bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining the Impact of Altered Protein Expression and Ubiquitination Levels on the Development of Resistance to Proteasome Inhibitors Using Proteomics Analysis
Authors: Dowling Paul , Ting Kay Reen , Meiller Justine , Kelly Shane , Clynes Martin , O'Gorman Peter
Published: October 2019

The range of new proteasome inhibitors currently in clinical or preclinical development hold tremendous promise for the treatment of haematological malignancies, in particular multiple myeloma, both in frontline and relapsed or refractory settings. However the development of resistance to proteasome inhibitors is a significant problem. To facilitate the analysis of this phenotype, a carfilzomib-resistant cell line was established and the effects on altered protein expression and ubiquitination levels determined. Label-free quantitative mass spectrometry was performed on whole cell extracts from the resistant model together with ubiquitin remnant motif antibody K-ε-GG enriched peptides for modification analysis. A significant number of proteins with altered expression and ubiquitination levels were positively identified. In particular, the proteasome maturation protein (POMP) was found to have a significantly up-regulated abundance level in the resistant phenotype, a protein essential for the assembly of standard proteasomes and immunoproteasomes (Figure 1). Abnormal ubiquitination levels were also detected on proliferating cell nuclear antigen (PCNA), significantly on Lysine-164 which is involved in DNA-repair pathways. Identifying the relevant biological pathways affected by proteasome inhibitors and the changes which mediate drug resistance may allow for optimal therapeutic use and potential new drug targets. POMP and PCNA may be important target molecules to help overcome the establishment of resistance to proteasome inhibitors in the treatment of multiple myeloma. Further work could examine broadening the therapeutic application of proteasome inhibitors that is currently confined to multiple myeloma and identifying the key proteins involved in common resistance mechanisms.

Disclosures

No relevant conflicts of interest to declare.
Cord Blood Unit Cryopreservation: Positioning Segments for Potency Assessment
Authors: Shoulars Kevin , Gentry Tracy , Noldner Pamela , Page Kristin M , Kurtzberg Joanne
Published: October 2019

Introduction: Banked, unrelated umbilical cord blood units (CBU) are a rich source of hematopoietic stem cells for hematopoietic reconstitution. Potency assays on attached segments have been used for CBU release from the bank to the transplant center. CBUs have historically been manufactured and cryopreserved in bags with three segments (made from the tubing used to fill the bag) arranged across the top of the cassette. Prior to cryopreservation, the CBU is overwrapped, placed in a metal cassette and frozen in a control-rate freezer and submerged under liquid nitrogen for long-term storage. After cryopreservation, segments can be removed from the cassette, without disturbing the bag, for HLA typing, potency assays, or other testing. Last year, in an effort to increase the number of segments available for testing, we changed the configuration of the cryobag to create a fourth segment. To fit in the cassette, the segments were arranged with two segments across the top and two segments bent at 90 degrees to fit between the 20% and 80% portions of the bag. When performing our annual stability testing, we realized that the two distal segments, positioned between the 20% and 80% compartments yielded lower potency results than the segments positioned across the top of the cassette. We investigated this finding as described below.

Method: Three large fresh CBUs were processed separately on a Sepax cell processor (Biosafe, Geneva, Switzerland) and evenly divided into two cryopreservation bags. For each CBU, one bag was created with three segments and the other bag with four segments. The cryopreservative, dimethylsulfoxide in dextran, was added at a final concentration of 10% to both bags prior to cryopreservation. Segments were positioned either across the top (3 segments) or in the bent configuration (4 segments) for each of the paired CBUs. All units were cryopreserved and stored in a Thermogenesis Bioarchive. After two weeks in storage, the CBUs were tested. The bags and segments were assayed for ALDHbr, viable CD34, CD45, glycophorin A and viability (7-AAD) along with colony forming units (CFUs), the potency assay used by our bank.

Results: The segments configured across the top of the bag correlated with results from the cryobag for ALDHbr (as a percent of viable CD45+, Table 1), CD34+ (as a percent of viable CD45+) and CFU. In contrast, segments placed between the 20% and 80% compartments were significantly lower than the bag for both ALDHbr (as a percent of viable CD45+) and CFU, although CD34+ (as a percent of viable CD45+) was equivalent.

Conclusions: Segments attached to CBUs can predict potency of the CBU by various parameters. However, proper placement of the segments within the cryopreservation cassette must be performed to ensure that potency measured on the segments reflects potency measured on the cord blood unit.

Disclosures

No relevant conflicts of interest to declare.
Intrathecal Rituximab for EBV-Associated Post-Transplant Lymphoproliferative Disorder with Central Nervous System Involvement Unresponsive to Intravenous Rituximab-Based Treatments: A Prospective Study
Authors: Liu Qifa , Wu Meiqing , Sun Jing , Zhang Yu , Huang Fen , Zhou Hongsheng , Fan Zhiping , Xuan Li , Yu Guopan , Guo Xutao , Dai Min , Feng Ru
Published: October 2019

Backgroud:

Although the introduction of rituximab as a first-line treatment has improved outcome of post-transplant lymphoproliferative disorder (PTLD), PTLD with central nervous system involvement (CNS-PTLD) still has a dismal prognosis because of low penetrance across the blood-brain barrier. In this prospective study, we reported intrathecal rituximab was efficacy and safety in the patients with CNS-PTLD who had failed to respond to the intravenous rituximab-based treatments.

Methods:

From June 2009 to November 2013, 32 cases of EBV-associated PTLD were recorded in the Southern Medical University Institute of Hematology. Fourteen patients diagnosed with CNS-PTLD were enrolled in this prospective study. For the patients who failed to response to the initial intravenous rituximab-based treatments, sequential dose-escalation schedule of intrathecal rituximab (initial dose of 20mg, increased by 10mg each week and maximum dose of 50 mg) was administrated weekly.

Results:

Three patients were responsive and 11 were unresponsive to the initial treatments within one week after the treatments. For the 11 patients who failed to respond to the initial treatments, 9 patients received intrathecal rituximab within 7-11 days and 2 patients refused the treatment. After two cycles of rituximab-based treatments, 10 patients achieved complete response (CR), 2 patients were partial response and 2 patients were non-response. With a median follow up of 664 (range 18 to 1545) days after the diagnosis of CNS-PTLD, 7 patients survived and 7 died. The causes of death included PTLD progressing (n=3), PTLD relapse (n=1), GVHD (n=1), CMV pneumonia (n=1) and pseudomonas aeruginosa sepsis (n=1). The 3-year probability of OS was 45.7% ±14.7% in CNS-PTLD, which was no significant difference as compared to PTLD without CNS involvement(55.6% ±11.7%, P=0.706).

Conclusion:

Intrathecal rituximab might be an effective and safe method for CNS-PTLD in the allo-HSCT recipients who were unresponsive to the intravenous rituximab-based treatments.

Disclosures

No relevant conflicts of interest to declare.
To Treat or Not to Treat: Metabolomics Reveals Biomarkers for Treatment Indication in Chronic Lymphocytic Leukaemia Patients
Authors: Piszcz Jaroslaw , Armitage Emily Grace , Alessia Ferrarini , Ruperez Francisco Javier , Kulczynska Agnieszka , Bolkun Lukasz , Kloczko Janusz , Kretowski Adam , Ciborowski Michal , Barbas Coral
Published: October 2019

In chronic lymphocytic leukaemia (CLL) clinical course of patients is heterogeneous. Some present an aggressive disease onset and require immediate therapy, while others remain without treatment for years. Current disease staging systems developed by Rai and Binet may be useful in forecasting patient survival time, but do not discriminate between stable and progressive forms of the disease in the early stages. Recently ample attention has been directed towards identifying new disease prognostic markers capable of predicting clinical aggressiveness at diagnosis. In this research we reached for LC-MS metabolic fingerprinting method to analyse serum of stable (n=51) and progressive (n=42) CLL patients and controls (n=45) with aim to discover metabolic indicators of disease status. A panel of markers discriminating aggressive from indolent patients was discovered. Ten of them were selected for validation on larger population (45 controls and 92 CLL) with an independent analytical technique. Linoleamide (p=0.002) in addition to various acylcarnitines (p=0.001-0.000001) showed to be significant markers of CLL in its aggressive form. Acetylcarnitine (p=0.05) and hexannoylcarnitine (p=0.005) were also distinguishable markers of indolent subjects. Forming a panel of selected acylcarnitines and fatty acid amides, it was possible to reach a highly specific and sensitive diagnostic approach (AUC=0.766).

Disclosures

No relevant conflicts of interest to declare.
Outcome of Patients with Hodgkin Lymphoma transformed from Chronic Lymphocytic Leukemia
Authors: Mocikova Heidi , Markova Jana , Gaherova Lubica , Smolej Lukas , Simkovic Martin , Kozak Tomas
Published: October 2019

Background. Hodgkin lymphoma (HL) that develops in the course of chronic lymphocytic leukemia (CLL) is a rare event. Optimal management of these patients (pts) is not defined. We analyzed outcome of 10 pts treated for Hodgkin´s variant (HV) of Richter syndrome.

Patients and methods. Overall 10 pts (8 males) with CLL and subsequent HV were treated between 1996 and 2015. Median age at diagnosis of HV was 68 (range 54 - 83) years. Prognostic markers of CLL at diagnosis: unmutated IGHV (5 pts), del(17p) or TP53 mutations (0 pt), del(11q) (2 pts), deletion of 13q14 region (4 pts), trisomy 12 (2 pts), ZAP 70 pos. (6 pts), CD38 pos.(4pts). The diagnosis of HV included various subtypes of HL: nodular sclerosis (3pts), mixed cellularity (3pts), lymphocyte rich (1pt), not othervise specified (3pts). EBV status of pts at diagnosis of HV of Richter syndrome: 6 pos., 1 negat. nad 3 not done. Initial and second-line treatments of CLL consisted of fludarabine- based regimens combined with rituximab or alemtuzumab and 3 pts received chlorambucil. Treatment of HV of Richter syndrome included: ABVD (7 pts) in combination with rituximab (2 pts), COPP (3 pts) and involved field radiotherapy of 30 Gy after chemotherapy (2 pts). Further treatment was required in 6 pts due to insufficient response with persistent CLL: rituximab alone (2 pts), R-bendamustine (1pt), R-DHAP (1 pt), cyclophosphamide and dexamethasone (1 pt) and gemcitabine (1 pt). None of these patients underwent autologous or allogeneic stem cell transplantation.

Results. The median time from CLL diagnosis to development of HV was 8.4 (range 2.6 - 16.5) years. Median overall survival from diagosis of CLL was 17.3 years and median survival after diagnosis of HV was 3.5 years. Out of 10 pts 5 are alive :3 in complete remission (CR), 1in partial remission of CLL (HL in CR), 2 in progression of CLL (HL in CR). Five pts died (3 lymphoma progressions, 1 second cancer, 1 unknown causality).

Conclusion. Our results indicate a long period for developing of HV of Richter syndrome and its subsequent poor outcome. Current HL based chemotherapy is not sufficient in HV of Richter syndrome and new treatment approaches should be considered.

Disclosures

No relevant conflicts of interest to declare.
Thrombotic Microangiopathy in Multiple Myeloma
Authors: Yui Jennifer , Van Keer Jan , Vij Ravi , Jhaveri Kenar , Kastritis Efstathios , Buadi Francis , Dispenzieri Angela , Dingli David , Russell Stephen J. , Leung Nelson
Published: October 2019

Background: Thrombotic microangiopathy (TMA) is a hematologic syndrome associated with endothelial injury, microvascular thrombosis, and hemolytic anemia resulting in end organ damage. TMA is rarely associated with multiple myeloma (MM) and is usually reported in the setting of allogeneic hematopoietic stem cell transplants (AHSCT), with associated graft versus host disease and calcineurin inhibitor use. We present 11 cases of TMA in MM patients not associated with AHSCT.

Methods: Cases were collected from multiple centers in patients with confirmed diagnosis of MM. TMA was diagnosed by the presence of thrombocytopenia and evidence of microangiopathic hemolytic anemia.

Results: Eleven patients with MM were identified to have developed TMA during the course of their disease. Two patients presented with TMA at the time of MM diagnosis. A third had been off treatment for over a year prior to presenting with a TMA which correlated with a significant increase in her free light chains. The other cases occurred during active treatment. All remaining patients received proteasome inhibitors, with three patients on bortezomib-based regimens and five patients on carfilzomib. One patient on a bortezomib-based regimen had been stable prior to developing a TMA in the setting of sepsis. In the remaining patients, the TMA developed rapidly after initiating the proteasome inhibitor. In all cases, the TMA improved after drug withdrawal. One patient was later rechallenged with bortezomib and again developed TMA.

Discussion: A review of the literature reveals only nine cases of TMA with MM. Of these, 4 patients had TMA as the initial presentation of MM, and 5 patients were on treatment, most often bortezomib-based regimens. Our series patients match those in the literature closely and demonstrate that TMAs can occur in MM outside of the context of AHSCT. In roughly one third of patients, TMAs were the initial presentation of MM and improved with treatment of the MM. In others, the use of proteasome inhibitors appears to be associated with development of TMA. In these cases, the temporal correlation, resolution of TMA after medication withdrawal, and the instance of recurrence of TMA with rechallenge of bortezomib, support a causal relationship between the medication and the TMA. Thus, a high clinical suspicion for TMA must exist for patients on proteasome inhibitors who develop worsening anemia and thrombocytopenia. Medication should be withdrawn if TMA is diagnosed.

Disclosures

Vij: Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Merck: Consultancy.
Free Light Chain Ratio As a Simple Prognostic Marker in a Single Institution Experience
Authors: Pena Camila , Voisin Javier , Peralta Alexis , Ortiz Manuela , Balboa Viviana , Delgado Florencia
Published: October 2019

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by bone marrow plasma cell infiltration and the secretion of monoclonal immunoglobulin (Ig) in the serum or urine. Median survival in MM patients is approximately 5 years and significant morbidity may be experienced. The course is progressive and although, always incurable, the prognosis is highly variable. The two more widely used staging systems in MM are by Durie and Salmon and the International Staging System (ISS). Others have been studied, including serum free light chain (sFLC) concentrations and ratio (sFLCr).

Methods: We measured sFLC in 27 newly diagnosed MM (21 Intact Ig MM, 5 light chain MM, 1 non secretory MM) at our center (Hospital del Salvador, Santiago de Chile) from October 2013 until June 2015. The sFLCr was calculated with the involved monoclonal light chain as the numerator. The median sFLCr was 17. Patients were divided into a "low group" (<17 sFLCr) and a "high group" (>17 sFLCr). We also analyzed these patients using the cut off (sFLCr of 50) previously reported by Garcia de Veas Silva et al. [Hematology reports 2015; 7 (s1) p23] The median follow-up was 16 months.

Results: During the period of study there were 8 deaths (29,7%). Seven (87,5%) of these deaths presented with an ISS score of 3 (table 1). Mortality rates were lower in the group of patients with "low" sFLCr (15,3%, 2 deaths in a group of 13 patients), as compared to patients with a "high" sFLCr (42,9%, 6 deaths in a group of 14 patients) (table 2).

Using the cutoff established by Garcia de Veas Silva et al, the mortality rate for patients with sFLCr >50 was 66,7% vs. 11,1% in for patients in the <50 sFLCr group (table 3).

Discussion: Although a short follow up period was available for analysis, we believe these results are promising. sFLCr can be used as an easy prognostic indicator in newly diagnosed, symptomatic MM, especially when high risk patients (>50 sFLCr) are identified. The introduction of biomarkers in the evaluation of MM patients will enable better risk assessment and rational follow up.

Table 1. International Staging System. Stratification of our study population. ISS N Patients N Deaths Mortality (%) 1 2 0 0 2 12 1 8,3 3 13 7 53,9

Table 2. Mortality rate in our study population using the median sFLCr as a cut off value. Mortality MM N Patients N Deaths Mortality (%) All 27 8 29,6 sFLCr>17 14 6 42,9 sFLCr<17 13 2 15,4

Table 3. Mortality rate in our population using the published cut off sFLCr value. Mortality MM N Patients N Deaths Mortality (%) All 27 8 29,6 sFLCr>50 9 6 66,7 sFLCr<50 18 2 11,1

Disclosures

Delgado: The Binding Site: Employment.
Quercetin Displays Anti-Myeloma Activity and Synergistic Effect with Dexamethasone in Vitro and In Vivo Xenograft Models
Authors: Guo Xing , He Donghua , Yang Li , Li Yi , Chen Qingxiao , Chen Jing , Zhang Enfan , Lin Xuanru , He Jingsong , Yang Yang , Han Xiaoyan , Zheng Gaofeng , Wu Wenjun , Zhao Yi , Huang He , Cai Zhen
Published: October 2019

Background: Multiple myeloma (MM) is a hematological malignancy with clonal plasma cell hyperplasia, which is still an incrurable disease since chemoresistance remains the major problem in MM management. Quercetin, a kind of dietary flavonoids, has shown its anticancer activity in many kinds of cancer cell lines and we tried to explore the effect of quercetin in MM.

Methods: In vitro, we examined the proliferation of MM cell lines(RPMI8226,ARP-1,MM1R) after treatment with quercetin combined with or without dexamethasone by MTT.Flow cytometry was used to detect apoptosis and cell cycle of MM cells induced by quercetin with or without dexamethasone.Then we detected mRNA and protein expression associated with apoptosis and cell cycle arrest by semiquantitative real time-polymerase chain (qRT-PCR)and western blot analysis. In vivo,a xenograft mice model of human myeloma was established and the mice received vehicle or quercetin alone or dexamethasone alone or quercetin combinded with dexamethasone, and the tumorburdern and the tumor tissue samples were analyzed by tumor volume and immunohistochemistry.

Results: Quercetin inhibited proliferation of MM cells by inducing apoptosis and cell cycle arrest in the G0/G1 or G2 phase(quercetin group vs control,p<0.05).Western blot showed that quercetin activated caspase3,caspase9,PARP-1 and increased cytochrome C release. C-myc and cyclinD1 expression were down-regulated and p21 were upregulated. Quercetinalso displays synergistic inhibition effect with dexamethasone in vitro (quercetin with dexamethasone vs quercetin only or dexamethasone only,p<0.05) and western bolt confirmed these results.In vivo,tumor burdern of xenograft mice modeltreated by quercetin was significantly lower than those of control(quercetin group vs control,p<0.05).

Conclusions: Quercetin inhibits proliferation of MM cells by inducing apoptosis and cell cycle arrest in the G0/G1 or G2 phase through downregulating c-myc and cyclinD1 and upregulating p21 .Quercetinalso displays synergistic inhibition effect with dexamethasone.Thus,quercetin combination with dexamethasone therapy may be an effective option for MM patients.

Disclosures

No relevant conflicts of interest to declare.
Involvement of T-Cells in the Bone Pathology of Multiple Myeloma
Authors: Simantirakis Emmanouel , Giannakoulas Nikolaos , Siapati Elena , Vassilopoulos George
Published: October 2019

Introduction: Multiple myeloma (MM) is a plasma cell dyscrasia that is heavily dependent on a permissive BM micro-environment. Components of the WNT pathway have been implicated in this process and specifically, WNT-inhibitors such as Dkk1, have been blamed for the osteolytic lesions of MM. WNT signaling is required for proper osteoblast development while the pathway is also involved in enhancing the self renewal potential of various stem cells. A source of WNT ligands in the BM are the T-cells; when stimulated by parathormone, T-cells secrete wnt10b that affects hematopoietic stem cell numbers. In the present study, we evaluated WNT signaling in the T-cells of patients with MM in order to test whether these bystander T-cells have an altered phenotype that could influence disease pathology.

Methods: A total of 18 MM patients was analyzed after informed consent was obtained; the patients underwent BM biopsy for diagnostic and staging purposes. T-cells were isolated from BM mononuclear cells using magnetic beads with a purity of around 90%. Normal controls were T-cells isolated from healthy subjects. RNA was isolated and analyzed by RT-PCR for expression of PTH receptor (PTH1R), wnt10b and the canonical WNT signal transducer, b-catenin (bCAT).

Results and discussion: BM concentration of T-cells was 15%, close to the normal values, indicating no major deviation from normal conditions. Activity of the WNT pathway was estimated by bCAT mRNA levels; relative to the control, bCAT levels in MM T-cells were uniformily suppressed. In a total of 8 evaluable samples, there was a 3x decrease in bCAT levels. Although WNT activity could be influenced by different factors, there is a possibility that it may be related to PTH activity and PTH receptor expression on patient T-cells. In all our cases, calcium was recorded within normal range and PTH values were also normal. However, PTH1R mRNA levels in MM T-cells were 14.2x lower compared to normal T-cells, a value that argues for a biological role in disease pathogenesis. A direct consequence of low PTH1R mRNA levels, according to our hypothesis, would lead to a decrease in WNT10b. Indeed, WNT10b levels were on average 90% of the normal with relative wide variation; in 3 samples, there was an average 3.2x increase while in 8, the levels were on average 21% of normal. On the clinical level, lower wnt10b activity, was more often associated with bone pathology while higher wnt10b activity was more often associated with extramedullary plasmacytomas. Our data show that T-cells may not be bystanders in the MM disease progression and certain manifestations sush as bone disease, could be associated with, aberrant T cell function. Ongoing studies are under way to link clinical and laboratory data.

Disclosures

No relevant conflicts of interest to declare.
STK405759 As a Novel Tubulin Active Agent for Multiple Myeloma Therapy
Authors: Rozic Gabriela , Lena Paukov , Jakubikova Jana , Adrian Duek , Avigdor Abraham , Nagler Arnon , Leiba Merav
Published: October 2019

Background: Despite advances in treatment, multiple myeloma (MM) remains incurable due to development of drug resistance in the bone marrow microenvironment. Microtubules (MTs) are dynamic protein biopolymers formed through polymerization of heterodimers of α- and β-tubulins. Disruption of microtubules induces cell cycle arrest in G2-M phase and formation of abnormal mitotic spindles. MTs are also involved in many processes in interphase cells, including intracellular trafficking, cell motility and angiogenesis. The important functions of MT in the cells make them an attractive target for anti-myeloma drug discovery.

Furan metotica is a novel class of anti-mitotic spindle drugs that inhibit kinetochore-microtubule binding and trigger a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We evaluated the activity of STK405759, a member of the furan metotica family, as a novel, potential antitubulin drug for MM treatment in preclinical models.

Methods: Cytotoxic activity of STK405759 was evaluated by XTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. Tubulin polymerization inhibition was evaluatedusing a biochemical cell free assay and by testingthe levels of soluble and polymerized tubulin in MM-treated cells using Western blot analysis. Efficacy and toxicity of the drug were checked in a murine MM xenograft model. Histochemistry was used to assay tumor apoptosis.

Results: STK405759 had a potent cytotoxic activity against a wide variety of MM cell lines and patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. In contrast, the viability of normal peripheral blood mononuclear cells derived from healthy donors and MM patients was not affected. Importantly, STK405759 induced cell death of RPMI MM cells co-cultured with HS-5 bone marrow stromal cells.

STK405759 inhibited tubulin polymerizationin a cell free system anddecreased the level of polymerized tubulin in MM treated cells.The STK405759 anti-tubulin activity was supported by demonstration of MM cell cycle arrest followed by activation of an apoptotic default pathway. Activation of pro-caspase-8 and poly (ADP-ribose) polymerase in the cleaved forms, as well as down-regulation of the Mcl-1 anti-apoptotic protein was detected in RPMI treated cells.

Combination studies of STK405759 with bortezomib, lenalidomide or dexamethasone showed significant synergistic and additive cytotoxicity in MM cells. In vivo studies revealed decreased MM tumor burden and prolonged survival of STK405759-treated mice compared to controls. STK405759 induced apoptosis of tumors cells from treated mice.

Summary/Conclusion: STK405759 is an active, microtubule-targeting agent with potent anti-myeloma activity. These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM.

Disclosures

No relevant conflicts of interest to declare.
Bendamustine-Bortezomib-Desametasone (BVD) in the Management of Relapsed and Refractory Multiple Myeloma : A REAL-Life Experience
Authors: Cerchione Claudio , Catalano Lucio , Pareto Anna Emanuele , Basile Santina , Marano Luana , Peluso Ilaria , Simeone Luigia , Vitagliano Orsola , Palmieri Salvatore , Rocco Stefano , Ferrara Felicetto , Pane Fabrizio
Published: October 2019

Introduction:

Bendamustine is a bifunctional alkylating agent, with low toxicity, proved to be effective in relapsed, refractory and in new diagnosed Multiple Myeloma (MM).

It has been evaluated efficacy and tolerance of Bendamustine, in combination with bortezomib-dexametasone (BVD) in patients with relapsed and refractory MM (rrMM), whose prognosis is particularly severe. A regional retrospective real-life analysis of patients with rrMM who had been treated with BVD as salvage therapy has been performed.

Methods:

35 patients (19 M/16 F), with rrMM, median age at diagnosis 57 years (r. 36-82), median age at start of treatment 62 years (r.37-83) treated with several lines of treatments (median 6, r. 2-11), every refractory to all the drugs previously received (also Bortezomib), received BVD (Bendamustine 90mg/sqm days 1,2; Bortezomib 1.3mg/sqm days 1,4,8,11, Desametasone 20mg days 1,2,4,5,8,9,11,12, Pegfilgrastim day +4) every 28 days, until progression.

ISS was equally distributed, and cytogenetic was evaluable in 9 patients, and in particular one del13q and one t(11;14). All the patients had previously been treated with schedule containing bortezomib and IMIDs, 90% of them with melphalan, 77% with cyclophosphamide, 34% with antracyclines and 30% had also received radiotherapy. 58% of them had undergone at least to a single auSCT. All patients were relapsed and refractory to last therapies received before BVD.

Results:

Bendamustine was well tolerated, with grade 3 transfusion-dependent anemia in 29% of patients, and 41% grade 3 neutropenia (no ospedalization was required, no septic shocks were observed). No severe extrahematologic toxicity was observed, only grade 1 gastrointestinal side effect (nausea), treated by common antiemetic drugs.

According to IMWG, after a median follow-up of 9 months (r.2-36), ORR was 54% (1 CR, 1 VGPR, 9 PR, 8 MR) with 7 PD and 9 patients in SD, which can be considered as an impressive result in this subset of rrMM patients. In particular, for 4 patients, BVD was, after having achieved a PR, a bridge to second auSCT, and for one patient a bridge to auSCT.

Median OS from diagnosis was 61.4 months (range 6-151), median OS from start of Bendamustine was 7.2 months (range 2-36).

Conclusion:

BVD has shown significant efficacy (ORR 54%) in a particular severe setting of patients, relapsed and refractory to all avaiable therapeutic resources, and in particular cases it could be considered as a bridge to a second autologous or allogenic BMT.

Disclosures

No relevant conflicts of interest to declare.
AML Relapse after Rituximab Treatment for Graft-Versus-Host Disease: Crucial Role for B Cells in Graft-Versus-Host and Graft-Versus-Leukemia
Authors: Gillissen Marijn Aletta , Levie Sophie E. , de Jong Greta , Yasuda Etsuko , Bakker Arjen Q. , van Helden Pauline M. , Jefferson Douglas M. , Huisman Cynthia , Spits Hergen , Hazenberg Mette D.
Published: October 2019

Patients with hematologic malignancies that develop graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) have a reduced risk of disease relapse compared to patients that do not develop GvHD, suggesting that GvHD and graft-versus-leukemia (GvL) responses are co-dependent. T cells are important in these processes, as T cell depletion from the graft reduces the risk of GvHD at the expense of disease relapse. Much less is known about the effect of B cell depletion on GvHD and GvL responses. Small patient series have demonstrated variable efficacy of Rituximab in the treatment of steroid-refractory chronic GvHD but the effect of depleting allo-reactive B cells on disease relapse remains to be determined. We here report on a patient with steroid-refractory GvHD whose AML relapsed after Rituximab treatment.

This 39-year old male received an allogeneic HSCT for chemotherapy-related AML (AML-t) that however relapsed 8 weeks after the transplantation. Upon rapid cessation of immunosuppressive therapy (cyclosporine, mycophenolic acid) and without additional chemotherapy he obtained full remission, at the expense of severe GvHD of the skin, liver and intestine that was corticosteroid-refractory. B cell depletion with Rituximab was successful as second-line treatment for GvHD but eliminated the GvL response and the patient died of AML relapse several months after.

To evaluate the B cell repertoire of this patient at the moment of maximal GvH and GvL responses, we isolated peripheral blood B lymphocytes that were transduced with Bcl-xL and Bcl-6 to create clonal B cell lines. These B cells were screened for binding to AML and host tissues. One clone was retrieved that specifically bound to AML cell lines and AML blasts freshly isolated from newly diagnosed AML patients. Antibodies from this clone induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Several other clones were retrieved that were specific for host tissues such as liver (HepG2 and H69 cell lines), skin (primary fibroblasts) and/or colon (CaCo cell line).

These data demonstrate the pivotal role of B lymphocytes in anti-leukemia and anti-host immune responses in an allogeneic HSCT recipient with relapsed AML-t.

Disclosures

No relevant conflicts of interest to declare.
Intensive Chemotherapy of 107 Primary Mediastinal B-Cell Lymphoma Patients. Single Center Experience
Authors: Mangasarova Jana Konstantinovna , Kravchenko Sergey Kirillovich , Magomedova Aminat Umaraskhabovna , Misyurina Anna , Baryakh Elena , Vorobiev Vladimir Ivanovich , Gemdzhian Eduard G. , Nesterova Ekaterina , Gilenko Alisa Igorevna , Gorenkova Liliya , Penskaya Ekaterina , Margolin Oleg Victorovich , Savchenko Valeri
Published: October 2019

Introduction. Primary mediastinal B-cell lymphoma (PMBCL) amounts from 0,9 to 4% of Non-Hodgkin Lymphomas. Predominantly suffer young women. All relapses in PMBCL occur as early events. Taking into account unfavorable prognosis in case of relapse or progression of PMBCL (2-year overall survival (OS) is about 15% [Kurvilla J., 2008]), it's necessary to achieve a complete remission (CR) in first line treatment.

Aim. To compare an efficacy of R-DA-EPOCH followed by R-DHAP and autologous peripheral blood stem cell transplantation (autoSCT) with BEAM conditioning regimen with modified (m) NHL-BFM-90 chemotherapy protocol in pts with partial remission (PR) of PMBCL.

Patients and Methods. In 107 pts (34 males, 73 females) diagnosis of PMBCL was established according to WHO criteria. All pts had stage II of disease according to Ann Arbor classification. 30 pts received R-DA-EPOCH, 77 pts - m-NHL-BFM-90 ± R. Median age of pts in R-DA-EPOCH group was 28 years old (from 20 to 67 years old); in m-NHL-BFM-90 ± R-group - 77 years old. Increased LDH concentration was revealed in 24 from 30 (80%) pts in R-DA-EPOCH group and in 75/77 (97%) pts from m-NHL-BFM-90 ± R group. Bulky mediastinal disease was in 30/30 (100%) pts in R-DA-EPOCH group and in 59/77 (76%) - m-NHL-BFM-90 ± R group. In case of CR after 6 courses according to computer tomography and PET scanning treatment was completed. In case of PR pts underwent 2 R-DHAP courses with autoSCT (BEAM conditioning regimen). All pts from m-NHL-BFM-90 ± R group independently from PET-results stopped treatment. All cases of treatment failure occurred till one year of observation. Kaplan-Meier method was used to calculate OS and relapse-free survival (RFS). OS was defined as time from diagnosis to death from any reason; RFS was defined as time from diagnosis until relapse or death from any cause.

Results: After 6 R-DA-EPOCH therapy CR was achieved in 24/30 (80%) pts, 6/30 (20%) pts had PR and underwent 2 R-DHAP courses followed by auto-SCT (BEAM). 2- RFS and OS was 100%. Median follow up was 16 months. In m-NHL-BFM-90 ± R group 10-year RFS and OS were 85% and 92%, respectively. Median follow up was 53 months.

Conclusion: High dose pulsed m-NHL-BFM-90-chemotherapy as well as R-DA-EPOCH allows getting high efficacy of primary mediastinal B-cell lymphoma therapy. Although future analysis will show more distant results of R-DA-EPOCH chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Effect of Severe Hypoalbuminemia on Myelosuppression and Other Toxicities of High Dose Melphalan and Autologous Stem Cell Transplantation in AL Amyloidosis Patients
Authors: Lee Shin Yin , Meehan Robert , Sloan John Mark , Quillen Karen , Brauneis Dina , Shelton Anthony C , Sanchorawala Vaishali
Published: October 2019

Background:

High-dose melphalan with autologous peripheral blood stem cell transplantation (HDM/SCT) has been shown to extend survival as well as to induce hematologic and clinical responses in selected patients with light chain (AL) amyloidosis. The most frequent toxicities of HDM are profound myelosuppression and gastrointestinal (GI) side effects. Studies have shown that 80% of melphalan is bound to plasma proteins (60% albumin bound) with ~20% free. We hypothesized that AL amyloidosis patients with severe nephrotic syndrome and profound hypoalbuminemia might have greater free melphalan fraction and more treatment-related toxicity.

Methods:

Patients with AL amyloidosis treated with HDM/SCT between 2011 and 2014 with severe hypoalbuminemia (SH), defined as a pre-transplant serum albumin of ≤2g/dL, were studied retrospectively. The stem cell transplant database was queried for patient demographic information, pre-transplant albumin level, HDM dose, renal function, pre-transplant 24-hour urine protein level, time to neutrophil and platelet engraftment, and treatment-related complications. Patients with AL amyloidosis treated between 2011 and 2012 without severe hypoalbuminemia, defined as serum albumin level of > 2g/dL (WSH), served as a control group.

Results:

Of the 84 patients with AL amyloidosis treated with HDM/SCT in this 4 year period, 16 (19%) with SH were identified. 41 patients were identified in the control group (WSH). There was no difference in the proportion of patients with all non-hematologic grade 3 or 4 adverse events between the groups. All patients suffered from expected grade 4 myelosuppression. The only statistically different non-hematologic grade 4 toxicity in SH was acute renal failure requiring temporary hemodialysis (n=4/16, 25% SH vs n=2/41, 5% WSH; p=0.05), with 1 subject eventually needing long term dialysis. There were no grade 4 mucositis or GI toxicities in either groups. The only statistically different grade 3 non-hematologic toxicity was lightheadedness (n=3/16, 19% SH vs n=0/41, 0% WSH; p=0.02).

Conclusion:

These data suggest that patients with severe hypoalbuminemia do not have more prolonged myelosuppression or GI toxicities when treated with HDM/SCT compared to those with higher serum albumin levels in AL amyloidosis. Grade 4 renal toxicity with acute renal failure requiring temporary hemodialysis (p=0.05) and grade 3 lightheadedness (p=0.02) occurred more commonly in SH than WSH group. In this retrospective study, we did not measure free melphalan concentrations in the blood. However, these data suggest that patients with severe hypoalbuminemia do not require adjustment of melphalan dosing.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Haploidentical/Mismatched Hematopoietic Stem Cell Transplantation for Nonresponders to Immunosuppressive Therapy Compared with First-Line Rabbit Antithymocyte Immunoglobulin Treatment Against Acquired Severe Aplastic Anemia
Authors: Liu Limin , Wang Xiuli , Jin Song , Hao Lin , Zhang Yanming , Zhang Xingxia , Depei Wu
Published: October 2019

We treated 86 patients with SAA, 31 patients who failed to respond to previous therapy underwent haploidentical hematopoietic SCT (haplo-HSCT, n=26) or mismatched unrelated donor HSCT (MMUD-HSCT, n=5). The other 55 patients were treated with immunosuppressive therapy (IST). At 6 months post-treatment, the treatment failure rates of HSCT and IST groups were 19.35% and 29.09% (P=0.320). Hematopoietic recovery time was shorter in the HSCT group than IST group (P > 0.05). The estimated OS at 3 years was 79.2% ± 7.7% in HSCT group and 89.7% ± 4.4% in the IST group (P=0.058). The estimated failure-free survival (FFS) at 3 years was 79.2% ± 7.7% in the HSCT group and 62.9% ± 8.0% in the IST group (P=0.391). The estimated FFS at 3 years in SAA that had progressed from non-SAA (NSAA) of HSCT was 71.6% ± 14.0% and 16.7% ± 13.6% of IST (P=0.021). Within the HSCT group, 38.71% of the patients developed grade II-IV acute GVHD, and 18.52% of the patients experienced moderate-severe chronic GVHD. These results suggest that haplo-HSCT/MMUD-HSCT and IST have similar treatment failure rate, OS and FFS. haplo-HSCT/MMUD-HSCT might provide a better chance of FFS than IST for SAA that had progressed from NSAA.

Disclosures

No relevant conflicts of interest to declare.
Non-Transmissible Measles Virus Vector Selectively Induces Human Primed and Naïve like Pluripotent Stem Cells from Hematopoietic Cells
Authors: Hiramoto Takafumi , Tahara Maino , Sakamoto Chika , Ono Hiroaki , Kohara Hiroshi , Takeda Makoto , Tani Kenzaburo
Published: October 2019

Our newly gene transfer vector could transduce multiple genes simultaneously into human hematopoietic cells with higher efficiency than general gene transfer vector. By using the vector, we could induce high quality pluripotent stem cells (naïve like pluripotent stem cells) from human cord blood derived CD34+ cells.

Measles virus (MV) which belongs to negative single strand RNA viruses has been known to have high affinity for human immune cells including monocytes, B cells and T cells. Although MV induced severe diseases, most of MV induced diseases can be controlled and curable by effective vaccination against H glycoprotein. Based on this background, we have newly developed novel measles virus vector to achieve the efficient and safe gene transfer to various cells. Our MV gene transfer vector is non-transmissible and transfer multiple genes simultaneously into human cells. Exogenous gene expressions from MV vector were controlled by changing the position in the vector genome.

The MV vector which encoded 5 genes (enhanced GFP, human OCT3/4, SOX2, KLF4, and L-MYC) (MV-ΔF-OSKL-EGFP) induced transgenes in almost all of hematopoietic cells besides natural killer cells. Quiescent T cells (CD45RA and CD197 expressed T cells), which include Naive and stem cell memory T cells were also transduced by MV-ΔF-OSKL-EGFP. For the purpose of transducing genes into hematopoietic cells safety and developing novel immunotherapy for cancer using gene modified T cells, our current results strongly suggested that the newly developed MV vector is considered to be more powerful vector than conventional gene transfer methods.

Then, we generated induced pluripotent stem cells (primed iPS cells) from terminal differentiated T cells and naive like pluripotent stem cells (NL-iPS cells) from cord blood derived CD34+ cells using MV-ΔF-OSKL-EGFP in same culture method and culture media. These primed iPS and GSL-iPS cells were demonstrated to express pluripotent markers (OCT3/4, NANOG) and differentiate into three germ line tissues. NL-iPS cells also expressed TFE3 proteins in the nuclei and SSEA1 in the cytoplasm. Interestingly, Enhanced GFP from MV-ΔF-OSKL-EGFP were lost in primed iPS cells and expressed strongly in NL-iPS cells. In the case of establishing iPS cells using this MV vector from CD34+ cells, we could establish only NL-iPS cells but not primed iPS cells, whereas we could not establish NL-iPS cells by using conventional vectors including Sendai vious vector in the same methods as MV vector.

In conclusion, our newly developed MV vector is considered to lead revolutionary advance in the research and clinical fields of gene therapy and stem cell.

Disclosures

No relevant conflicts of interest to declare.
Dinamic Evaluation of Quality of Life and Late Effects in a Cohort of Acute Myeloid Leukemia Long Term Survivors
Authors: Capelli Debora , Giostra Liviana , Maravalle Denise , Leoni Pietro , Olivieri Attilio
Published: October 2019

Late effects and quality of life (QoL) in Acute Myeloid Leukemia (AML) long term adult survivors represent an unexplored field of interest. We prospectively evaluated late effects and dynamic QoL in 44 and 29 cured AML patients (17 <60 years, and 12 >60 years), treated at our department between 1997 and 2010 (7 Allogeneic, 16 Autologous Transplant, 21 chemotherapy alone). We administered EORTC QLQ-C30 and FACT-AN questionnaires at two different time points with a median interval of 29 months (range: 12-34 months). We stratifyed QoL scores by age at diagnosis, performance status (PS), Sorror Index, kind of leukemia treatment, comorbidity at diagnosis.

We observed a worsening of emotional (-9.03; p= 0.04) and cognitive (-6.94; p= 0.05) EORTC scale scores, while FACTG (+2.9; p=0.03), emotional (+1.1; p= 0.04) and Functional (+2.25; p=0.001) well being FACT scores increased.

Multivariate analysis showed that older patients had worse EORTC QLQ-C30 physical and emotional scale scores and higher values of pain symptoms in comparison to younger counterpart, with RR of 20.1 (p = 0.001), 22.7 ( p <0.04) and 18.4 (p=0.03) respectively. Elderly patients also had lower Total Outcome Index and FACT-An subscale scores (RR: 11.9, p= 0.02; and 8.77, p= 0.04 respectively). Sorror index > 2 was related to lower EORTC QLQ-C30 social scale and dyspnea scores (RR: 32.5; p=0.001 and 21.7; p=0.001 respectively) and FACT-An functional well being values (RR=3.9; p=0.001).

We evaluated late effects occurring in 44 patients, since the third month after the end of treatment, with a median follow-up of 70 months (range: 12-166 months). The most frequent grade II-IV late toxicity was cardiac (3 arythmia, 9 cardiomyopathy) with 89% incidence in patients with Sorror HCT-CI score>2 at diagnosis vs 8.8% in the remaining patients and 0%, 20% and 55.5% in patients receiving respectively Daunorubicin, Idarubicin and at least two different anthracyclines. Sorror Index>2 was the only factor significantly predicting cardiotoxicity at the multivariate analysis with a RR of 82.7 (p=0.001). Twelve patients developped a transient hemochromatosis secondary to transfusions, treated with phlebotomy in 3 cases. Three patients (2 males and 1 female) had been fertile; all female patients developped menopause after Transplant. Four patients had secondary neoplasia consisting of Multiple Myeloma, breast cancer, myelodisplasia and axillary sarcoma.

Our study underlines the role of Sorror Index at diagnosis in defining patients eligibility to cardio-prophylactic therapy. The analysis of larger series of cured AML patients are strongly needed in order to define guidelines for reducing long term treatment AML toxicity.

Disclosures

No relevant conflicts of interest to declare.