bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of Molecular Response in CML Patients with Atypical BCR-ABL Tanscripts. Recommendations By the EUTOS Collaboration
Authors: Ernst Thomas , Schäfer Vivien , White Helen E , Saussele Susanne , Lange Thoralf , Gerrard Gareth , Cross Nicholas CP , Hochhaus Andreas 
Published: October 2019

Abstract

Approximately 2‐3% of CML patients (pts) have variant BCR‐ABL fusions transcripts that cannot be monitored by RQ‐PCR using standard methodologies. Most rare variants are accounted for by seven variant fusions (e1a2/3, b2/3a3, e6a2, e8a2, e19a2). Within the European Treatment and Outcome Study (EUTOS) we sought to establish and validate robust RQ-PCR methods for each of these abnormalities. RNA was extracted from leukocytes obtained from 20 mL peripheral blood. Samples were received either locally or by mail and spent between 1 to 3 days in transit. Multiplex reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to identify BCR-ABL1 transcripts (Cross et al., Leukemia 1994). In all cases, gel electrophoresis showed an unusual band. PCR products were then sequenced to characterize BCR-ABL1 rearrangements. Monitoring of residual disease was carried out by RQ-PCR with specific forward and/or reverse primers and probes using a serial dilution of individual BCR-ABL1 or GUSB plasmid DNA calibrators. GUSB transcripts were quantified as internal control and results were expressed as the BCR-ABL1/GUSB ratio with no conversion according to the international scale (IS) as it is recommended for only typical major (M) BCR-ABL1 and cannot be applied to other transcripts in CML. 43 pts from 8 prospective studies and pts outside of studies were investigated. A total of 435 samples (1-72 per patient, median 8) were analyzed. Pts expressed 6 different atypical BCR-ABL1 transcripts (e19a2, n=18; e1a2, n=10; b2a3, n=5; b3a3, n=5; b2a3&b3a3, n=2, e8a2, n=3). At the start of the observation period, ratios BCR-ABL1/GUSB ranged between 0.01% and 179.1% (median 13.75%). Follow-up results were determined in comparison to the starting level and expressed as relative log reduction. 9 pts were in deep molecular remission considering a minimum GUSB transcript level of 20,000/reaction. During the observation period, six pts relapsed with a significant increase of BCR-ABL1/GUSB ratios. We conclude that very few pts are diagnosed with unusual BCR-ABL1 transcripts, but their characterization is important for proper assessment of treatment response in affected patients, and to avoid false negative assessment of residual disease status. Based on the individual PCR strategies, results cannot be expressed with precision on the International Scale and thus the common molecular milestones and triggers for treatment discontinuation are difficult to apply. We suggest that residual disease in these cases should be assessed primarily by considering trends in BCR-ABL/reference gene ratios over time.

Disclosures
Saussele: NOVARTIS PHARMA: Consultancy, Honoraria; BMS: Honoraria. Cross:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.
The Crosstalk Between Leptin Receptor Activation and iNKT Mediated Anti-Tumor Immunity in Multiple Myeloma
Authors: Favreau Mérédis , Menu Eline , Vanderkerken Karin , Faict Sylvia , Maes Ken , Van Valckenborgh Els , De Bruyne Elke , Govindarajan Srinath , Drennan Michael , Leleu Xavier , Zabeau Lennart , Tavernier Jan , Venken Koen , Elewaut Dirk 
Published: October 2019

Abstract
In recent years, researchers have showed a growing interest in the mechanistic relationship between bone marrow adipose tissue and adjacent tumors. However, the impact of bone marrow adipocytes on development of hematological malignancies, particularly multiple myeloma is unknown. With aging, bone marrow changes occur and fatty deposits can occupy up to 70% of the BM cavity. Interactions of bone marrow adipose tissue with bone cells and other immune cells, possibly suggest indirect ways in which bone marrow adipocytes may affect MM disease progression. Leptin, an adipokine released by adipocytes and crucial in energy homeostasis, displays immune modulatory properties but its role in anti-tumor immunity remains unclear. In this study we aimed to investigate the intriguing relationship between leptin receptor activation and invariant natural killer T (iNKT) cell mediated anti-tumor immunity in multiple myeloma. The murine immunocompetent 5T33MM model, mimicking the human disease closely, and human samples of newly diagnosed patients were used to investigate this research topic.
A marked increase in serum leptin levels and an upregulation of the leptin receptor expression on iNKT cells was observed in multiple myeloma, in both mice and newly diagnosed patients. In vitro functional analysis demonstrated a direct role for leptin in the downmodulation of the iNKT cell function and an indirect role by acting on the MM cells itself, potentiating the immunosuppressive effect on iNKT cells. We next evaluated the in vivo effects of blocking the leptin receptor together with activating iNKT cells with the prototypic glycolipid a-galactosylceramide (a-GalCer), in the 5T33 myeloma model. Remarkably, strong protection was seen in the combined regimen (a-GalCer and leptin receptor blockade), which was found to be linked to an amplified and sustained activation of iNKT cells, preventing them to become hypoactive. These findings suggest that leptin has a crucial immune suppressive role in myeloma development. Overall, our data reveal the leptin receptor axis as a novel target to treat multiple myeloma.

Disclosures
Leleu: TEVA: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.
ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma
Authors: Kumar Subodh , Nanjappa Purushothama , Talluri Srikanth , Shammas Masood A , Munshi Nikhil C 
Published: October 2019

Abstract
Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated.
In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application.

Disclosures
No relevant conflicts of interest to declare.
Bortezomib-Containing Regimens for the Treatment of Newly Diagnosed AL Amyloidosis: Experience of Two Centers in Canada
Authors: Alahwal Hatem , Song Kevin W. , Duggan Peter , Sutherland Heather J. , Neri Paola , Broady Raewyn , Bahlis Nizar J. , Jimenez-Zepeda Victor 
Published: October 2019

Abstract
Introduction Bortezomib is a novel, dipeptide boronic acid molecule that has shown anti-tumor activity against Multiple Myeloma (MM) and more recently has been demonstrated to be efficacious in the treatment of Light-chain Amyloidosis (AL). Bortezomib-based regimens have changed the landscape of the treatment of AL Amyloidosis due to high levels of response as well as the adequate tolerance. Based on the above mentioned, we aimed to assess the role of Bortezomib-containing regimens (BCR) for the treatment of newly diagnosed AL amyloidosis. Methods 80 consecutive patients have been treated with bortezomib-containing regimens at BCCA and TBCC in Calgary, AB and Vancouver, BC, Canada from 01/2010 to 01/2016. Among these cases, 51 (63.8%) have received CyBorD, 19 (23.7%) bortezomib (1.3 mg/m2) and dexamethasone and the rest Bortezomib in different combinations (Table 1). The primary objective of the study was to assess hematological and organ response. Results Clinical characteristics are shown in Table 1. Median age at diagnosis was 66 years and 51.1% of patients were male. At the time of analysis, 56 patients are still alive and 14 patients have already progressed. A HR was seen in 65/72 evaluable patients (ORR, 90%) after a median of 6 cycles (range, 1-24). Fourteen patients (17.5%) did not have measurable disease based on light chain levels. CR (34.7%) and VGPR (26.4%) were seen in 61% of cases. Organ response was achieved in 49% of cases. Cardiac response was observed in 40% of cases. Median OS and PFS have not been reached (Estimate, 67 and 41 months, respectively). (Fig 1a) However, OS was shorter in the group with severe heart involvement (NT-pro-BNP>5000 or BNP>1100) (p=0.03) (Fig 1b). In conclusion, BCR are efficacious for the treatment of newly diagnosed AL Amyloidosis, providing with a high degree of organ and hematological responses. The impact of BCR on advanced heart dysfunction cases remains unclear but it seems that these patients still require a different approach maintaining a dismal prognosis.
Overall survival and advanced cardiac disease Overall survival and advanced cardiac disease Figure 1 PFS for the whole AL group treated with BCR Figure 1. PFS for the whole AL group treated with BCR

Disclosures
Song: Janssen: Honoraria; Otsuka: Honoraria; Celgene: Honoraria, Research Funding. Neri:Celgene and Jannsen: Consultancy, Honoraria. Bahlis:Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria; BMS: Honoraria. Jimenez-Zepeda:Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene, Janssen, Amgen, Onyx: Honoraria.
C75 Fatty Acid Synthesis (FAS) Inhibitor Has Potent Immunosuppressive Activity
Authors: Senyuk Vitalyi , Mahmud Dolores , Oh Annie L. , Patel Pritesh R. , Rondelli Damiano 
Published: October 2019

Abstract
Fatty acid synthesis (FAS) or oxidation (FAO) are important regulatory pathways in immune response. In fact, FAS plays a pivotal role in antigen presentation and T cells activation and FAO leads to fatty acid degradation which has been previously shown to regulate hematopoietic stem cell maintenance. Here we hypothesized that FAS can be a new target to suppress T cell alloimmune responses in solid organ or stem cell transplantations. Therefore, we tested if the FAS inhibitor C75 could suppress T cell alloreactivity without impairing normal hematopoiesis.
The immuno-suppressive (IS) effect of moderate FAS inhibition was demonstrated in mixed leukocyte cultures (MLC) where C75 at 10 mkg/ml significantly reduced T cell proliferation and prevented the expansion of CD3+CD25+ and CD3+CD69+ T cells. In T cells stimulated by alloantigen, C75 also induced the downregulation of NF-kB gene expression and the upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene involved in ubiquitination and degradation of NF-kB protein.
When compared to other standard IS agents, such as anti-thymocyte globulin (ATG), Cyclosporine A, Rapamycin or inhibitor of FAO Etomoxir, C75 showed similar anti-T cell activity.
The same dose of C75 (10 mkg/ml) did not cause apoptotic death of human CD34+ cells in vitro, nor affected CD34+ cell clonogenicity in vitro. In fact, C75 increased the number of BFU-E and CFU-GM colonies (P < 0.05). We observed that the expression of de novo DNA methyltrasferases DNMT3A and DNMT3B, which are important regulators of stem cell renewal, was strongly reduced in CD34+ cells co-cultured for 3 days with allogeneic T cells. On the contrary, in the presence of C75 the expression of DNMT3A and DNMT3B was not different from baseline control. To test the in-vivo effect of C75 we utilized a xenograft model of stem cell rejection where 2 x 105 human CD34+ cells and HLA incompatible T lymphocytes were injected in immunodeficient nonobese diabetic/ltsz-scid/scid - IL2 receptor gamma chain knockout (NSG) mice at 1:1 ratio. Four weeks after transplantation, control NSG mice showed complete rejection of huCD45+CD34+ cells and the expansion of T cells in the marrow and spleen. NSG mice treated with intra-peritoneum injections of C75 every 3 days for 2 weeks, instead, showed 10-15% human CD45+ myeloid cells in the marrow and spleen at week 4 after transplant, suggesting at least a partial effect on preventing rejection of incompatible stem cells.
We showed here that moderate FAS inhibition may represent a novel immunosuppressive strategy and our findings will prompt preclinical investigations exploiting the effect of FAS inhibitors alone or in combination with standard IS agents in models of allogeneic transplantation or bone marrow failure.

Disclosures
No relevant conflicts of interest to declare.
Patients with Hematologic Malignancies Can Benefit More from Allele-Level HLA Matching Than Antigen-Level after Single Unit Umbilical Cord Blood transplantation: a Retrospective Single Center Clinical Analysis
Authors: Guangyu Sun , Zimin Sun , Xiaoyu Zhu 
Published: October 2019

Abstract
Introduction:The increasing number of unrelated umbilical cord blood (UCB) has been used as an alternative graft because greater degrees of donor-recipient HLA mismatch are tolerated. Generally, lower resolution
HLA typing (antigen-level) for HLA-A and -B and at the allele level for HLA-DRB1 is used to select UCB units; HLA-C and -DQB1 are not typically considered. A few studies have suggested that HLA-matching based upon allelic typing for 8 HLA loci(HLA-A, -B, -C and -DRB1 )should be taken into account when searching UCB units; and the NMDP also encourages extended high-resolution typing of umbilical cord blood units to facilitate further study of the impact of HLA. Nevertheless, few studies have taken into consideration that if HLA-matching based upon allelic typing for 10 loci (HLA-A, -B, -C,-DRB1 and -DQB1) can benefit more patients than traditional HLA typing method (antigen-level for HLA-A,-B and allele-level for HLA-DRB1). In this study, we aim to compare the clinical outcomes between patients using allele-level HLA typing and those using traditional HLA typing method, such as engraftment, relapse and survival after unrelated cord blood transplantation (UCBT).
Patients and methods: 309 patients with hematologic malignancies who underwent UCBT in Anhui Provincial Hospital from May 2008 to December 2015 were analyzed. All patients received a single UCB unit after intensified myeloablative conditioning regimens and a combination of cyclosporine A (CsA) and mycophenolate mofetil (MMF) was given for graft-versus-host disease (GVHD) prophylaxis. Of 309 patients at enrollment, 95 patients used traditional HLA typing method (antigen level for HLA-A,-B and allele level for HLA-DRB1, at least 4/6 HLA loci matching must be met) and 214 patients used allele-level HLA typing method (allele-level for HLA-A,-B,-C,-DRB1 and -DQB1, at least 5/10 HLA loci matching must be met). The follow-up assessment was conducted before May, 31, 2016.
Results: Patients using allele-level HLA typing method had a statistically significant higher cumulative incidence of platelet engraftment (P=0.039), and the risk of grades 2-4 acute GVHD was statistically significant lower than patients using traditional HLA typing method (P=0.01). The risk of transplant-related mortality (TRM) by 1 year was also lower than patients using traditional HLA typing method (P=0.043), while there was no increase in the risk of relapse (P=0.338). Conclusion: Using allele-level HLA typing method when searching UCB units (at least 5/10 HLA loci matching must be met) can improve the engraftment of platelet, reduce the risk of grades 2-4 acute GVHD and TRM, which can take the place of traditional HLA typing method to search UCB units.

Disclosures
No relevant conflicts of interest to declare.
Stem Cell Transplantation for Thalassemia Major from Alternative Donors: A Single Center Experience from India
Authors: Philip Chepsy C , Mathew Amrith , Kakkar Naveen , Sobti Praveen , John M Joseph 
Published: October 2019

Abstract
Allogeneic stem cell transplant (aSCT) which remains the only curative option for patients with β thalassemia major (TM) is often limited by a lack of ideal donors. The role of alternative (related phenotypic donor- RD and Unrelated donor -UD) donor hematopoietic cell transplantation in thalassemia is not well established. Use of conventional myeloablation with busulphan and cyclophosphamide in alternative RDs (either phenotypically identical or 1-antigen-mismatched donors) has been associated with higher graft failure and acute graft versus host disease (aGvHD) prompting novel approaches (Gaziev et al., 2000, 2013). We undertook a retrospective analysis of patients who underwent aSCT from alternative donors in TM.
From February, 2009 to June, 2016, 51 HLA matched transplants for TM was done at our center. Among them 19 (37.3%) used alternate donors. In this cohort, the median age was 9 years (range: 2-18) and there were 13 (68.4%) males. Thirteen (68.4%) were Pesaro Class III and 8 (42.1%) were in the Class III higher risk group as defined earlier (Mathews et al., 2007). All 19 received a treosulfan based regimen. PBSC (Peripheral Blood Stem Cell) was the source of stem cells in 78% and bone marrow in the rest. All patients received a cyclosporin (CSA) plus short course methotrexate GvHD prophylaxis regimen. There were 4 (12%) deaths. The major cause of death was fungal infection (3 had invasive fungal infection of whom 1 developed candidemia following a very late UD deferral).
Of the alternative grafts 7 (36.8%) received RD grafts and 13 (68.4%) from UD. We compared the results of RD with HLA-matched sibling (matched sibling donors [MSDs]) aSCT in 32 patients and UD in 12 patients (Table 1). One (14.3%) patient developed aGvHD (Grade 2-4). The entire RD group had sustained engraftment. Rejection incidence was 0% in the RD, 8.3% in UD and 3.4% in MSD groups, with respective thalassemia-free survival probabilities at one year of 68.6% ±18.6%, 83.3% ±15.2%, 90.0% ±5.5% (P = .549) Fig 1.
In conclusion, the present data shows that a treosulfan based reduced toxicity myeloablative regimen with a PBSC graft has the potential to allow patients to safely undergo aSCT from phenotypically identical RD.

Disclosures
No relevant conflicts of interest to declare.
Nrf2 Regulates Sensitivity of Leukemias to Artemisinins
Authors: Bai Jennifer , Chen Xiaochun , Civin Curt I. 
Published: October 2019

Abstract
The artemisinins are a class of drugs widely used to treat severe malaria due to their potency and few adverse effects. In addition to antimalarial activity, artemisinins have been shown to have potent anti-cancer activity, specifically in leukemia. Therefore, artesunate (AS), the WHO-recommended treatment for severe malaria, and a new dimeric artemisinin derivative (ART-838) are promising agents to repurpose for leukemia treatment. Reactive oxygen species (ROS) have been implicated in the mechanism of artemisinin activity in both malaria and cancer; however, the full mechanisms of artemisinin activity in cancer have not yet been elucidated. In 2014, Ariey et al, published a paper on malaria identifying the Kelch (K13) propeller domain as a molecular marker for artemisinin resistance. Since Keap1 ̶ the human homolog of K13 ̶ is involved in ROS regulation, it may also play a role in artemisinin action in cancer. To investigate the role of Keap1-Nrf2 signaling cascades in mediating artemisinin action and resistance in leukemia, qPCRs and Western blots were performed to quantify endogenous levels of selected Keap1 pathway molecules (Keap1, Nrf2, Nqo1, and Gsta1) in 23 human acute leukemia cell lines. However no significant correlation was observed between the levels of these molecules (mRNA or protein) and AS/ART-838 sensitivity. Using an antioxidant response element (ARE) luciferase reporter that can be activated by Nrf2, a significant increase in ARE activity was observed in K562 cells upon AS or ART-838 treatment. In addition to increased transcription upon drug treatment, the ARE downstream proteins Nqo1 and Gsta1 were upregulated upon AS or ART-838 treatment. K562 cell lines with Keap1 and Nrf2 stably knocked down (KD) were generated. The Nrf2 KD K562 cell line had increased sensitivity to AS and ART-838. In contrast, KD of Keap1 did not result in a substantial change in drug sensitivity to either artemisinin. In conclusion, these studies suggest involvement of Nrf2 in human leukemia cell resistance to artemisinins. Further studies on Nrf2 may help us better understand the molecular actions of the artemisinins in order to optimize their efficacy as single agents or in drug combinations for leukemia treatment.

Disclosures
No relevant conflicts of interest to declare.