bluuebunny/crossref_metadata_2025_split huggingface dataset:
PF230 THE RNA HELICASE DDX17 PROTECTS ACUTE MYELOID LEUKEMIA CELLS AGAINST DNA DAMAGE
Authors: Jagdhane P. , Garg S. , Xia J. , He L. , Misiak D. , Mortusewicz O. , Helleday T. , Müller‐Tidow C. , Koehn M. , Pabst C. 
Published: June 2019

Background:Alternative splicing (AS) is essentially regulated by RNA‐binding proteins (RBPs) and is one of many deregulated cellular processes in Acute Myeloid Leukemia (AML). The alternative usage of exons substantially increases the coding potential of mRNAs and frequently leads to the expression of tumor‐promoting mRNA variants in cancer modulating apoptosis, proliferation, invasion and migration of cancer cells. This underlines the great plasticity of AS in cancer and justifies the need to elucidate the function of nuclear RBPs in specific cancer types. We found that the RNA helicase DDX17 is significantly more highly expressed in primary human AML specimens with high versus low Leukemia Stem Cell (LSC) frequency.Aims:In this study, we aimed to understand the functional role of DDX17 in normal hematopoietic stem and progenitor cells (HSPCs) and in AML cells. In particular, we sought to determine whether DDX17 exerts its function in the hematopoietic system primarily via AS or AS‐unrelated mechanisms.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether DDX17 was essential for primary human cord blood CD34+ HSPCs and leukemia cells. Furthermore, mRFP tagged DDX17 was overexpressed through lentiviral transduction to track protein localization by immunofluorescence (IF) imaging. Lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) was used to determine how DDX17 KD affected gene expression and alternative exon usage. Pull down experiments combined with mass spectrometry revealed binding partners in presence and absence of chemotherapeutic agents. Finally, we performed homologous recombination (HR) and drug sensitivity assays to determine the role of DDX17 in DNA damage.Results:Knockdown of DDX17 induced DNA damage and severely impaired the function of healthy HSPCs and TP53 wild type AML cells. TP53 null cells maintained their proliferative capacity during short term in vitro expansion in absence of DDX17, but were significantly sensitized towards DNA damaging agents. In line, overexpression of DDX17 protected the cells against double strand break (DSB) inducing drugs. IF imaging revealed direct recruitment of DDX17 to DNA damage sites, while RNA‐Seq showed suppression of multiple DNA repair genes.Summary/Conclusion:RNA‐Seq, IF imaging, proteomics, and HR assays suggest an essential role of DDX17 in DNA repair via different mechanisms including direct recruitment to DNA damage sites, alternative splicing, and regulation of DNA repair genes on the transcriptional level.
PF298 LISOCABTAGENE MARALEUCEL TREATMENT OF PATIENTS WITH RELAPSED/REFRACTORY B‐CELL NON‐HODGKIN LYMPHOMA AND SECONDARY CENTRAL NERVOUS SYSTEM LYMPHOMA: INITIAL RESULTS FROM TRANSCEND NHL 001
Authors: Abramson J. , Palomba M.L. , Arnason J. , Lunning M. , Solomon S. , Farazi T. , Garcia J. , Xie B. , Newhall K. , Dehner C. , Siddiqi T. 
Published: June 2019

Background:No clinical studies have yet evaluated chimeric antigen receptor (CAR) T cell therapies in patients with relapsed/refractory (R/R) B‐cell non‐Hodgkin lymphoma (NHL) who have secondary central nervous system (CNS) lymphoma.Aims:To report data from patients with secondary CNS lymphoma receiving lisocabtagene maraleucel (liso‐cel; JCAR017), an investigational, anti‐CD19 CAR T cell product administered as a defined composition of CD4+/CD8+ CAR T cells, in the phase 1 TRANSCEND NHL 001 study.Methods:Eligible patients had confirmed B‐cell NHL with R/R disease after ≥2 prior lines of therapy. Patients with secondary CNS lymphoma were eligible as long as they also had systemic disease or, if it developed on study, patients could continue to receive liso‐cel. After lymphodepleting chemotherapy, liso‐cel was administered at 1 of 2 dose levels; 50 × 106 total CAR+ T cells (dose level 1) or 100 × 106 total CAR+ T cells (dose level 2). Efficacy was evaluated per the Lugano criteria. Patients achieving a complete response could be retreated with liso‐cel upon progressive disease.Results:At the data cutoff, 9 patients with secondary CNS lymphoma at initial treatment (n = 6), retreatment (n = 2), or cycle 2 (n = 1) received liso‐cel. Four patients were treated at dose level 1 and 5 at dose level 2. The median (range) age was 60 (47–73) years and the number of prior lines of therapy was 3 (2–7). Median (range) time to peak CAR+ T cell expansion was 12.5 (7–112) days. One of 9 patients had grade 2 cytokine release syndrome (CRS) and 1 of 9 patients had a neurological event (NE; grade 3 decreased level of consciousness). No retreatment patients had CRS or NE, however, 1 retreatment patient had an NE of grade 2 temporal edema during the initial treatment with liso‐cel. Five patients received prophylactic levetiracetam. One patient received corticosteroids and tocilizumab. Other toxicities were predominantly cytopenias. There were no treatment‐related deaths. Four patients responded to liso‐cel; all with a best response of complete response, of which 2 are ongoing at 270 and 545 days post‐liso‐cel. All 4 responses occurred after initial liso‐cel treatment; no retreated patients responded.Summary/Conclusion:In the ongoing TRANSCEND NHL 001 study, liso‐cel continues to demonstrate the ability to be safely delivered to patients with R/R B‐cell NHL, including those with secondary CNS lymphoma, a population of patients with a highly unmet medical need. No excess NE was noted in this population. This cohort continues to be evaluated.
PF299 EARLY RESPONSE OBSERVATION OF PEDIATRIC PATIENTS WITH BURKITT'S LYMPHOMA TREATED WITH CHIMERIC ANTIGEN RECEPTOR (CAR) T‐CELLS
Authors: Zhang W.‐Q. , Hu B. , Jing L. , Yang J. , Du J. , Wang S. , Ren Y.‐L. , Liu Y. , Zhang Y.‐H. 
Published: June 2019

Background:Pediatric patients with relapsed or refractory Burkitt's lymphoma (BL) present as a major challenge due to their poor outcomes despite use of high‐intensity chemotherapy and rituximab. CAR‐T has shown efficacy in treating refractory or relapsed leukemia and non‐Hodgkin lymphoma (NHL) in adult patients.Aims:To assess the safety and efficacy of CAR‐T in the treatment of refractory or relapsed BL in pediatric patients.Methods:Our trial (ChiCTR18000144) enrolled and treated 5 patients with BL. The inclusion criteria includes: 1) The patient who didn’t get complete remission(CR)after at least 5 courses of standard chemotherapy plus CD20 mAb; and 2) The patient who didn’t get partial remission (PR) or showing progression after 2 courses of second‐line chemotherapy; and 3) High expression of at least one B cells markers (CD19/CD20/CD22) examined by immunohistochemistry. The highest expressed marker was chosen as first round CAR‐T target. The therapeutic doses of CAR‐T range from1∗10^6 to 3∗10^6 per kilogram of body weight each course. The effects and adverse events of CART cells after infusion were observed. The change of CAR‐T cells and cytokines were measured weekly. Tumor response was evaluated at day 30 and day 60 post infusion and every two months afterwards.Results:Five patients aged 6 to 10 years with a median age of 8 years, were enrolled, including 4 male (80%) and 1 female (20%). St Jude staging at the time of diagnosis: 3 cases (60%) in stage III, 2 cases (40%) in stage IV, 1 case of CNS invasion (20%), 1 case of bone marrow invasion (20%) and none in stage I or stage II.Five patients were treated with 1 to 3 rounds of CAR‐T. The overall response rate (OR) was 100%. Three cases (60%) achieved CR by one round of CD19 CAR‐T cell therapy, while two cases (40%) achieved PR with CD19 CAR‐T treatment and achieved CR after subsequent treatment with CD22 or CD22 and CD20 CAR‐T.During the treatment, fever, gastro‐intestine discomfort, rash, capillary leak syndrome, Immunopneumonia, tremor, hypotension, Immunocytopenia abnormal coagulation, liver dysfunction, and other toxic effects occurred. One patient (20%) was diagnosed of cytokine‐release syndrome(CRS) grading I and the other four grading III. All cases recoverd fully after active symptomatic and supportive treatment (including application of glucocorticoids in CRS grading III). There were no death events.The last follow‐up was January 30, 2019. The longest follow‐up duration was 382 days, the shortest 62 days, and the median was 249 days. Currently, all patients were in event‐free survival.Summary/Conclusion:CD19/CD20/CD22‐CAR‐T therapy shows robust efficacy in pediatric patients with refractory and relapsed BL and the toxic and adverse effects are moderate and can be well controlled.
PF322 APPROACH TO DOUBLE HIT LYMPHOMA (DHL) DIAGNOSIS AND TREATMENT IN EUROPE – A CROSS‐SECTION STUDY OF EHA LYMPHOMA WORKING PARTY (EHA LYG)
Authors: Aurer I. , Kersten M.J. , Dreyling M. , Federico M. 
Published: June 2019

Background:DHL is a new lymphoma entity defined in the WHO 2018 classification. Diagnosis of DHL requires the use of FISH for MYC, BCL2 and BCL6, a technic that is not routinely used in the diagnostics of diffuse large‐B cell lymphomas (DLBCL). Treatment recommendations for DHL differ from those of DLBCL, but the level of evidence for them is low.Aims:EHA LYG performed this study to identify common practices in DHL diagnosis and treatment in different European countries.Methods:Lymphoma experts from different European countries were contacted and asked to fill out a questionnaire regarding recommendations and common practices in diagnosis and treatment of suspected DHL and DLBCL.Results:Almost half of European countries do not have national recommendations for DHL screening. Routine FISH screening is recommended in less than 20%, approximately 20% recommend screening all double‐expressors irrespective of cell of origin (COO), and 10% only GC‐type double‐expressors. COO is universally defined according to the Hans algorithm. MYC cut‐off values for defining double‐expressors are mostly 40% (but sometimes 70%), for BCL2 between 40% and 60% and for BCL6 60%. In one third of countries is screening recommended also in cases with clinical high‐risk features. FISH for DHL is routinely available to all DLBCL patients in approximately 40% of countries, in another 40% only to patients treated at referral hospitals, and not available at all in 20%. Only rarely is the availability of testing dependent on the type of insurance. Approximately a third of countries have nation‐wide recommendations for treating DHL. However, most patients with diagnosed DHL who are able to tolerate more aggressive therapies receive regimens different from R‐CHOP, mostly DA‐R‐EPOCH and Burkitt lymphoma‐type regimens, less frequently R‐CHOEP14. In approximately one third of the countries are alternative regimens used only in referral centers.Summary/Conclusion:The responses to our questionnaire suggest that current practices in recognizing DHL in Europe, even in relatively rich countries, result in missing of a substantial number of patients, especially in those treated outside of referral centers and with lower‐risk features. This fact must be taken into account when describing the natural history of DHL. Most patients diagnosed with DHL receive more aggressive treatment than those with DLBCL.
PF372 INHIBITION OF BROMODOMAIN AND EXTRA‐TERMINAL (BET) PROTEINS INCREASES SENSITIVITY TO VENETOCLAX IN CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: Carrà G. , Lingua M.F. , Maffeo B. , Guerrasio A. , saglio G. , Taulli R. , Morotti A. 
Published: June 2019

Background:The development of drugs able to target BTK, PI3k‐delta and Bcl2 has dramatically improved Chronic Lymphocytic Leukemia therapies. However, resistant cases to these drugs have already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures.Aims:In this study, we investigated thecooperative role of Venetoclax and the JQ1 inhibitor in order to prevent and eventually overcome the development of resistance to Bcl‐2 inhibitor.Methods:MEC‐1/EHEB cells lines and primary CD19 positive lymphocytes from CLL patients were treated with BET inhibitor JQ1 and venetoclax. Cell lines were screened for cell viability upon treatment with Venetoclax, JQ1and the combination of the two. Responses were measured as apoptosis induction (Annexin V), cell growth arrest and cell‐cycle accumulation. Furthermore, Western Blot analysis RT‐PCR and Immunofluorescence were used to determine expression of Brd members and BCL‐2 pathway components.Results:We demonstrated that treatment with BCL2 inhibitor is associated with a rapid emergence of resistant clones. Compared with each agent alone, cotreatment with JQ1 and venetoclax improved apoptosis in CLL cell lines and of the in vitro isolate primary CD19+ lymphocytes. Additionally, cotreatment could prevent the development of resistant clones. Lastly, JQ1 is also able to restore sensitiveness to venetoclax in venetoclax‐resistant CLL cell lines.Summary/Conclusion:Our findings indicated that cotreatment with JQ1 and BCL2 inhibitor synergistically induced apoptosis and eventually, prevent the development of resistant clones. Further knowledge is key to development of combinatorial treatment strategies with this two drugs.
PF377 ACALABRUTINIB WITH OBINUTUZUMAB IN TREATMENT‐NAIVE AND RELAPSED/REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA: 3‐YEAR FOLLOW‐UP
Authors: Woyach J.A. , Rogers K. , Bhat S. , Blachly J. , Jianfar M. , Frigault M.M. , Hamdy A. , Izumi R. , Munugalavadla V. , Quah C. , Wang M.H. , Byrd J.C. 
Published: June 2019

Background:Acalabrutinib is a highly selective, potent, covalent Bruton tyrosine kinase inhibitor.Aims:This Phase 1b/2 trial evaluated acalabrutinib with the CD20 antibody obinutuzumab in patients with treatment‐naive or relapsed/refractory chronic lymphocytic leukemia (CLL).Methods:Patients with treatment‐naive or relapsed/refractory (≥1 prior therapy) CLL were eligible. In 28‐day cycles, acalabrutinib was given orally at 100 mg twice daily or 200 mg once daily (n = 15; all switched to 100 mg twice daily) until progressive disease; obinutuzumab was given in standard fashion for 6 cycles starting with Cycle 2. The primary endpoints were overall response rate (ORR) and safety. Minimal residual disease (MRD) was assessed using flow cytometry (sensitivity 10–4).Results:Nineteen treatment‐naive and 26 relapsed/refractory patients were treated. The median age of all patients was 61 years (range, 42 to 76). Patient characteristics, disposition, efficacy and MRD are in the Table. Common adverse events (any grade) were upper respiratory tract infection (71%), increased weight (71%), maculopapular rash (67%), cough (64%), diarrhea (62%), headache (56%), nausea (53%), arthralgia (51%), and dizziness (47%). Common Grade 3/4 adverse events were decreased neutrophil count (24%), syncope (11%), decreased platelet count, increased weight and cellulitis (9% each). There were 2 (4%) Grade 3 bleeding events (hematuria, muscle hemorrhage) and 1 (2%) Grade 3 atrial fibrillation event.Summary/Conclusion:Acalabrutinib plus obinutuzumab was well tolerated and yielded high response rates that were durable and deepened over time in treatment‐naive and relapsed/refractory patients with CLL.image
PF392 MONOCLONAL GAMMOPATHY AND HYPOGAMMAGLOBULINEMIA AS INDEPENDENT PROGNOSTIC FACTORS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A RETROSPECTIVE MULTICENTRIC EXPERIENCE
Authors: Corbingi A. , Innocenti I. , Tomasso A. , Pasquale R. , Visentin A. , Varettoni M. , Autore F. , Morelli F. , Trentin L. , Reda G. , Laurenti L. 
Published: June 2019

Background:Chronic Lymphocytic Leukemia (CLL) is an indolent B‐cell lymphoproliferative disorder. Several prognostic factors such as IGHV mutation status and chromosomal aberrations as trisomy 12, del11q, del13q or del17p have been detected so far. More recent genetic mutations such as BIRC3, SF3B1, NOTCH1 and TP53 stratifies even further the prognosis and outcome in CLL patients (pts). However all data above reported, because of expensive techniques and experience typical of big laboratories, are not available to all medical centers while data concerning the presence of hypogammaglobulinemia, IgM or IgG monoclonal gammopathy are easily achievable, even though the prevalence and the impact on natural history of CLL pts is controversial and contradictory.Aims:The aim of the study is to evaluate the prevalence and the outcome of monoclonal IgM/CLL, IgG/CLL and hypogammaglobulinemia compared with CLL pts with normal immunoglobulin (Ig) levels.Methods:We collected from four different Italian centers 1359 pts diagnosed with typical CLL from 1987 to 2017 with a baseline assessment of serum Ig, immunofixation, chromosomal aberrations and clinical features; evaluating their impact on time to progression (TTP), time to treatment (TTT) and overall survival (OS).Results:Once assessment was made, pts included in our study who met eligibility criteria were 1359. Median age was similar in all the groups and data gathered from the four centers showed an overlapping rate about prevalence and time‐dependent‐parameters in CLL pts and their related subclasses.The overall prevalence of monoclonal gammopathy is 14% of whom 128 pts (9%) with IgG/CLL, 68 pts (5%) with IgM/CLL. Hypogammaglobulinemia was detected in 172 pts (13%) while 991 pts (73%) had no evidence of paraprotein. Among the groups, serum IgG paraprotein showed a correlation with a poorer and shorter overall survival (OS) and an inferior clinical outcome.Summary/Conclusion:Our study provided data about prevalence and prognosis of monoclonal gammopaty and hypogammaglobulinemia in CLL patients. Serum Ig paraprotein was present in 14% of all CLL pts with a worst outcome in IgG/CLL group. Thus, making of serum Ig, a simple data to obtain, useful to assess CLL prognosis.image
PF397 IDENTIFICATION OF PROTEIN‐PROTEIN INTERACTIONS NECESSARY FOR MAINTENANCE OF BCR‐ABL CORE COMPLEX
Authors: Gregor T. , Bosakova M.K. , Rynes J. , Trantirkova S.F. , Mayer J. , Trantirek L. , Krejci P. 
Published: June 2019

Background:Approximately 50% of chronic myeloid leukemia (CML) patients in deep remission experience a return of clinical CML after withdrawal of tyrosine kinase inhibitors (TKIs), suggesting signaling of catalytically inactive BCR‐ABL. This allows for survival of cancer cells responsible for CML relapse after discontinuation of the TKI therapy. Elucidation of protein‐protein interactions within the BCR‐ABL signaling complex is crucial for understanding of the mechanism of residual signaling of TKI‐inhibited BCR‐ABL.Aims:This study aims on detailed characterization of BCR‐ABL interactions with members of its core complex, i.e. the signaling mediators Grb2, SOS1, SHC1, cCbl, CrkL, SHIP2, Sts1 and p85a.Methods:Peptide microarrays were used to map binding interfaces between BCR‐ABL and its interactors in high resolution. The microarray data were validated by in vivo co‐immunoprecipitation and proximity ligation assays carried out with BCR‐ABL deletion mutants and their respective interactors. Reporter assays were employed to analyze the downstream signaling of BCR‐ABL in cells.Results:We demonstrate that TKIs inhibit catalytic activity of BCR‐ABL, but do not completely abrogate its downstream signaling. Further, treatment with TKIs does not entirely dissolve the BCR‐ABL signaling complex consisting of Grb2, SOS1, SHC1, cCbl, CrkL and SHIP2. The interacting interfaces between BCR‐ABL and members of its core complex were mapped in high resolution. The data show that CrkL binds to proline‐rich regions located in C‐terminal, intrinsically disordered region of BCR‐ABL, that deletion of pleckstrin homology (PH) domain of BCR‐ABL diminishes interaction with SHC1, and that SHIP2 associates with the src‐homology (SH)3‐SH2‐tyrosine kinase (TK) domain of BCR‐ABL. We further demonstrate that BCR‐ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of at least three core complex members, the Grb2, SOS1 and cCbl. Introduction of Y177F substitution to BCR‐ABL blocks association with Grb2, SOS1 and cCbl, and reduces residual signaling of TKI‐inhibited BCR‐ABL.Summary/Conclusion:The protein‐protein interactions within the BCR‐ABL core complex were characterized in detail here. Our findings support the concept of targeting BCR‐ABL signaling in CML by inhibition of Y177 interaction interface.
PF448 EFFECTIVE STABILIZATION OF VIRAL VECTORS IN LIQUID USING AN ALGORITHM‐BASED DEVELOPMENT APPROACH
Authors: Reinauer E. , Kemter K. , Hasler J. , Rodenstein C. , Altrichter J. , Martin S. 
Published: June 2019

Background:With both the approvals of CD19 CAR‐T cell therapies and promising clinical data of gene therapy trials such as in hemophilia or sickle cell disease, recent years have seen significant progress of gene and cell therapy in the field of hematological malignancies as well as rare hematologic genetic disorders. Hence, replication‐deficient recombinant viral vectors such as adenovirus serotype 5, adeno‐associated virus and members of the poxvirus family represent a rapidly growing field of vaccine development and gene therapy.Viral vectors are known as complex supra‐molecular ensembles of macromolecules produced by living organisms (nucleic acids, proteins, polysaccharides and in the case of lipid enveloped viruses of phospholipids) which are prone to a variety of complex chemical and physical degradation pathways, in particular due to stress induced by manufacturing, storage and distribution. This represents a significant hurdle for the development of stable vector‐based pharmaceuticals such as vaccines or gene therapeuticsAims:Here, we studied whether LEUKOCAREs formulation platform may also efficiently protect viral vectors in thermal stress experiments. As a representative example of viral vectors we used adenovirus serotype 5 (Ad5).Methods:In an algorithm‐based development approach, we used Design of Experiment (DoE) to select the most effectively stabilizing formulations for Ad5. We stored Ad5 at 37 °C and 25 °C as accelerated aging temperature to identify the most effective stabilizing excipients after short term storage as well as at 5 °C for real time storage. Based on these results, the best selected formulations were further iteratively optimized and used in long term storage at 5 °C.Results:By analysis of the infectious virus titers and mathematical combination of these results with the DoE matrix, the linear influence of each amino acid used in the DoE matrix was determined. The accelerated aging conditions were shown to be predictive for real‐time aging. Several of the excipients indicated a neutral influence, whereby we observed the importance of a well‐balanced combination of the components with each other regarding concentration and stabilizing interactions. The predictive potential of this approach was confirmed by two iteratively composed formulations tailored for Ad5 during long‐term storage.Summary/Conclusion:The pre‐selection strategy of effectively stabilizing excipients by means of an algorithm‐based development approach and the applied accelerated aging model is highly efficient and enables the generation of best‐in‐class stability formulations for Ad5 viruses and viral vectors in liquid. Moreover, this approach could have beneficial impact when applied early in downstream processing, which could enable significant reduction of viral vectors manufacturing costs for gene therapy.