition. Importantly, the combination of proteasome and autophagy inhibitors shows great potential to kill apoptosis-resistant lymphoma cells. Proteasome inhibitor with or without CQ induced cell death in DLBCL cells cannot be blocked completely by either caspase inhibitors or knockdown of Bax/Bak proteins, suggesting that cell death occurs via a caspase-independent pathway. In summary, proteasome inhibitors induce autophagy and confer DLBCL cells resistance by eliminating I-κBα and possibly other pro-apoptotic proteins. Addition of autophagy inhibitors to bortezomib has the potential to induce increased killing in patients with resistant lymphoma.

Disclosures:
Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.
Hematopoietic Growth Factors In Aplastic Anemia Patients Treated with Immunosuppressive Therapy: Systematic Review and Meta-Analysis - 2010 Update.
Authors: Gurion Ronit , Gafter-Gvili Anat , Vidal Liat , Paul Mical , Ben-Bassat Isaac , Yeshurun Moshe , Shpilberg Ofer , Raanani Pia 
Published: October 2019

Abstract
Abstract 1164

Background:
Immunosuppressive therapy (IST) is the treatment for patients with severe aplastic anemia (SAA) not eligible for transplantation. It is controversial whether there is a role for hematopoietic growth factors (HGF) as an adjunct to IST in these patients.

Objectives:
A meta-analysis evaluating the role of HGF in this setting was published by our group in 2009. Since then, results of the largest conducted clinical trial by the Aplastic Anemia Working Party of the EBMT have been reported. We therefore updated our meta-analysis in order to evaluate if in 2010 there is still a role for the addition of HGF to IST in patients with SAA.

Methods:
Systematic review and meta-analysis of randomized controlled trials comparing treatment with IST and HGF to IST alone in patients with SAA. An updated search in The Cochrane Library, MEDLINE, conference proceedings and references was conducted in July 2010. Two reviewers independently assessed the quality of the trials and extracted data. Outcomes assessed were: all-cause mortality, overall hematologic response, infections and clonal evolution (transformation to myelodysplastic syndrome or acute leukemia). Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled.

Results:
Our search yielded 7 trials, randomizing 619 patients, including the 205 patients included in the EBMT trial recently published. Trials were conducted between the years 1991 and 2008. The IST regimen for most trials consisted of anti-thymocyte globulin, cyclosporine and steroids. The HGF in 6 trials was G-SCF and in 1 trial GM-CSF and erythropoietin. The addition of HGF to IST, compared with IST alone yielded no difference in all cause mortality at 100 days (RR 1.33, 95% CI 0.56–3.18) and at 5 years (RR 0.91, 95% CI 0.64–1.30, Fig.1). There was no difference in overall hematologic response at 12 months between the two arms (RR 1.16, 95% CI 0.91–1.47). There was no increase in the incidence of clonal evolution in the HGF arm compared to the control (RR 1.45, 95% CI 0.42–5.07). In addition there was no difference in the number of infections between both arms (RR 0.98, 95%CI 0.82–1.17).

Conclusions:
The addition of HGF to IST in SAA does not influence all-cause mortality, long term response, or the incidence of infections. The cumulative data in our updated meta-analysis is consistent with the results of our previous report. Therefore, HGFs should not be recommended routinely as an adjunct to IST for patients with SAA.

Disclosures:
Shpilberg: Roche: Consultancy, Honoraria.
Clinical Grade Isolation of Regulatory T Cells From G-CSF Mobilized PBSC Improves with Initial Depletion of Monocytes.
Authors: Mahmud Dolores , Park Youngmin , Mahmud Nadim , Rondelli Damiano 
Published: October 2019

Abstract
Abstract 1176
We have recently demonstrated that G-CSF mobilized peripheral blood stem cells (PBSC) CD4+CD25+FoxP3+ cells (Tregs) prevent anti-CD34+ hematopoietic stem cells T cell alloreactivity in-vitro and co-transplantation of CD34+ cells and Tregs does not affect human stem cell engraftment in NOD/SCID mice (Mahmud D et al. Biol Blood Marrow Transplant, 2010). Since only a small number of Tregs can be isolated from normal peripheral blood we examined whether PBSC can be a useful source of Tregs for future clinical trials. Five leukapheresis products from healthy donors who received rh G-CSF at 10 ug/kg daily for 5 days were processed using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA). CliniMACS CD34 reagent was initially used to isolate CD34+ cells. To isolate Tregs a 2-step procedure was initially utilized. A cocktail of clinical grade CD14, CD8 and CD19 reagents was mixed with the CD34- cells and depletion of monocytes, cytotoxic T cells and B cells was obtained by using the Depletion 2.1 program. The CD4+ cells were then enriched in Tregs by positive selection of CD25+ cells using a clinical grade CD25 reagent (Miltenyi). Because PBSC contain large amount of myeloid cells, and particularly monocytes, this clinical scale 2-step strategy was compared with a 3-step method that included an initial negative selection of CD14+ monocytes, followed by negative selection of CD8+ and CD19+ cells and a positive selection of CD25+ cells. Prior to isolation, the average proportion of CD4+CD25+ cells in PBSC was 0.77±0.26% in 5 separate PBSC products. After the 2-step process the proportion of CD4+CD25+ cells was 35±33% (n=3) vs 72±1% after the 3-step process. Therefore, utilizing the 3-step approach a better yield of Tregs was observed (10 vs 60%). Intracellular expression of FoxP3 was on average 74% in CD4+CD25+ cells obtained with a 3-step process. Contamination of different cell subsets in the final products enriched in Tregs was largely superior following the 2-step as compared to 3-step isolation method. Contaminating monocytes were, on average, 43 vs 5.7%, and contaminating CD8 and CD19+ cells were 12 vs 1.7% and 0.9 vs 0.3%, respectively. In the two procedures using a 3-step approach the final absolute number of Tregs isolated from products containing on average 30 × 109 mononuclear cells, was 95 and 93 × 106, respectively. These findings obtained using clinically available reagents and device, suggest that depletion of monocytes may improve the purity of Treg cell population isolated from PBSC. PBSC may represent a valuable source of Tregs for future clinical trials.

Disclosures:
No relevant conflicts of interest to declare.
RHPS4, An Anti-Telomere Agent, Specifically Targets the Subgroup of AML Cells with Loss or Gain of Parts of Chromosomes.
Authors: Pallis Monica , Clarke Simone , Grundy Martin , Richardson Jaineeta , Ojo Dotun , Anderson Susan , Russell Nigel H. , Seedhouse Claire 
Published: October 2019

Abstract
Abstract 1195
Cytogenetic abnormalities vary greatly in AML and those with gains and losses of chromosome parts, terminal deletions and whole chromosome losses could theoretically derive from telomere dysfunction. Cells with these abnormalities are characterised by telomere shortening and abnormal function characterised by activation of both the negative telomere regulator TRF1 and also of hTERT. The G quadruplex ligand RHPS4 (Pharminox Ltd, Biocity, Nottingham, UK) binds telomeres and induces DNA damage at higher doses in the short term and critical telomere shortening in longer term growth assays. We have previously reported cytostatic and cytotoxic effects for this agent in AML cell lines, whilst others have found that normal haematopoietic progenitor cells are not targeted. We have now examined effects of RHPS4 on the in vitro growth and survival of primary AML cells, comprising 9 samples with normal and 8 with abnormal karyotypes (5 complex, one -7, one 5q- and one with additional material on chromosomes 2 and 9). Cells were incubated with RHPS4 in standard 14 day colony assays and for 7 and 21 days in a novel suspension culture medium supplemented with supernatant from cultured normal human osteoblasts. In the 7 day cultures, no effects were observed on cell survival. In the longer term growth assays, a submicromolar IC50 for RHPS4 was observed in 7/8 samples with aberrant cytogenetics in contrast with only 1/9 with normal cytogenetics (P=0.003, Fisher's exact test). Furthermore, RHPS4 significantly sensitised samples with abnormal cytogenetics to daunorubicin reducing the median IC50 from 27nM to 10nM (n=8, p<0.05).
A qPCR method was used to measure telomere length of untreated DNA from 15 of the samples; the qPCR assay had previously been performed in a separate patient cohort alongside a Southern technique and comparable values were attained for the two different methods. There were no significant differences in telomere lengths between the normal and abnormal cytogenetic groups suggestive that telomere length is not a determinant of RHPS4 sensitivity in these assays. Samples with adverse cytogenetics have previously been associated with an up-regulation of basal DNA damage markers and may therefore be sensitive to compounds that can exacerbate levels of damage. We therefore studied the levels of p-ATM by immunocytochemistry in RHPS4-treated cells and found a significant increase compared to untreated cells - hence RHPS4 may be acting via a DNA damage mechanism.
We conclude that telomere targeting has exciting potential as therapy for a subset of AML patients including those with adverse cytogenetics who have a poor response to conventional chemotherapeutics.

Disclosures:
No relevant conflicts of interest to declare.
Jak2 Regulates Bcr-Abl In CD34+ Cells From Imatinib Mesylate-Resistant CML Patients.
Authors: Samanta Ajoy K. , Perazzona Bastianella , Chakraborty Sandip N. , Sun Xiaoping , Modi Hardik , Bhatia Ravi , Arlinghaus Ralph B. 
Published: October 2019

Abstract
Abstract 1220
Despite the success of imatinib mesylate (IM), complete molecular remission is difficult to achieve in IM-treated CML patients. After continuing treatment with IM, a population of leukemic stem/progenitor cells remains, which are insensitive to IM. Our previous studies have shown that Jak2 regulates Bcr-Abl signaling and maintains levels of Bcr-Abl in CML cell lines, fresh cells from blast crisis patients, and mouse hematopoietic cells transformed by wild-type and imatinib-resistant mutants of BCR-ABL (Samanta et al. ASH 2009 meeting, submitted). We hypothesize that Jak2 plays similar role in Bcr-Abl+ CD34+ stem/progenitor cells, and that inhibition of Jak2 will induce apoptosis by inhibiting critical signaling pathways. In our current studies we have examined Bcr-Abl CD34+ progenitor cells from CML blast crisis patients. These cells are resistant to IM but quite sensitive to cell death induction by the selective Jak2 inhibitor TG101209 at 5–10 μM concentrations for 48 h. Similar results were obtained with cells from IM-resistant accelerated phase and IM-resistant chronic phase patients. Western blotting of CD34+ cells from an IM-resistant blast crisis CML patient established that a four h treatment with TG101209 at 10 μM severely reduced levels of Bcr-Abl protein and levels of phosphotyrosine 177 within Bcr-Abl.
To begin to assess whether Jak2 effects on Bcr-Abl signaling also occur in early stage Bcr-Abl induced disease, we assayed CD34+ cord blood cells transformed by Bcr-Abl in short-term culture (Chu S et al. Cancer Res 2007; 67: 7045–7053). The results showed that a six h treatment with TG101209 (10 μM) reduced levels of: Jak2 protein; activated Jak2 (lower levels of pTyr 1007/8); Tyr 177 within Bcr-Abl; and the Bcr-Abl protein itself. These results support our earlier findings that Jak2 regulates Bcr-Abl signaling in CML cell lines (Samanta AK, et al. Cancer Res 66 (13):6468-72, 7/2006; Samanta AK, et al., Oncogene 28 (14):1669-81, 4/2009; Tao W., et al., Oncogene 27(22):3194-200, 5/2008). Furthermore, these results suggest that Jak2 plays a critical role in progenitor CML cells from early stage and late stage patients with CML disease.

Disclosures:
Bhatia: Novartis: Consultancy.
Survival and Response Outcomes to Different Treatment Schedules in CML Patients Starting Therapy with Imatinib. Results from the CML Spanish Registry (RELMC)
Authors: Casado Luis Felipe , Massague Isabel , Giraldo Pilar , Perez-Encinas Manuel , De Paz Raquel , Martínez-López Joaquín , Bautista Guiomar , Osorio Santiago , Requena Maria José , Palomera Luis , Peñarrubia Maria Jesús , Calle Carmen , Maestro Begoña , Steegmann Juan Luis 
Published: October 2019

Abstract
Abstract 1237

Background:
The RELMC is a multicentric, 17-hospitals-based cancer registry whose aim is to describe the treatments received by patients with CML, their outcomes, and the variables that influence treatment choices.

Aim:
To study the response and survival outcomes, in newly diagnosed CML patients treated with Imatinib (Im) as first line treatment.

Patients and methods:
249 newly diagnosed CML patients have been included. They are distributed in the following subgroups according to treatments received Im400. 166 patients received only Im400.

Result:
A summary of response and outcome is included in Table 1.
Complete cytogenetic response with regards to the best response, the CCyR rate was lower in patients with Im400-HDIm-2GTKI (60%) and Im400-2GTKI (62%). The rates were 84% in Im400, 83% in Im400-HDIm and 85% in HDIm; P Chi2 8,381(a) p=0,079. The CCyR cumulative incidence was also lower in patients with Im400-HDIm-2GTKI and Im400-2GTKI in comparison to the other groups, although second line response was faster in patients who changed to 2GTKI after Im400. The frequency of CCyR as best response in the Hasford high risk patients was low in all groups (66%,50%,50%,50&55%).
Major molecular response MMR as best response was lower in patients with Im400-HDIm-2GTKI (50%) and Im400-2GTKI (47%). The rates were 83%, 81% and 77% in the Im400, Im400-HDIm and HDIm groups respectively; P Chi2 19,4(a)p=0,001. The MMR cumulative incidence was higher in the HDIm group, lower in those treated with Im400-HDIm-2GTKI, and intermediate and similar in the other three groups. MMR as best response in the Hasford high risk patients was also low in all groups (60%, 75%, 50%, 50% & 33%).
Complete molecular response regarding best response, the CMR rate was lower in patients with Im400-HDIm-2GTKI (37,5%), Im400-2GTKI (31,6%) and Im400-HDIm (34%). In the other groups, the rate was 48% (Im400), and 72% (HDIm); P Chi2 17,4(a) p=0,002. The CMR cumulative incidence was higher in the HDIm group, and nil in those treated with Im400-HDIm-2GTKI.
Salvage therapy after suboptimal response (SR) or Failure (F). Two-thirds of patients with SR or F were able to obtain an optimal response and avoid transformation with a timely therapy change. All but one of the options (Im400-HDIm-2GTKI group) were similarly effective.

Survival:
6 patients progressed (2,4%) (4 AP, 2 BC), and died; 6 patients changed to allo BMT and were censored; 6 patients died of non-CML related causes.

Conclusion:

Disclosures:
Palomera: Janssen Cilag: Honoraria. Steegmann:Bristol-Myers Squibb: Honoraria, Participated in advisory boards, Research Funding; Novartis: Participated in advisory boards, Research Funding.
Treosulfan, Etoposide and Cyclophosphamide as Non TBI-Conditioning Regimen Followed by Allogeneic SCT for Acute Lymphoblastic Leukemia
Authors: Kröger Nicolaus , Stelljes Matthias , Bornhäuser Martin , Bethge Wolfgang A. , Arnold Renate , Martin Hans , Schmid Christoph , Kolbe Karin , Goekbuget Nicola , Hoelzer Dieter , Zander Axel R. , Beelen Dietrich 
Published: October 2019

Abstract
Abstract 1327
Total body irradiation (TBI) is an essential part of the conditioning regimen for acute lymphablastic leukaemia (ALL) patients who undergo allogeneic stem cell transplantation. Due to high toxicity and mortality induced by TBI, we investigate in a multicenter prospective phase II study the toxicity and efficacy of a non TBI-based regimen consisting of treosulfan (3 × 12 g/m2), etoposide (30 mg/kg), and cyclophosphamide (120 mg/kg) in patients with ALL who underwent allogeneic stem cell transplantation. Major inclusion criteria were complete remission, indication for allogeneic stem cell transplantation according GMALL, non eligibility for TBI or patient's wish to avoid TBI. Graft-versus host prophylaxis consisted of cyclosporine A and short course methotrexat and in case of unrelated donors also of anti-lymphocyte globulin (ATG). This preliminary analysis is based on 36 patients (female: n=17; male: n=19) with a median age of 42 years (range: 18–60y). Remission-status at transplantation was either 1. CR (n=27), 2.CR (n=7) or >2.CR (n=2). Donors were HLA-identical sibling (n=6), matched unrelated (n=21) or mismatched unrelated (n=9). 39% of the patients were Philadelphia chromosome positive. Primary graft failure was observed in 1 pts. The median time to achieve leukocyte (> 1×109/l)) and platelet (>20 × 109/l) engraftment was 22 (range, 11–47) and 24 days (range, 11–95), respectively. The toxicity was moderate including VOD (6%) and liver toxicity grade III (8%) and IV (3%). Acute graft versus host disease grade II-IV and grade III/IV was noted in 22% and 11%, respectively. Chronic graft versus host disease at 1 year was seen in 27%, which was extensive in 7% of the patients. After a median follow up of 12 months, the cumulative incidence of non-relapse mortality (NRM) at 1 year was only 9% (90% CI: 1–17%) and for relapse 37% (90% CI: 21–52%). The estimated 1-year disease free survival was 56% (90% CI:40-71%) and significantly better for patients transplanted in 1.CR vs 2. or higher CR (68% vs 15%, p=0.05). The estimated 1-year overall survival was 81% (90% CI: 69–92%). This preliminary results of a treosulfan, non TBI based conditioning regimen followed by allogeneic stem cell transplantation shows favourable toxicity profile with low non-relapse mortality. Longer follow up is needed to determine long-term freedom from disease.

Disclosures:
Kröger: MEDAC: Research Funding. Off Label Use: Treosulfan is not approved as conditioning regimen for stem cell transplantation.
Higher Non-Relapse Mortality with BuCyVP Compared to BuCy In Allogeneic Hematopoietic Stem Cell Transplant
Authors: Duong Hien , Bolwell Brian J. , Rybicki Lisa , Kalaycio Matt , Andresen Steven , Sobecks Ronald , Dean Robert , Sweetenham John William , Tench Shawnda , Copelan Edward A. 
Published: October 2019

Abstract
Abstract 1340
We analyzed results of allogeneic hematopoietic stem cell transplantation from 1/2000 to 1/2009 in 520 adult patients, receiving either bone marrow or peripheral stem cells, in order to identify factors associated with non-relapse mortality (NRM). Median age at transplant was 47 years (range 18–70). Median follow up was 43 months (range 5–122 months). Two hundred eighty four (55%) patients were male. Diagnoses included: acute myeloid leukemia 210 (40%), acute lymphoblastic leukemia 68 (13%), myelodysplastic syndrome 66 (13%), non-Hodgkin or Hodgkin lymphoma 64 (12%), chronic myelogenous leukemia 45 (9%), or other (13%). High scores (≤3) on hematopoietic cell transplant-specific comorbidity index were identified in 162 (31%) patients. For preparative regimens, 187 (36%) received BuCy (busulfan 1mg/kg oral given every 6 hours × 16 doses days -8 to -4, followed by cyclophosphamide 60mg/kg × 2 days -3 to -2), 91 (18%) received BuCyVP (busulfan 1mg/kg oral every 6 hours × 16 doses days -9 to -5, followed by etoposide 60mg/kg days -5 to -4, then cyclophosphamide 60mg/kg days -3 to -2), 225 (43%) received TBI-based therapy, and 17 (3%) received other therapy. Two hundred ninety five (57%) underwent related transplant. A majority (67%) received stem cells derived from bone marrow.
In univariable analysis, male recipient (p=0.07), high comoborbidity score (p=0.004), more prior chemothepary regimens (p=0.037), BuCyVP (p<0.001) or TBI-based preparative regimen (relative to BuCy, p=0.045) and unrelated donor (p=.001) were associated with high NRM. In multivariate analysis, male recipient (p=0.008), high comorbidity score (p=0.002), and BuCyVP compared to BuCy (p<0.001) remained independent risk factors for NRM. There were no significant differences in the proportions of identified cause of death between regimens. Figure 1 illustrates the sustained influence of preparative therapy on NRM:
Factors associated with poor overall survival (OS) and relapse-free survival (RFS) on multivariate analysis included male recipient (p=0.001, p=0.003, respectively), older age at transplant (p=0.048 for RFS), high comorbidity score (p<0.001, p<0.001), more prior chemotherapy regimens (per 1 regimen increase, p=0.22, p=0.02 respectively), and BuCyVP compared to BuCy (p=0.007, p=0.021).
Long-term follow-up of a large cohort of patients indicates that compared to BuCy, BuCyVP is associated with significantly higher NRM and lower OS and RFS.

Disclosures:
Sweetenham: Pfizer: Research Funding.