bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA and Kir Mismatch Transplant Affect Cytopenia Beyond Day 28 after Thalassemia Transplant: A Primary Manifestation of Chronic Graft Versus Host Disease
Authors: Zhou Xiaohui , Li Chunfu , Liao Jianyun , Liu Xiangjun
Published: October 2019

Background Cytopenia beyond day 28 post-transplant (CB-28PT) following hematopoietic stem cell transplantation (HSCT) with β-thalassemia major (TM) rarely was reported. The exact mechanism for the development of CB-28PT is not well known.

Aim To find out causes of CB-28PT cytopenia.

Method We retrospectively analyzed data (HLA mismatch status, HLA antibody status of patients, KIR gene mismatch status, KIR-ligand matching status, donor/patient CMV status, donor/patient age and sex) of 93 TM patients underwent HLA 8/8 fully matched or 7/8 matched unrelated donor HSCT. All the patients used sole NF-08-TM protocol with median follow-up time of 19 (r: 2-44) months.

Results Results show a significant association between DRB1 mismatch and CB-28PT (P = 0.012). In addition, presence of Class I HLA antibody in the patient’s sera seems increase the chance of CB-28PT. Finally, the matching between inhibitory KIR2DL1 and their corresponding ligand HLA-C2 has a protective effect for CB-28PT.

Conclusion We propose that CB-28PT may be a primary manifestation of cGVHD in pediatric TM patients undergoing HSCT positive influenced by HLA DRB1 mismatch, HLA class I antibody and negatively affected by KIR-ligand match.

Disclosures

No relevant conflicts of interest to declare.
Generating Chimeric Antigen Receptors Utilizing Novel Anti-CD3 Nanobeads
Authors: Schultz Liora M. , Czerwinski Debra K , Fu Aihua , Levy Shoshana , Levy Ronald
Published: October 2019

The processes of ex vivo transduction of T cells to express chimeric antigen receptors (CARs) and of CAR+ T cell expansion influence the phenotype, function and ultimate fate of the final CAR+T cell product infused into patients. CAR constructs, despite expression of endogenous activation signals, require exogenous T cell activation during CAR transduction to allow optimal lenti-viral or retroviral-mediated integration of the CAR gene of interest into T cells. Clinical CAR therapy trials utilize anti-CD3 antibody-mediated activation or combined CD3 and CD28 stimulation using CD3, CD28 specific magnetic beads. We introduce novel magnetic nanoparticle beads generated from iron oxide nanoparticles conjugated to streptavidin and bound to biotinylated T cell activating antibodies for the purpose of CAR transduction. The small size of these nanobeads confers the advantage of decreased steric hindrance and enhanced capability of bead surface antibodies to access T cell surface antigen for binding and stimulation. We achieve efficient CAR transduction using anti-CD3 nanobead-mediated T cell stimulation and demonstrate CD19 specific CAR-mediated cytotoxicity of CD19+ tumor using an annexin V and 7AAD cytotoxicity assay. Evaluation of T cell phenotype following anti-CD3 nanobead-mediated T cell activation demonstrates preferential activation of naïve T cells as compared to central and effector memory cells. Addition of anti-CD28 costimulation is not necessary to achieving or inhibiting this preferential naïve T cell activation. Naïve T cells exhibit greater replicative capacity and anti-tumor function as compared to both effector and central memory T cells for adoptive transfer. We anticipate that preferential generation of naïve T cell derived CAR+ T cells achieved by introducing anti-CD3 nanobead stimulation can further improve the outcomes of clinical trials using CAR therapy.

Disclosures

Fu: NVIGEN Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Retrospective Observational Study on Multiple Myeloma Cases Admitted to Kfsh – Dammam Between 1st of June 2006 till the End of December 2013
Authors: Abduljalil Omar Zeid , Mohiuddin Abid , Al Hashmi Hani Hassan
Published: October 2019

Introduction: Multiple myeloma (MM) is characterized by the neoplastic proliferation of a single clone of plasma cells producing a monoclonal immunoglobulin. It accounts for approximately 1 percent of all cancers and slightly more than 10 percent of hematologic malignancies in the United States. There is a scarcity on the information of myeloma in the kingdom of Saudi Arabia.Hence was this observational study from a single center in Dammam, Saudi Arabia. . Method:

Retrospective chart review of consecutive patients Diagnosed with Multiple myeloma in the period between 1st of May 2006 until end of December 2013 at King Fahd Specialist Hospital in Dammam (KFSHD). Purpose:

to describe the presenting characteristics of MM patients in our region, treatment patterns prescribed with the outcome of therapy that include progression free survival and overall survival. Result:

63 patients with multiple myeloma were diagnosed and treated during this period. The percentage of patients with ECOG (≥2) is 52.3 % . 60 % of the patients had stage III international staging system (ISS). Only 17.4% received Triple agents as primary therapy as most of our patients received duplet therapy. 46% received High Dose therapy and Autologous stem cell transplantation. the PFS was 41.1 % and the OS of 69.1% at 95 months. Conclusion:

Our results showed that we had rather younger patient population with a median age of 60 compared to the average western myeloma patient population. Despite the relatively young age those patients unfortunately had relatively poor performance status and high ISS stage which correlate with worse clinical outcome. However, the response to therapy and the outcome of our patients were superior to the outcome of any cohort of patients with similar disease characteristic. This observation worth further studying which could be due to an unknown disease or treatment related factors that might affect positively the patient’s outcome.

Disclosures

No relevant conflicts of interest to declare.
Interventions to combat Vitamin D Deficiency in patients undergoing Hematopoietic Stem Cell Transplantation
Authors: Pereira Andrea Z , Piovacari Silvia MF , Lucio Fabiana , Tanaka Marcia , Barrere Ana Paula N , Silva Juliana Bernardo , Ribeiro Andreza Alice Feitosa , Hamerschlak Nelson
Published: October 2019

Introduction: Patients undergoing Hematopoietic Stem Cell Transplantation(HSCT) may have low vitamin D (VD) level because of decreased exposure to sunlight, the major cause of VD Deficiency (VDD), from prolonged hospital stays, limited outdoor activity, and sunscreen use, and decreased oral intake caused by gastrointestinal treatment toxicity. Besides that gastrointestinal graft-versus-host disease (GVHD) limit absorption of VD. Some medications received during the HSCT can increased the VD catabolism, and alterate renal and kidney function.

Objectives: To evaluate the reduction of number of patients with VD Deficiency in patients undergoing HSCT after educational classes for the multi-professional team (physicians, dietitians and nurses).

Methods: We analyzed 72 patients undergoing HSCT May 2012 to January 2014 in the Hematology-Oncology and Bone Marrow Transplantation Center at Albert Einstein Hospital in São Paulo, Brazil. The serum levels of vitamin were measured in the first day of hospitalization of the patients adults (>= 18 years) who would be undergoing HSCT. All types of HSCT patients were included.We used in our study the VDD was defined and recommended by the Institute of Medicine as a 25(OH)D <=20 ng/ml, VD insufficiency of 21-29 ng/ml, and VD normal >=30 ng/ml.In the 2012 the multi-professional HSCT Team had 3 classes about VD and, everyone were informes about the VD research protocol.

Results: 72 adult patients were observed in this study, aged between 18 and 74 years, with the majority (77.8%) with less than 65 years. Of the total, 59.7% were men and 41.7% had normal body mass index. 100% of the lymphoma patients had VDD. In 2012(n:33), 60% patients had VDD and in 2013 (n:39), 40% (p<0,05).

Conclusions: When all of members of HSCT team were informed about the benefits of high VD levels in patients undergoing HSCT by classes and research protocol, we can reduce VDD.

Disclosures

No relevant conflicts of interest to declare.
Concurrent Tumors in Patients with Hematologic Malignancies
Authors: Sumoza Luis David , Messinger Marina , Sumoza Patricia , Aguirre Roxana , Harsha Poola , Avila Dayra
Published: October 2019

Background: Secondary tumors has been described in a variety of patients with either hematologic malignancies or solid neoplasm, and most of the time is related to previous chemotherapy and radiation therapy exposure, but little is found in the literature about synchronous or metachronous neoplasm that can be found in patient with a newly diagnosis of hematologic malignancy; we report 45 cases, presented in a single institution from 2007 to present.

Methods: A retrospective review of records using our tumor registry data, from patient with hematologic malignancies at John H. Stroger Hospital of Cook County, was performed and 45 patients with either synchronous or metachronous neoplasm were identified.

Results: Lung cancer was the most common malignancy representing 22.2%, Colorectal cancer 20%, Prostate cancer 17.7%, Breast cancer 11.1%, Urothelial cancer (Kidney and Bladder) 8.8%, Myelodisplastic syndrome, Pancreatic cancer, Thyroid cancer, Non-Hodgking lymphoma, Acute Myeloide Leukemia, Vulvar cancer, Testicular cancer and Skin Cancers 2.2% each one respectively.

Results: We found that in our heterogeneous population of patient with hematologic malignancies, the incidence of synchronous or metachronous neoplasm practically follows a very similar pattern of the general population, despite that many of this patients have been exposed to chemotherapy and radiation therapy.

Disclosures

No relevant conflicts of interest to declare.
A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue
Authors: Scoville Steven D , Keller Karen , Mundy-Bosse Bethany , Caligiuri Michael A. , Freud Aharon G.
Published: October 2019

Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population.

Disclosures

No relevant conflicts of interest to declare.
Improved Immune Recovery after Transplantation of TCRαβ/CD19 Depleted Allografts from Haploidentical Donors in Pediatric Patients
Authors: Lang Peter , Feuchtinger Tobias , Teltschik Heiko-Manuel , Schwinger Wolfgang , Schlegel Patrick , Schumm Michael , Lang Anne-Marie , Lang Barbara , Schwarze Carl Philipp , Ebinger Martin , Urban Christian E. , Handgretinger Rupert
Published: October 2019

Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease.

Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach > 100 CD3+/µl, > 200 CD19+ cells/µl and > 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial.

Disclosures

No relevant conflicts of interest to declare.
Dysregulation of SHFM1, a Novel Target for Prevention of Genomic Instability in Myeloma, Is Associated with Epigenetic Changes at Specific CpG Sites
Authors: Shammas Masood , Kumar Subodh , Pal Jagannath , Nanjappa Puru , Samur Mehmat , Gkotzamanidou Maria , Shi Jialan , Munshi Nikhil C.
Published: October 2019

We have previously demonstrated that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. We, therefore, hypothesized that mechanisms underlying dysregulation of HR, drive genomic instability and have potential to serve as novel markers of progression and target to make cancer cells static. In an attempt to identify molecular intermediates underlying increased HR, we conducted a high-throughput shRNA screen using HR activity in MM cells as an endpoint, and identified split hand/split foot malformation locus 1 - SHFM1 - as a novel regulator of HR in myeloma. We demonstrate that suppression of SHFM1 using specific lentiviral shRNA knockdown inhibits HR activity in myeloma as well as other (esophageal) cancer cells. Suppression of HR activity is also associated with reduction in spontaneous DNA breaks thus having a positive impact on genomic integrity. We have also observed that suppression of SHFM1 gene lead to reduction in acquisition of new genomic changes by both copy number alteration as well as SNPs. Evaluation of expression profile in 330 MM patients and 18 normal plasma cells indicated that the gene is overexpressed in myeloma, relative to normal plasma cells (P = 0.0059). Expression of this gene in myeloma also significantly correlates with its copy number (R=0.43; P<0.00000004). To understand the molecular mechanism of its overexpression in myeloma, we evaluated DNA methylation changes associated with altered expression of this gene. We investigated CpG sites in the promoter region of SHFM1 gene using primers with sodium bisulfite converted DNA-specific design. The primers were designed such that they avoid annealing to CpG sites at the region of interest to the maximum extent possible. Samples were bisulfite converted and amplification of all samples using specific primer pairs was performed, and then massively parallel sequenced and analyzed. We observed methylated CpG at two SHFM1 gene locations in PBMC but unmethylated in all 12 out of 12 myeloma cell lines at first location and in 9 out of 12 myeloma cell lines at second cite (Fisher’s exact test for comparisons of PBMC vs. average methylation ratios in caner samples for two locations was P=1.38E-13 and 0.05, respectively).

In summary our results highlight SHFM1 as an important gene modulating genomic instability and its control by methylation provides an insight into epigenomic control of DNA stability.

Disclosures

No relevant conflicts of interest to declare.
Leukemia-Associated Oncometabolite 2-Hydroxyglutarate Regulates Epigenetic Modifications in Response to Oxygen Availability in Cells without IDH1/2 Mutations
Authors: Intlekofer Andrew M. , Dematteo Raymond G. , Finley Lydia W. , Cross Justin R. , Thompson Craig B.
Published: October 2019

Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) result in production of the 'oncometabolite' D-2-hydroxyglutarate (D-2HG). D-2HG blocks differentiation of leukemia cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases and TET family DNA methylcytosine dioxygenases, which function as important regulators of chromatin structure and gene expression. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although leukemia-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. However, biologic sources and/or activities of L-2HG remain unknown. Possible physiologic roles for both 2-hydroxyglutarate enantiomers are suggested by the existence of evolutionarily conserved 'waste disposal' enzymes, known as D2HGDH and L2HGDH, whose sole function is to eliminate D-2HG or L-2HG, respectively. Indeed, children born with genetic deficiency of either enzyme develop severe developmental disorders known as D- and L-2HG acidurias, characterized by systemic elevation of 2HG. We sought to investigate physiologic sources and functions of D-2HG and L-2HG by manipulating D2HGDH and L2HGDH in cells without IDH1/2 mutations. Using this approach, we found that under conditions of oxygen limitation, cells potently and selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG production is not mediated by IDH1 or IDH2, but instead appears to occur via promiscuous substrate usage by lactate dehydrogenase A (LDHA), and to a lesser extent, malate dehydrogenase 2 (MDH2). Hypoxic induction of L-2HG increases repressive histone modifications, including H3K9me3 and H3K27me3, chromatin marks associated with an undifferentiated cellular state. Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that might prevent a cell from undergoing differentiation when it lacks the capacity for oxidative metabolism. As hematopoietic stem cells (HSCs) reside in hypoxic niches, oxygen-regulated L-2HG might influence self-renewal of HSCs and represent a physiologic system exploited by IDH1/2 mutant-derived D-2HG in leukemia.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Iron Overload in the Face of Anemia
Authors: Shah Yatrik
Published: October 2019

Several distinct congenital disorders can lead to tissue-iron overload with anemia including β-thalassemia and sickle cell disease. We show that intestinal absorption of iron is highly increased and significantly contributes to tissue iron accumulation in these disorders. The present work describes a novel pathway by which oxygen sensing transcription factors are highly upregulated in iron overload anemias and are subsequently essential for the increase intestinal iron absorption. Oxygen signaling is mediated through well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)1a and HIF2a. In the intestine, HIF2a directly activates divalent metal transporter 1 (DMT1), duodenal ferric reductase (DcytB), and Fpn1, which are iron transporters critical for adaptive changes in iron absorption. We demonstrate that HIF2a and its downstream target gene, DMT1 are essential for iron accumulation in mouse models of β-thalassemia and sickle cell disease. Furthermore, studies of thalassemic mouse model with established iron overload demonstrated that loss of intestinal HIF2a and DMT1 signaling led to decreased tissue iron accumulation in the livers. Interestingly, disrupting intestinal HIF2a not only improves tissue iron accumulation, but a marked improvement of anemia was also observed. These novel findings suggests that inhibition of HIF2a signaling pathway could be a novel and robust treatment strategy for several conditions that cause iron overload with anemia.

Disclosures

No relevant conflicts of interest to declare.
The Somatic Cancer Genome
Authors: Golub Todd R.
Published: October 2019

New genome sequencing technologies have led to an explosion of cancer genome sequence information, resulting in new insights into the genetic basis of cancer. This talk will review recent advances in somatic cancer genome analysis, with particular emphasis on hematological malignancies including diffuse large B-cell lymphoma and multiple myeloma. New analytical approaches have led to both a refined ability to distinguish true drivers of cancer from mere passenger mutations and to a better understanding of what scale of cancer genome sequencing will be required to “complete” a cancer genome atlas of mutations. While many of the recurrently mutated genes in cancer have known function, many remain uncharacterized. As such, a new kind of follow-up research activity is required – one that can bring systematic, high throughput biology methods to bear on elucidating mutant allele function and their potential as therapeutic targets. Efforts to pilot such a “Target Accelerator” will be presented, with particular emphasis on use of the Connectivity Map/Library of integrated cellular signatures (LINCS) resource to systematically connect mutant alleles to pathways and to potential therapeutic strategies.

Disclosures

Golub: Foundation Medicine, Inc.: Consultancy, Equity Ownership.
Inhibition of the NAMPT-Triggered Deacetylation of HCLS1 Protein: A New Therapeutic Option in Chronic Myeloid Leukemia
Authors: Samareh Bardia , Zimmer Inna , Zikic Andrew , Klimenkova Olga , Loghmani-Khouzani Houra , Suttorp Meinolf , Kanz Lothar , Welte Karl , Skokowa Julia
Published: October 2019

G-CSFR-triggered phosphorylation and activation of the hematopoietic-specific lyn-substrate 1 (HCLS1) protein is important for the granulocytic differentiation of hematopoietic stem cells (HSCs) (Skokowa J. et al., Nat Med 2012). G-CSF also activates protein deacetylation via Nicotinamide phosphoribosyltransferase (NAMPT)/NAD+/sirtuin 1 (SIRT1) pathway (Skokowa J. et al., Nat Med 2009). SIRT1 is NAD+-dependent protein deacetylase playing an important role in the regulation of gene transcription. Interestingly, having strong pro-differentiation effects on myeloid progenitors, both HCLS1 and NAMPT are markedly hyper-activated in myeloid leukemias, inducing proliferation and survival of undifferentiated blasts.

Knowing this, we investigated whether HCLS1 could be activated by NAMPT/NAD+/SIRT1 pathway via deacetylation in HSCs. Indeed, we found that HCLS1 is deacetylated in HSCs by NAMPT/SIRT1 and that G-CSF treatment of these cells induced deacetylation and phosphorylation of HCLS1 via NAMPT/SIRT1. Moreover, we found that deacetylation of HCLS1 is important for myeloid differentiation and proliferation of HSCs.

We further demonstrated that HCLS1 protein was activated by deacetylation in blasts of chronic myeloid leukemia (CML) patients, as compared to HSCs of healthy individuals. This was in line with elevated mRNA and protein levels of NAMPT and HCLS1. We further investigated, whether inhibition of NAMPT-triggered deacetylation of HCLS1 has any effect on the survivial and differentiation of CML blasts in vitro. We found that treatment of CML blasts with the specific NAMPT inhibitor FK866 resulted in increased acetylation of HCLS1. In addition, treatment of CML blasts with the FK866 in a dose of 10nM resulted in marked inhibition of proliferation and elevated cell death, which was consistent between different patients and was comparable to the effects of Imatinib treatment. In CFU assay in the presence of FK866 (10nM) no CFU colonies were grown. Even at a dose of 3nM FK866 drastically suppressed total colony counts. This inhibitory effect was further amplified by combination of FK866 with Imatinib. Addition of 10nM of FK866 to CFU culture medium was not toxic for CD34+ cells of healthy donors.

Taken together, we demonstrated that NAMPT and HCLS1 have dose-dependent effects on hematopoietic cells and that NAMPT activates HCLS1 functions by SIRT1-triggered deacetylation. Inhibition of NAMPT in CML blasts markedly reduced their survival, proliferation and differentiation. In conclusion, we suggest a possible therapeutic application of NAMPT inhibition in the treatment of CML patients via deactivation of HCLS1 function by acetylation. This therapy could be applied in combination with Imatinib, or in Imatinib-resistant CML patients.

Disclosures

No relevant conflicts of interest to declare.
Qbleed Score in an African American Cohort
Authors: Benyounes Amin , Candelario Nellowe , Braitman Leonard , Leighton John C. , Tester William , Varadi Gabor
Published: October 2019

Introduction/Background:

The Qbleed score is an algorithm that calculates the absolute risk of gastrointestinal and intracranial bleed in patients on anticoagulation that was published in the British Medical Journal in July 2014.

It was developed using a UK general practice database. African Americans were not represented in the development of the algorithm. We intended to investigate if the score is applicable to African Americans.

Materials and Methods:

Our retrospective cohort study was conducted at Albert Einstein Medical Center in Philadelphia, USA. The study's duration was 7 years between 1 January 2007 and 31 December 2014. Inclusion criteria to our study were African Americans admitted to the hospital for gastrointestinal bleeding (GIB) event requiring an esophagogastroduodenoscopy (EGD) or for an intracranial hemorrhage (ICH) event. Study participants on anticoagulants were identified by chart review. The Qbleed score of African Americans on anticoagulation admitted for GIB and ICH was calculated at the time of study entrance and compared with a control group; the control group was African Americans on anticoagulation that did not experience any of the above bleeding events.

Results:

1288 study participants were admitted for a GIB event requiring EGD. Of these, 107 (8.3%) were on anticoagulants.

The average Qbleed value in this group was 1.36 compared to 0.78 in the control group. On average, this group had 0.57 higher Qbleed score than controls (95% confidence interval 0.13 to 1.01, p=0.01). The receiver operating curve statistic value was 0.6698 (95% confidence interval 0.60 to 0.74).

1169 study participants were admitted for an ICH event. Of these 59 (5%) were on anticoagulants.

The average Qbleed value in this group was 0.85 compared to 0.48 in the control group. On average, this group had 0.36 higher Qbleed score than controls (95% confidence interval 0.16 to 0.57, p=0.0004). The receiver operating curve statistic value was 0.65 (95% confidence interval 0.56 to 0.74).

Conclusion:

In our African American cohort the absolute risk of gastrointestinal and intracranial bleeding calculated by the Qbleed score was significantly higher in patients who experienced a bleeding event. Our study suggests that the application of the Qbleed score to African Americans allows the identification of patients with higher risk of bleeding while on anticoagulation.

Disclosures

No relevant conflicts of interest to declare.
Upregulation of Indoleamine 2,3-Dioxygenase Enzymes in Leukemic Mesenchymal Stromal Cells (MSCs) Can Influence MSC/Acute Myeloid Leukemia Cell Cross Talk
Authors: Ciciarello Marilena , Corradi Giulia , Baldazzi Carmen , Testoni Nicoletta , Lemoli Roberto Massimo , Cavo Michele , Curti Antonio
Published: October 2019

Mesenchymal stromal cells (MSCs), an essential element of both normal and leukemic hematopoietic microenvironment, are multipotent cells with a unique immune-modulating ability. Thus, MSCs play a crucial role for both the proliferation and differentiation of hematopoietic stem cells (HSCs) and induce an immune-tolerant milieu. Indoleamine 2, 3-dioxygenase (IDO1 and IDO2) enzymes catabolize tryptophan to kynurenines and play a key role in the induction of immune tolerance in different settings, including acute myeloid leukemia (AML). Furthermore, IDO1/IDO2 pathway is a well described mechanism by which MSCs exert their mmunomodulatory properties. We hypothesized that: 1) MSC-dependent mechanisms are involved in leukemia initiation, maintenance and progression; 2) the expression of IDO1 and IDO2 by MSCs is part of a MSC-dependent mechanism able to create a tumor-supportive milieu. To this aim, we isolated MSCs from the bone marrow of AML patients (AML-MSCs) at diagnosis. We first analyzed their phenotypic and functional properties compared to that of healthy donor-derived MSCs (HD-MSCs). We found that AML-MSCs showed a reduced proliferative capacity but normal immunophenotype, differentiative and immunomodulatory capacity as compared to HD-MSCs. Furthermore, AML-MSCs did not show the chromosomal abnormalities identified in the primary blast counterpart (FISH analysis). We next investigated IDO1/2 expression and functions in MSCs. We demonstrated that IDO enzymes are expressed in AML-MSCs as well as in HD-MSCs. IDO1 is efficiently upregulated by different inflammatory stimuli, and IDO1 protein expression parallels mRNA in both HD-MSCs and AML-MSCs. Interestingly, IDO2 mRNA is expressed at low basal level in all analyzed conditions in HD-MSCs, while it is upregulated, in particular after IFN-gamma stimulation, in AML-MSCs, although the level of induction varies between different patients. When T-cell proliferation was tested in MSC co-cultures, w/or w/out IDO1/2 inhibitor, 1-methyltryptophan, we found that MSC immunomodulatory potential is IDO-dependent both in HD-MSCs and AML-MSCs. Finally, we found that in co-culture assay with primary AML blasts, MSCs stimulated blast proliferation and this effect is, at least in part, IDO-mediated. These data suggested that IDO enzymes, in particular IDO2, may be differentially expressed in AML-MSCs as compared to HD-MSCs and IDO inhibition has an impact on MSC/AML cell cross talk. These findings may help to discover novel niche-target prognostic/therapeutic factors and to provide novel applications for drugs already under active clinical investigation (i.e. IDO-inhibitors).

Disclosures

Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.
Prognostic Role of Minimal Residual Disease before and after Hematopoietic Stem Cell Transplantation in Childhood Acute Lymphoblastic Leukemia
Authors: Rossi Bartolomeo , Campeggio Mimma , Magrin Elisa , Zecca Marco , Rubert Laura , Fagioli Franca , Quarello Paola , Lovisa Federica , Basso Giuseppe
Published: October 2019

Background and Objectives

Currently, more than 80% of children with acute lymphoblastic leukemia (ALL) can be cured through intensive and risk-adapted chemotherapy protocols. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered beneficial for approximately 10% of the patients who are at very-high risk at frontline therapy and for the majority of patients after relapse. In this retrospective study, we aimed to assess the prognostic role of minimal residual disease (MRD) before HSCT and at different time points after transplantation in children with ALL.

Patients and Methods

We analyzed 64 pediatric ALL patients given HSCT: 22/64 were in first complete remission (1CR) and 42/64 in second complete remission (2CR). Genomic DNA was obtained from bone marrow aspirates collected at diagnosis/relapse, before HSCT (pre-HSCT) and at the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). MRD was measured by quantitative real-time PCR assays based on patient-specific junctional regions and interpreted according to the EuroMRD guidelines. The association between MRD and survival was assessed by chi-square test.

Results

All evaluated patients were analyzed for MRD before transplantation (pre-HSCT). MRD was negative in 26/64 patients and positive in 38/64 patients. As for cases with positive MRD, 17/38 showed MRD levels ≥1x10-3 and 21/38 <1x10-3. Any detectable MRD positivity at pre-HSCT was significantly associated with a poor prognosis: 28/38 patients with positive MRD are dead, whereas 22/26 with negative MRD are alive in CR (P < 0.001). Among the 42 patients in 2CR, 14/42 had negative pre-HSCT MRD and 28/42 were MRD positive. The negativity of MRD before transplantation was found to be significantly associated with a good prognosis: 13/14 patients with negative MRD are alive in CR, while 23/28 with positive MRD are dead (p < 0.001). Post-HSCT1 MRD was analyzed in 53 patients: 17/53 were MRD positive and 36/53 were MRD negative. Based on MRD status, the prognosis was significantly different: 26/36 patients with negative post-HSCT1 MRD are alive in CR, whereas 14/17 of patients with positive MRD are dead (P < 0.001). Post-HSCT3 MRD was assessed in 41 patients and 19/41 were found positive. Persistence of MRD was associated with a poor prognosis also at this time-point (P = 0.001).

Conclusions

These results confirm that MRD assessment has a critical role both before and after transplantation. Negative MRD before transplantation is strongly associated with a good prognosis, particularly in 2CR patients. Since persistence of MRD after HSCT is significantly associated with a worse outcome, these patients could benefit from early discontinuation of immunosuppression, adoptive T-cell therapy and use of new drugs.

Disclosures

No relevant conflicts of interest to declare.
Expression of Programmed Cell Death 1 and Programmed Cell Death Ligand 1 in Extranodal NK/T-Cell Lymphoma, Nasal Type
Authors: Jo Jae-Cheol , Choi Yunsuk , Cha Hee Jeong , Lee Eun Hee , Kang Eun Kyoung , Kim Hawk
Published: October 2019

Background

Programmed cell death ligand 1 (PD-L1) is expressed on extranodal NK/T-cell lymphoma, nasal type (ENKL) tumor cells. The programmed cell death 1 (PD-1) and PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in ENKL. The aim of this study was to investigate the expression of PD-1 and PD-L1 and to determine the clinicopathological impact of PD-1 and PD-L1 positivity in ENKL.

Methods

We performed PD-1 and PD-L1 immunostaining in 79 ENKL biopsy samples and retrospectively analyzed medical records of all 79 patients from 4 tertiary referral hospitals. The demographic features, performance status, stage, LDH, primary sites, nodal sites, hemoglobin, white blood cell, platelet, creatinine, international prognostic index (IPI), and prognostic index for T-cell lymphoma (PIT) were recorded.

Results

The expression rates of PD-1-positive and PD-L1-positive ENKL were 7.6% and 88.6%, respectively (Figure 1A & 1B). PD-L1-negative ENKL (n=9) was significantly associated with high intermediate or high risk IPI (n=7, P=0.002) and group 3 or 4 PIT (n=6, P=0.043). Patients with PD-1-positive ENKL (n=6) had a trend toward better overall survival (OS) compared with that in patients with PD-1-negative ENKL (P = 0.090, Figure 2A). In contrast, there was no significant difference in OS between PD-L1-positive and -negative ENLK (P = 0.428, Figure 2B).

Conclusions

This is the first report describing the clinicopathological features and survival outcome according to expression of PD-1 and PD-L1 in ENKL. PD-1 expression rate is very low, and PD-L1 negativity is associated with poor risk groups of IPI and PIT in ENKL

Disclosures

Kim: Alexion Pharmaceuticals: Research Funding; Il-Yang: Research Funding; Celgene: Research Funding; Novartis: Research Funding.
TP53 Mutated MDS Patients Respond Equally to Hypomethylating Agents but Have Significantly Shorter Response Duration Compared to Patients with Wild Type TP53
Authors: Takahashi Koichi , Kantarjian Hagop M. , Patel Keyur , Bueso-Ramos Carlos E , Kadia Tapan , Jabbour Elias , DiNardo Courtney , Daver Naval , Pierce Sherry , Futreal Andrew , Garcia-Manero Guillermo
Published: October 2019

Background: Prognostic impact of TP53 mutations has been well described in MDS. However little is known about predictive impact on response to hypomethylating agents (HMA).

Aim: To determine predictive impact of TP53 mutation on response to frontline HMA therapy in MDS.

Methods: Bone marrow samples from 168 patients with untreated MDS were screened for TP53 mutation by next generation sequencing platform. All patients were treated with upfront 5-azacitidine or decitabine based HMA therapy. 13 of them had longitudinal follow up of TP53 mutation after HMA therapy.

Results: 38 patients (23%) had TP53 mutations. At baseline, TP53 mutated patients were significantly more neutropenic (P = 0.02), thrombocytopenic (P = 0.008), and had higher bone marrow blast (P = 0.006). TP53 mutation was significantly associated with complex karyotype (P < 0.001), monosomal karyotype (P < 0.001), and del 17p/-17 (P < 0.001). There was a trend toward mutual exclusivity between splicing pathway gene mutations and TP53 mutation (P = 0.07). Complete response (CR) and overall response (OR) to HMA therapy was observed in 34% and 45% of TP53 mutated patients, respectively, and there was no statistical difference from wild type patients (P = 0.38 and P = 0.13). Time to achieve response was also similar between TP53 mutated and wild type patients (P = 0.2). However, TP53 mutated patients had significantly shorter CR duration compared to wild type patients (6.3 months versus 28.5 months, P = 0.001). 11 out of 13 patients who had longitudinal follow up of TP53 mutation were found to have the same persistent TP53 mutation when they lost response to HMA therapy. TP53 mutated patients had worse overall survival (P <0.001) and prognostic impact of TP53 mutation was significant after adjusting for complex karyotype or IPSS-R risk.

Conclusion: MDS patients with TP53 mutations equally respond well to HMA therapy compared to WT patients. However, duration of response is significantly shorter than WT patients, which translates into worse overall survival. Longitudinal follow up showed persistence of the same TP53 clone after HMA therapy. Novel therapeutic strategy to improve duration of response in TP53 mutated MDS is urgently needed.

Disclosures

DiNardo: Novartis: Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding.
MYC Regulation Via the LIN28B/Let-7 Axis in Multiple Myeloma
Authors: Manier Salomon , Powers John , Sacco Antonio , Glavey Siobhan , Huynh Daisy , Reagan Michaela R. , Salem Karma , Moschetta Michele , Mishima Yuji , Leleu Xavier , Roccaro Aldo M , Daley George Q. , Ghobrial Irene M.
Published: October 2019

Background. LIN28B regulates developmental processes and cellular reprogramming by suppressing let-7 microRNAs (miRNAs). A role for LIN28B has been reported in cancers, however the LIN28B/let-7 axis has not been studied in multiple myeloma (MM).

Methods. LIN28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Knockdown (KD) of LIN28B was performed on MM cell lines (U266, MOLP-8) using 2 shRNA and validated using 2 sgRNA by CRISPR knockout (KO). Downstream regulations were assessed by qRT-PCR and western blots, as well as RNA sequencing. For RNA sequencing, control and Lin28B CRISPR cells were used for library preparation (NEBNext kit) and sequencing on a HiSeq 2000. Proliferation of KD and KO cells were evaluated in vitro and in vivo in a xenograft mouse model. An LNA-GapmeR technology was used to develop a let-7 mimic in vivo in SCID mice.

Findings. Two independent GEP datasets (GSE16558; GSE2658) were analyzed for LIN28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high LIN28B levels correlated with a shorter overall survival (p = 0.0226), along with an enrichment of let-7 target genes by Gene Set Enrichment Analyses (GSEA). LIN28B KD cells had a significantly increased expression level of let-7 family members and were associated with down-regulation of let-7 target genes Myc and Ras at the protein level. We further confirmed downstream regulation of MYC and RAS in a LIN28B CRISPR KO model in MM cells (MOPL-8). We next validated the role of LIN28B in MM in vivo by using a xenograft tumor model showing a decreased tumor burden in LIN28B KD mice compared to scramble control (p =0.0045). In addition, we performed a RNA sequencing from the CRISPR LIN28B KO and control cells and observed a central role by GSEA for both MYC and E2F cell cycle pathways in LIN28B-engineered cells. LIN28B activity in regulating MYC and cell proliferation was further defined to be dependent on let-7 by performing a rescue experiment in MM1S cells. Moreover, we explored the possibility to therapeutically regulate MYC expression through let-7 with an LNA-GapmeR containing a let-7b mimic, in vivo, and showed that high levels of let-7 expression represses tumor growth in SCID mice by regulating MYC expression compared to control GapmeR treated mice (p = 0.0026).

Conclusions. These findings reveal the essential role of LIN28B/let-7 in regulating two essential oncogenic pathways in MM, MYC and RAS. Interference with this pathway may represent an efficient option for targeting MYC in cancer.

Disclosures

No relevant conflicts of interest to declare.
Decision-Tree Algorithm Optimize Hematopoietic Progenitor Cell-Based Prediction in Peripheral Blood Stem Cell Mobilization
Authors: Wu Chia-Yun , Chiou Tzeon-Jye , Liu Chun-Yu , Lin Feng-Chang , Lin Jeong-Shi , Hung Man-Hsin , Hsiao Liang-Tsai , Gau Jyh-Pyng , Liu Jin-Hwang , Yen Hsiu-Ju , Hung Giun-Yi , Hsu Hui-Chi , Tzeng Cheng-Hwai , Yang Muh-Hwa , Chen Po-Min , Yu Yuan-Bin
Published: October 2019

Background and Objectives

Enumeration of hematopoietic progenitor cells (HPC) using an automated hematology analyzer provides rapid, inexpensive, and less technically dependent prediction of peripheral blood stem cell (PBSC) mobilization. This study aimed to incorporate HPC enumeration along with other predictors for optimizing a successful harvest.

Materials and Methods

Between 2007 and 2012, 189 consecutive patients who proceeded to PBSC harvesting with a preharvest HPC ≥ 20 x 106 /L were recruited. A failed PBSC mobilization was defined as < 2 x 106 CD34+ cells/kg. Variables predicting a successful harvest identified by multivariate logistic regression and correlation analysis were subjected to classification and regression tree (CART) analysis.

Results

A total of 154 (81.5%) patients successfully achieved mobilization of CD34+ cells (median 8.18 x 106 CD34+ cells/kg). Five independent host predictors including age ≥ 60, a diagnosis of solid tumor, prior chemotherapy cycles ≥ 5, prior radiotherapy, and mobilization with G-CSF alone or high-dose cyclophosphamide, as well as laboratory markers including HPC and mononuclear cell (MNC) counts, were used for CART analysis. The number of host predictors with a cutoff at two, HPC cutoff at 28 x 106/L and MNC cutoff at 3.5 x 109 /L were best discriminative for successful prediction. In the decision tree algorithm, patients predicted as good mobilizers (0 to 2 risk factors) had a higher success rate (150/169, 88.8%) than that (4/20, 20.0%) of those predicted as poor mobilizers (3-5 risk factors). Moreover, patients predicted as good mobilizers and further with a HPC enumeration ≥ 28 x 106/L had a high probability of achieving successful mobilization (138/148, 93.2%).

Conclusion

Our CART algorithm incorporating host predictors, HPC enumeration and MNC count may improve prediction and thus increase the success of PBSC mobilization. Further prospective validation is necessary.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
CTLA-4 Polymorphisms and Haplotype Correlate with Survival and aGVHD after Allogeneic Stem Cell Transplantation from Related HLA-Haplotype-Mismatched Donor
Authors: Qin Xiao-Ying , Wang Yu , Li Guo-Xuan , Qin Yazhen , Xu Lan-Ping , Wang Fengrong , Chen Huan , Han Wei , Wang Jing-Zhi , Zhang Xiao-Hui , Chang Ying-Jun , Liu Kaiyan , Jiang Zheng-Fan , Huang Xiao-jun
Published: October 2019

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective treatment for patients with hematological malignancies. Disease relapse remains a major cause of transplant failure.T cell homeostasis is critical to determine the potency of the GVT effect. Cytotoxic T lymphocyte antigen-4 (CTLA-4 or CD152) is a T cell activation negative regulator. Recent studies have shown the association of the CTLA-4 polymorphisms with the outcome after HLA-identical sibling allogeneic HSCT.

Patients and Methods: In this study, we focused on four CTLA-4 polymorphisms, and analyzed the impact of donor genotypes and haplotypes on the conditions of 154 acute leukemia patients after related HLA-haplotype-mismatched transplantation. The four SNP genotypes (-1661, -318, CT60 and +49) were determined by TaqMan SNP genotyping assays.

Results: Recipients of donors with +49 GG showed significantly lower OS (69.1% vs. 85.6%, P=0.024) and higher incidence of III-IV aGVHD (10.0% vs. 2.1%, P=0.032) than those with GA + AA(Fig.1,Fig.2). Multivariate analyses showed that +49GG was an independent risk factor for OS (HR:0.457,95%CI=0.227-0.920,P=0.028). Patients receiving mDLI showed significantly lower OS with +49 GG donor than those with AG+AA (P=0.011).The haplotype analysis revealed only three haplotypes in the donor population -1661/-318/CT60/+49 i.e.,ACGG,ACAA and GTGA,the frequencies were 64.3%, 19.5%, and 16.2%, respectively.Donors with and without the ACGG/ACGG haplotype had the same effect on transplant outcome as those with +49 GG and +49 AG+AA.

Conclusion: The CTLA-4 +49 GG and the haplotype ACGG/ACGG reduced the overall survival and increased the aGVHD after allo-HSCT from the related HLA-haplotype-mismatched donor,knowledge of the CTLA-4 polymorphism and haplotype may provide useful information for donor selection and individual application of immunosuppressive agents and immunotherapy.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Disclosures

No relevant conflicts of interest to declare.